The Role of Human α7-Nicotinic Acetylcholine Receptors in

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Medical Education Dissertations Medical Education

11-2018

The Role of Human α7-Nicotinic Acetylcholine Receptors in The Role of Human 7-Nicotinic Acetylcholine Receptors in

Mediating Neuroprotective Action of Curcumin Mediating Neuroprotective Action of Curcumin

Eslam Mohammed Gaber ElNebrisi

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Recommended Citation Recommended Citation Gaber ElNebrisi, Eslam Mohammed, "The Role of Human α7-Nicotinic Acetylcholine Receptors in Mediating Neuroprotective Action of Curcumin" (2018). Medical Education Dissertations. 3. https://scholarworks.uaeu.ac.ae/med_ed_dissertations/3

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UAEU • rt 1i! )) 0 .l..:l1.a.J I ~ J.SU I L:.IIJ Lo V I Ci.sUJ ~ '\:)' United Arab Emirates University

United Arab E1nirates University

College of Medicine and Health Sciences

THE ROLE OF HUMAN a7-NICOTINIC ACETYLCHOLINE RECEPTORS IN MEDIATING NEUROPROTECTIVE ACTION OF

CURCUMJN

Eslam Mohammed Gaber ElNebrisi

This thesis is submitted in partial fulfilment of the requirements for the degree of

Doctor of Philosophy

Under supervision of Professor Murat Oz

November 2018

vii

Abstract

Curcumin is a polyphenolic compound isolated from the rhizomes of Curcuma

longa. Curcumin has been demonstrated to have antioxidant, anti-inflammatory, and

anticancer properties. Moreover, it has been shown to exhibit beneficial effects in the

treatment of several neurodegenerative diseases including Alzheimer’s and

Parkinson’s diseases. However, the molecular and cellular targets mediating the

pharmacological actions of curcumin remain largely unknown. In this study, the

effects of curcumin application on the functional properties of the nicotinic

acetylcholine receptor, a prototype for ligand gated ion channels were investigated.

Using the two-electrode voltage clamp technique, the results showed that curcumin

co-application caused a significant potentiation of the action of human α7-nicotinic

acetylcholine receptors (α7-nAChR) expressed in Xenopus oocytes. Importantly,

curcumin was found less effective on other nicotinic receptor subunit combinations

and other members of ligand-gated ion channels. Curcumin significantly decreased

desensitization of α7-nAChR suggesting that it acts as a type II PAM. High affinity

binding site for curcumin on α7-nAChR was also verified by molecular docking study.

In the second part of this study, the neuroprotective effects of curcumin in an

animal model of Parkinson’s disease was investigated. Stereotaxic micro-neurosurgery

was successfully established (for the first time in our university) and used to induce

the toxin based (6-hydroxydopamine; 6-OHDA) animal model of Parkinson’s disease.

This was followed by testing the behavior of the animals, tissue collection,

immunohistochemistry, striatal fiber density measurement, and stereological data

analysis. Systemic administration of curcumin alleviated 6-OHDA-induced motor

abnormalities and protected against substantia nigra pars compacta dopaminergic

neuronal loss, through an α7-nAChRs-mediated mechanism. The protective effects of

curcumin were completely reversed by the administration of the α7-nAChR-selective

antagonist methyllycaconitine (MLA).

In summary, the results of this study suggest that α7-nAChR-mediated activation

is an important mechanism for the neuroprotective effects of curcumin in toxin-based

(6-hydroxydopmine; 6-OHDA) animal model of Parkinson’s disease.

Keywords: Curcumin, α7-nAChR, Xenopus oocytes, Parkinson’s disease, 6-OHDA –

neuroprotection

viii

Title and Abstract (in Arabic)

خصائص تفعيل في في مستقبلات الأسيتايل كولين النيكوتينية 7دور وحدات ألفا

الكركم الوقائية للجهاز العصبي

الملخص

تتميز نبتة م. يعتبر الكركم أحد مركبات البولبفينول والمستخلص من جذمور نبات الكرك

لسرطان. وزيادة على ذلك، لمضاد كذلك، و صائص: مضادة للالتهابات، الأكسدةخبعدة الكركم

أمراض ج عدد من الأمراض مثلعرفت هذه النبتة بخصائص دوائية واستخدمت بفاعلية لعلا

ولقد وضعت عدة فرضيات لآلية عمل الكركم التنكس العصبي كداء الزهايمر وداء باركنسون.

وفي هذا السياق ية للنبات.إما عن طريق القنوات الأيونية أو النواقل لكل هذه الخصائص الدوائ

المرتبطة بربيطة أيونية محددة.قمنا بدراسة تأثير مادة الكركم على القنوات الأيونية

وظيفة مستقبلات الأسيتايل كولين النيكوتينية المكونة من لقد بدأنا بدراسة تأثير الكركم على

ثبيت الجهد خدام تقنية ت( و ذلك باستXenopusو المستنسخة في بويضات ضفادع ) 7وحدات ألفا

كهروفسيولوجية استتنتجنا أن مادة التجارب الالكهربائي باستخدام قطبين كهربائيين. من خلال

. 7المكونة من وحدات ألفا مستقبلات الأسيتايل كولين النيكوتينية الكركم لها تأثير محفز على

ية المرتبطة القنوات الأيونموعة وهذا التأثير خاص فقط بهذه المستقبلات دون غيرها من بقية مج

مستقبلات مستقلبات مادة الكركم على ات وثم قمنا بدراسة تأثير مشتق .بربيطة أيونية محددة

لهذه يضا أوقد أعطت تأثير محفز 7المكونة من وحدات ألفا الأسيتايل كولين النيكوتينية

المستقبلات ولكن بدرجة أقل من مادة الكركم.

مادة ل الوقائي تأثيرالفي الجزء الثاني من البحث بدراسة قمنا المعطيات، فإنناوبناءا على هذه

المرض وتطور الكركم على نموذج داء باركنسون. وذلك باستخدام الجرذان وتحريض حدوث

وحقن مادة -للمرة الأولى بجامعة دولة الإمارات العربية المتحدة-من خلال جراحة المخ الدقيقة

قد تبع ذلك اختبار سلوك الحيوانات، جمع الأنسجة، و .هيدروكسيدوبامين-6 الأعصاب السامة

أثبتت النتائج أن الكركم يتمتع . والكيمياء المناعية، وتحليل البيانات المجسمة لجمع النتائج

الأسيتايل كولين النيكوتينية المكونة من عن طريق مستقبلات بخصائص وقائية للجهاز العصبي

( MLAالتأثيرات الوقائية للكركم بإعطاء دواء ميثايلكاكونيتين )تم عكس كما 7وحدات ألفا

.7المضاد الانتقائي لوحدات الألفا

ix

وذات هو آلية مهمة باستخدام الكركم 7 وحدات ألفاتشير نتائج هذه الدراسة إلى أن تنشيط

.لمرض باركنسونفي النموذج الحيواني تأثير وقائي على الجهاز العصبي

المكونة من وحدات ألفا نةمستقبلات الأسيتايل كولين النيكوتي ،كمرالك الرئيسية: البحثم مفاهي

وقاية الجهاز العصبي ،هيدروكسيدوبامين-6 ،داء باركنسون ،7

x

Acknowledgments

First and foremost, I would like to thank my advisors Professor Murat Oz and

Professor Safa Shehab. Professor Oz, you have been encouraging me since I was a

Master student, you were abundantly helpful and offered me invaluable patience,

support and guidance. I would like to thank you for encouraging my research and for

allowing me to grow as a research scientist.

Prof. Shehab, Thank you for the opportunity to work for and learn from you. Thanks for

the patient guidance, encouragement and advice you provided throughout my time as

student in your lab. I consider myself extremely lucky to have a supervisor who cared

so much about my work, and who responded to my questions and queries so promptly.

I must express my deepest gratitude and thanks to Ahmed, my husband, for

supporting me spiritually throughout my study journey. I was continually impressed

by his patience and constant encouragement he provided me with. My lovely kids;

Bara’a, Anas, Rayan, and Yazan, for being patient enough to delay so many activities,

trips, and holidays till I graduate. My special appreciation and thanks to my beloved

family; my father and mother and all my brothers and sisters for their continual prayers

& endless love. I would like to thank my mother and father-in-law, for their daily

prayers every morning.

My thesis committee guided me through all the three years. I would like to convey

my special thanks to the members of the Advisory committee; Dr. Ojha, for his timely

suggestion and valuable contribution at every stage of the research, including both in-

vitro and in-vivo parts. Dr. Bassem Sadek, whom without his knowledge and

assistance this study would not have been successful.

xi

My special thanks are extended to Professor Bassam Ali, the coordinator of the

post-graduate studies for his full support in the past one year. In addition, I must also

appreciate all the guidance and support I have received from Dr. Maryam Al Shamsi,

the former-Assistant Dean of Research of the College of Medicine and Health

Sciences.

I would like to express my sincere gratitude to Professor Eric PK Mensah-Brown

for inspiring me to think bigger, for never-ending support, and for the great effort in

reviewing my thesis.

I would like to extend my thanks to my dearest friends Arwa Al Nahdi, Nermin

Essa, and Shaima Fikri who have given their heart whelming full support all the time.

My thanks also go out of CMHS to AAU, who so kindly participated in this

research by giving generously of their time and collaborating for molecular docking

experiments.

I am grateful to all of those with whom I have had the pleasure to learn during my

study, especial thanks go to Dr. Nassruddin Hammadi for providing me necessary

technical assistance in Animal House and his prompt inspiration. I would like to thank

Dr. Syed Muhammad Nurulain, Mrs. Petrilla Jayaprakash, Dr. Hayate Javid, Mrs.

Anjana Valappil, and Mrs. Sumisha Rehmathulla, for their kind help and co-operation

throughout my work in Prof. Oz and Prof. Shehab laboratories.

Above all, before all and after all, all praise be to Allah for the strength that keeps

me standing and for the hope that keeps me believing that I can do this and still more.

xii

Dedication

To my beloved husband, parents, siblings and

all my supportive family

xiii

Table of Contents

Title ............................................................................................................................... i

Declaration of Original Work ...................................................................................... ii

Copyright .................................................................................................................... iii

Advisory Committee ................................................................................................... iv

Approval of the Doctorate Dissertation ....................................................................... v

Abstract ...................................................................................................................... vii

Title and Abstract (in Arabic) ................................................................................... viii

Acknowledgments ........................................................................................................ x

Dedication .................................................................................................................. xii

Table of Contents ...................................................................................................... xiii

List of Tables........................................................................................................... xviii

List of Figures ........................................................................................................... xix

List of Abbreviations................................................................................................ xxii

Chapter 1: Introduction ................................................................................................ 1

1.1 Curcumin .................................................................................................... 1

1.1.1 Natural Curcumin Analogues and Metabolites .................................... 2

1.1.2 Pharmacokinetics and Pharmacodynamics of Curcumin ...................... 4

1.1.3 Molecular Targets of Curcumin ............................................................ 6

1.1.4 Biological Properties of Curcumin ....................................................... 7

1.1.5 Effects of Curcumin on Different Ion Channels and

Receptors ............................................................................................ 12

1.2 Acetylcholine Receptors .......................................................................... 13

1.2.1 Nicotinic Acetylcholine Receptors ..................................................... 14

1.3 Parkinson’s Disease .................................................................................. 24

1.3.1 Background ......................................................................................... 25

1.3.2 Pathophysiology .................................................................................. 25

xiv

1.3.3 Diagnosis............................................................................................. 27

1.3.4 Treatment ............................................................................................ 31

1.3.5 Prognosis ............................................................................................. 34

1.4 The Neuroprotective Role of Nicotine and Nicotinic Receptors

Against Nigrostriatal Damage .................................................................. 34

1.4.1 Dopaminergic and Cholinergic Systems Correlation and

Dopamine Release .............................................................................. 35

1.4.2 Immune Modulation via Nicotinic Receptors ..................................... 41

1.4.3 Effect of Nicotinic Receptors on L-Dopa Induced

Dyskinesia .......................................................................................... 46

1.4.4 Molecular Neuroprotective Mechanisms of Nicotinic

Acetylcholine Receptors ..................................................................... 52

1.5 Animal Models of Parkinson’s Disease ................................................... 55

1.5.1 The Neurotoxin Model ........................................................................ 55

1.5.2 Genetic Models ................................................................................... 64

1.6 Fundamental Methods of Assessing Structure and Function of

Nigrostriatal Pathway ............................................................................... 67

1.6.1 Dopaminergic Neurons in the Substantia Nigra Pars

Compacta ............................................................................................ 67

1.6.2 Dopaminergic Terminals in the Striatum ............................................ 69

1.6.3 Striatal Dopamine ............................................................................... 69

1.6.4 Lewy Body Aggregates ....................................................................... 69

1.6.5 Behavioral/Motor Assessment ............................................................ 69

Chapter 2: Aims and Objectives ................................................................................ 70

2.1 In-vitro Electrophysiological Study ......................................................... 70

2.2 In-vivo Study ............................................................................................ 70

Chapter 3: Materials and Methods ............................................................................. 72

3.1 Electrophysiological In-vitro Study ......................................................... 72

3.1.1 Female Xenopus Oocytes .................................................................... 72

3.1.2 Chemicals ............................................................................................ 73

3.1.3 Other Materials ................................................................................... 74

3.1.4 Experimental Setup ............................................................................. 75

xv

3.1.5 Preparation of Required Solutions...................................................... 78

3.1.6 Drug Application ................................................................................ 80

3.1.7 Isolation and Maintenance of Oocyte from Xenopus

Laevis ................................................................................................. 81

3.1.8 Oocyte Preparation.............................................................................. 83

3.1.9 Synthesis of cRNA .............................................................................. 85

3.1.10 In-vitro cRNA Synthesis ................................................................... 86

3.1.11 Microinjection of cRNA into Oocytes .............................................. 87

3.1.12 Two Electrode Voltage Clamp.......................................................... 90

3.1.13 Parameters Tested by Electrophysiological Recording .................... 93

3.1.14 Statistical Analysis .......................................................................... 100

3.2 Molecular Docking Experiments............................................................ 101

3.3 In-vivo Study: Animal Model of Parkinson’s Disease ........................... 102

3.3.1 Animals ............................................................................................. 102

3.3.2 Drugs ................................................................................................. 102

3.3.3 Surgical Procedure ............................................................................ 103

3.3.4 Apomorphine-Induced Rotational Behavior ..................................... 107

3.3.5 Histology ........................................................................................... 107

3.3.6 Measurement of Striatal Fiber Density ............................................. 108

3.3.7 Stereological Analysis ...................................................................... 109

3.3.8 Statistical Analysis ............................................................................ 110

Chapter 4: Results .................................................................................................... 111

4.1 Results .................................................................................................... 111

4.1.1 Effects of Curcumin on α7-nicotinic Acetylcholine

Receptors .......................................................................................... 111

4.1.2 Concentration Response Curve ......................................................... 114

4.1.3 Effects of Curcumin on α7-nAChRs are not Mediated by

G-proteins ......................................................................................... 116

4.1.4 Effects of Curcumin on α7-nAChRs are not Mediated by

Protein Kinases ................................................................................. 118

4.1.5 Effects of Curcumin on α7-nAChRs are not Dependent

on Intracellular Ca2+ ......................................................................... 120

xvi

4.1.6 Effects of Curcumin are not Dependent on Changes in

Membrane Potential.......................................................................... 121

4.1.7 Effects of Curcumin at Different Concentrations of

Acetylcholine .................................................................................... 123

4.1.8 Effects of Curcumin on the Desensitization of Nicotinic

Receptors .......................................................................................... 126

4.1.9 Effects of Curcumin on the Specific Binding of [125I]α-

bungarotoxin ..................................................................................... 128

4.1.10 Effects of Curcumin on the Current Mediated by

Different Nicotinic Receptor Subunits and Other

Members of Ligand-Gated Ion Channels ......................................... 130

4.1.11 Effects of Other Curcumin’s Analogues and

Metabolites on the Current Mediated by α7 Nicotinic

Acetylcholine Receptors ................................................................... 132

4.1.12 Docking of Curcumin and Curcumin Derivatives into

the Human α7-nAChR Transmembrane Domain ............................. 134

4.2 In-vivo Results ........................................................................................ 137

4.2.1 Apomorphine-Induced Rotation Test ............................................... 137

4.2.2 Morphological Analysis .................................................................... 140

Chapter 5: Discussion .............................................................................................. 148

5.1 Discussion .............................................................................................. 148

5.1.1 Effects of Curcumin on α7-Nicotinic Acetylcholine

Receptor ............................................................................................ 148

5.1.2 Effects of Curcumin on α7-Nicotinic Receptor are not

Mediated by G-proteins and Protein Kinases, and are

not Dependent on Intracellular Ca2+ Levels, and

Membrane Potential.......................................................................... 149

5.1.3 Effects of Curcumin at Different Concentrations of

Acetylcholine .................................................................................... 151

5.1.4 Effects of Curcumin on the Specific Binding of [125I]α-

bungarotoxin ..................................................................................... 152

5.1.5 Effects of Curcumin on Desensitization of Nicotinic

Receptors .......................................................................................... 153

5.1.6 Docking of Curcumin and Curcumin Derivatives into

the Human α7-nAChR Transmembrane Domain ............................. 154

xvii

5.1.7 Neuroprotective Properties of Curcumin in Parkinson’s

Disease .............................................................................................. 156

Chapter 6: Conclusions ............................................................................................ 170

References ................................................................................................................ 172

List of Publications .................................................................................................. 223

Appendix .................................................................................................................. 224

xviii

List of Tables

Table 1: Curcumin analogues and metabolites ............................................................ 3

Table 2: Motor and non-motor symptoms of Parkinson’s disease ............................. 28

Table 3: UK Parkinson’s disease society Brain Bank clinical

diagnosing criteria ........................................................................................ 30

Table 4: Treatment options of Parkinson’s disease.................................................... 32

Table 5: Chemicals required for the experiments ...................................................... 73

Table 6: Other materials and devices used in the study ............................................. 74

Table 7: Calcium free MBS solution composition ..................................................... 78

Table 8: Antibiotic materials ...................................................................................... 79

Table 9: ND96 solution composition ......................................................................... 79

Table 10: Normal Ringer’s solution composition ...................................................... 80

Table 11: The initial concentration of all subunits .................................................... 88

Table 12: Binding energies of curcumin and curcumin derivatives,

generated from their docking into the human α7-nAChR

transmembrane domain, along with the docking scores of

two known type II PAMs......................................................................... 135

Table 13: nAChRs and PAMs in clinical trials for treatment of PD

(“Home - ClinicalTrials.gov,” n.d.) ......................................................... 166

Table 14: Curcumin in clinical trials for treatment of various

neurodegenerative disorders (“Home - ClinicalTrials.gov,”

n.d.) .......................................................................................................... 168

xix

List of Figures

Figure 1: The source and chemical structure of curcumin ........................................... 1

Figure 2: Molecular targets of curcumin ...................................................................... 7

Figure 3: Therapeutic potential of curcumin ................................................................ 8

Figure 4: Curcumin structural features ...................................................................... 10

Figure 5: The activated forms of the acetylcholine receptor classes ......................... 13

Figure 6: Neuronal nicotinic acetylcholine structure ................................................. 15

Figure 7: Proposed mechanism of activation and desensitization ............................. 17

Figure 8: Types of Allosteric modulators .................................................................. 19

Figure 9: Molecular activation routes of the α7-nAChRs .......................................... 20

Figure 10: Distribution of nicotinic acetylcholine receptors human

brain .......................................................................................................... 22

Figure 11: Proposed mechanism of α7-nAChRs in Parkinson’s

disease ...................................................................................................... 23

Figure 12: Structures of the basal ganglia .................................................................. 24

Figure 13: Lewy body in affected dopaminergic neurons ......................................... 26

Figure 14: Pathophysiology of Parkinson’s disease................................................... 27

Figure 15: Schematic presentation of pathophysiology and treatment

of PD ........................................................................................................ 33

Figure 16: Effect of nicotine treatment on MPTP- lesioned primates ....................... 37

Figure 17: Striatal [3H]dopamine release of wild type and α7-

nicotinic receptor null mutant mice .......................................................... 38

Figure 18: Stimulation of dopamine release in in Drosophila

melanogaster ventral nerve cord (VNC) .................................................. 39

Figure 19: Data of Acetylcholine stimulated dopamine release before

and after bathing with different nicotinic and muscarinic

antagonists ................................................................................................ 40

Figure 20: The role of microglia in health and disease .............................................. 41

Figure 21: Staining of Primary human macrophages with fluorescein

isothiocyanate (FITC)-labelled α-bungarotoxin (α -Bgt,

1.5 mgml21) ............................................................................................. 42

Figure 22: RT–PCR analysis of α7-nAChRs expression on microglia

using N9 and primary cultured microglial cells ....................................... 44

Figure 23: Nicotine inhibits H2O2-induced astrocyte apoptosis

through protection of mitochondrial membrane potential ....................... 45

Figure 24: Nicotine administration reduces L-dopa-induced

dyskinetic-like movements in rats and monkeys ..................................... 48

Figure 25: Effect of TC-8831 and amantadine in combination with L-

DOPA in MPTP-lesioned monkeys ......................................................... 50

Figure 26: Effect of ABT-126 on LID in MPTP treated monkeys ............................ 51

xx

Figure 27 : α7-nicotinic acetylcholine-mediated molecular signaling

mechanism ................................................................................................ 54

Figure 28: Chemical structures of 6-hydroxydopamine (6-OHDA)

and dopamine ........................................................................................... 56

Figure 29: Molecular Mechanisms for different animal models of PD ..................... 58

Figure 30: Chemical structures of MPTP and MPP+ ................................................ 61

Figure 31: Dopaminergic neuronal distribution in striatum and

substantia nigra ......................................................................................... 68

Figure 32: An adult female Xenopus laevis (Professor Murat Oz’s

laboratory) ................................................................................................ 72

Figure 33: Two-electrode voltage-clamp (TEVC) recording set-up

from Xenopus oocytes .............................................................................. 76

Figure 34: The oocyte impaled with two microelectrodes ......................................... 77

Figure 35: Steps of oocyte isolation and preparation ................................................. 82

Figure 36: Frog’s ovarian lobe ................................................................................... 84

Figure 37: Stages of oocyte development .................................................................. 85

Figure 38: Agarose gel analysis of mRNA ................................................................ 86

Figure 39: Microelectrode set-up for cRNA injection ............................................... 89

Figure 40: cRNA injection set-up (Professor Murat Oz’s Laboratory) ..................... 89

Figure 41: Schematic of glass microelectrode assembly ........................................... 91

Figure 42: Schematic illustration of two-electrode voltage clamp

setup using Xenopus oocytes .................................................................... 92

Figure 43: Typical experimental protocol for electrophysiological

recording from oocyte .............................................................................. 94

Figure 44: Radioligand binding assay ........................................................................ 99

Figure 45: Time course of the experiment ............................................................... 104

Figure 46: Stereotaxic surgery to lesion nigrostriatal pathway ................................ 105

Figure 47: The three sites of 6-OHDA intra-striatal injection ................................. 106

Figure 48: The effects of acetylcholine and α-bungarotoxin in

oocytes expressing α7-nAChR ............................................................... 112

Figure 49: Effects of curcumin on α7-nicotinic acetylcholine

receptors ................................................................................................. 113

Figure 50: Effect of curcumin on α7-nicotinic acetylcholine receptors

is time- and concentration-dependent .................................................... 115

Figure 51: Effects of curcumin on α7-nAChR are not mediated by G-

proteins ................................................................................................... 117

Figure 52: Effects of curcumin on α7-nAChR are not mediated by

prtein kinases .......................................................................................... 119

Figure 53: Effects of curcumin on α7-nAChR are not dependent on

intracellular Ca2+ levels .......................................................................... 121

Figure 54: Effects of curcumin are not dependent on changes in

membrane potential ................................................................................ 122

xxi

Figure 55: Effects of curcumin at different concentrations of

acetylcholine .......................................................................................... 124

Figure 56: Acetylcholine concentration response curve .......................................... 125

Figure 57: Effect of curcumin on the desensitization of nicotinic

receptors ................................................................................................. 127

Figure 58: Effects of curcumin on the specific binding of [125I]α-

bungarotoxin .......................................................................................... 129

Figure 59: Effects of curcumin on the current mediated by different

nicotinic receptor subunits and other members of LGICs ..................... 131

Figure 60: Effects of curcumin analogues and metabolites on

Acetylcholine-mediated current ............................................................. 133

Figure 61: The binding mode of curcumin (cyan sticks) obtained

from docking into the human α7-nAChR transmembrane

domain (gray sticks) ............................................................................... 136

Figure 62: Motor performance of the rats was assessed using

apomorphine-induced rotation test (0.25 mg/kg)

expressed as full body turn per minute over 30 min .............................. 138

Figure 63: Apomorphine-induced rotation test in 6-OHDA injected

rats before and after MLA I.P injection ................................................. 139

Figure 64: Photographs of TH immunoreactive fibers ............................................ 141

Figure 65: Striatal TH-immunoreactive fiber density expressed as a

percentage of the fiber density on the lesioned side to the

non-lesioned side .................................................................................... 142

Figure 66: Photomicrographs of coronal sections of SN for TH

immunohistochemistry ........................................................................... 145

Figure 67: Stereological assessment of total numbers of TH-positive

cell bodies in the SN at all three levels; rostral, middle,

and caudal ............................................................................................... 147

Figure 68: Neuroprotective mechanisms of curcumin in PD ................................... 158

Figure 69: Drug-induced rotation test in rats that had 6-OHDA

injection in the right striatum ................................................................. 160

Figure 70: Hypothetical model of Ca2+- dependent cell survival

mechanism .............................................................................................. 165

xxii

List of Abbreviations

ABC Avidin–biotin-complex

AD Alzheimer's disease

ACh Acetylcholine

AP Antro-posterior

AUC Area under the curve

Ba2+ Barium

ANOVA Analysis of variance

BAPTA 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid

BDNF Brain-derived neurotrophic factor

BDMC Bisdemethoxycurcumin

Bcl-2 B-cell lymphoma 2

BDMC Bisdemethoxycurcumin

Ca2+ Calcium

CaCCs Ca2+ activated Cl- channels

CaM Calcium effector protein calmodulin

CD Cyclodextrin

CDK Cycline-dependent kinase

CMC Carboxy methyl cellulose

CNS Central nervous system

COMTi Catechol-O-methyl transferase inhibitor

COX-2 Cycloxygenase-2

CPu Caudate–putamen

CREB cAMP response element-binding

CUR-SL Curcumin-loaded silica liposomes

CUR-FL Curcumin-loaded flexible liposomes

DA Dopamine

DAB Diaminobenzidine

DAT Dopamine transporters

DBS Deep brain stimulation

DhβE Dihydro-β-erythroidine hydrobromide

xxiii

DMC Demethoxycurcumin

DMSO Dimethyl sulfoxide

DV Dorso-ventral

ECD Extracellular domain

ERK/MAPK Extracellular signal-regulated mitogen-activated protein kinase

FGF-2 Fibroblast growth factor-2

GABA G-aminobutyric acid

GDPβS Guanyl-5'-yl thiophosphate; guanosine 5'-(trihydrogen

3-thiodiphosphate 5'-O-(2-thiodiphosphate); 71376-97-1;)

Go-6983 3-[1-[3-(Dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-

(1Hindol-3-yl)-1H-pyrrole-2,5-dione

GPe Globus pallidus external segment

GPi Globus pallidus internal segment

GPx Glutathione peroxidase

HO-1 Hemoxygenase-1

H2O2 Hydrogen Peroxide

HPLC High-performance liquid chromatography

Im Membrane current

ICD Intracellular domain

IGF Insuline-like growth factor

IL Interleukin

iNOS Inducible nitric oxide synthase

IP Intraperitoneal

ISO Isoprenaline

I-V Current-voltage relationships

JAK2 Janus kinase 2

KN-62 l-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-∼-tyrosyl]

-4- phenylpiperazine

KT-5720 (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-

methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg,39,29,19-kl]

pyrrolo[3,4- i][1,6]benzodiazocine-10-carboxylic acid, hexyl ester

LB Lewy body

xxiv

L-dopa Levodopa

LEC Liposome-encapsulated curcumin

LID L-dopa-induced dyskinesias

LN Lewy neurites

LPS Lipopolysaccharides

LRRK2 Leucine rich repeat kinase 2

mAChR Muscarinic acetylcholine receptor

MBS Modified barth’s solution

MDMA 3,4-methylenedioxymethamphetamine

MFB Medial forebrain bundle

ML Medio-lateral

MLA Methyllycaconitine

MPP+ 1-methyl-4-phenylpyridinium

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

mRNA messenger RNA

NAc Nucleus accumbens

nAChR Nicotinic acetylcholine receptor

NADPH Nicotinamide adenine dinucleotide phosphate hydrogen

NAM Negative allosteric modulator

NEM N-ethylmaleimide

NF-kB Nuclear factor-kappaB

NGF Nerve growth factor

NMS Non-motor symptoms

PAM Positive allosteric modulator

PB Phosphate buffer

PBS Phosphate buffered saline

PCA P-chloroamphetamine

PCR Polymerase chain reaction

PD Parkinson’s disease

PET Positron emission tomography

PINK1 Phosphatase and tensin homolog- induced novel kinase 1

PKA Protein kinase A

xxv

PKC-412 tyrosine kinase inhibitor

PLGA Polylactic-co-glycolic acid

pLGIC Pentameric ligand-gated ion channels

PPAR Peroxisome-proliferator activated receptor

PTEN Phasphatase and tensin homolog

PTX Pertussis toxin

RNA Ribonucleic acid

ROS Reactive oxygen species

SAM Silent allosteric modulator

SN Substantia nigra

SNpc Substantia nigra pars compacta

SNpr Substantia nigra pars reticulata

SOD Superoxide dismutase

STAT Signal transducer and activator of transcription

STN Subthalamic nucleus

TGF Transforming growth factor

TH Tyrosine hydroxylase

THC Tetrahydrocurcumin

TMD Transmembrane domain

TNF- α Tumor necrosis factor- α

Vc Command potential

Vm Membrane potential

VMAT Vesicular monoamine transporter

VTA Ventral tegmental area

5-HT Serotonin or 5-hydroxytryptamine

ΔΨm Mitochondrial membrane potential

1

Chapter 1: Introduction

1.1 Curcumin

Curcumin is a polyphenolic compound, the main ingredient of turmeric (Curcuma

longa), and a member of the ginger family (Zingiberaceae) (Aggarwal and Sung,

2009). The plant grows largely in India, China and other tropical countries (Aggarwal

et al., 2007). Vogel and Pelletier were the first to report the isolation of a “yellow

coloring-matter” from the rhizomes of Curcuma longa (turmeric) and named it

curcumin in 1815. Vogel found that turmeric is a mixture of many components and

successfully isolated a pure curcumin oil in 1842. In 1910, Milobedeska and Lampe

characterized its structure as diferuloylmethane, or 1,6-heptadiene-3,5-dione-1,7-bis

(4-hydroxy-3-methoxyphenyl)-(1E, 6E) (Figure 1), and three years later they

synthesized curcumin (Gupta et al., 2012). Curcumin exhibits keto–enol tautomerism,

where enol forms predominant in an alkaline medium while keto forms in acidic and

neutral media (Priyadarsini, 2014).

Figure 1: The source and chemical structure of curcumin.

(A) The root of turmeric. (B) Crystallized powder of curcumin. (C) The enol and keto

forms of curcumin [Modified from (Zhang et al., 2013)].

Curcuma Longa

Rhizome

Turmeric

Curcumin (Enol form)

Curcumin (Keto form)

(A) (C) (B)

2

Curcuminoid (the yellow pigmented fraction of turmeric) forms 3-5% of turmeric

and is composed mainly of three derivatives; curcumin (diferuloylmethane, curcumin

I), demethoxycurcumin (DMC, curcumin II), and bisdemethoxycurcumin (BDMC,

curcumin III) (Anand et al., 2008; Goel et al., 2008). Recently, cyclocurcumin has

been isolated as a curcuminoid component of turmeric by Kiuchi et al. (Kiuchi et al.,

1993). Among all, curcumin is the most abundant (~77%) followed by DMC (~17%)

and BDMC (~3%) (Anand et al., 2008; Goel et al., 2008). Curcumin is hydrophobic

in nature and not soluble in neutral solvent e.g: water, but soluble in organic solvents

e.g: dimethylsulfoxide (DMSO), ethanol, and acetone (Priyadarsini, 2009).

1.1.1 Natural Curcumin Analogues and Metabolites

As mentioned earlier, curcumin I, curcumin II, curcumin III, and cyclocurcumin

collectively known as curcuminoid, are considered the natural turmeric analogues

(Table 1.A - D).

There are numerous curcumin metabolites that have been reported and are

available commercially, including tetrahydrocurcumin (THC), dimethyl curcumin,

didemethyl curcumin, Vanillylidenacetone, Di-(tert-Butyl-dimethylsilyl) Curcumin,

O-tert-Butyl-dimethylsilyl Curcumin, and curcumin-d6 (Table 1.E - K).

http://www.scbt.com/search/redirect.php?location=datasheet-500729-casnumber-1134639-23-8.html&searchPhrase=curcumin&datasheet=sc-500729&tableName=&productType=&page=1


3

Table 1: Curcumin analogues and metabolites

b. Bisdemethoxycurcumin c. Demethoxycurcumin d. Cyclocurcumin

e. Tetrahydrocurcumin f. Demethyl curcumin g. Didemethyl curcumin

h. Di-(tert-Butyl-

dimethylsilyl) Curcumin

i.Vanillylidenacetone j. O-tert-Butyl

dimethylsilyl Curcumin

k. Curcumin-d6

a. Curcumin






4

1.1.2 Pharmacokinetics and Pharmacodynamics of Curcumin

Curcumin has a low bioavailability in plasma and tissue due to its poor absorption,

rapid metabolism, as well as rapid systemic elimination. Various studies have been

conducted on the pharmacokinetics and pharmacodynamics of curcumin, the first of

which was reported by Wahlstrom and Blennow 1978 in Sprague-Dawley rats.

Curcumin had poor absorption from the gut, and nearly 75% of curcumin was excreted

in the feces with negligible amounts being detected in blood plasma (Wahlström and

Blennow, 1978). Holder et al. experimented with deuterium- and tritium-labeled

curcumin given intravenously (I.V) and intra-peritoneally (I.P), and observed that

majority of curcumin was excreted in bile and then in feces (Holder et al., 1978).

Ravindranath in 1980, used three different doses (10, 80, and 400 mg/kg) of tritium-

labeled curcumin, and detected curcumin in tissue of interest after 12 days of

administration. The percent of absorbed curcumin (60-66%) remained constant with

no difference between doses indicating that increasing the dose did not increase

absorption, thus there is a dose-dependent limitation of curcumin’s bioavailability

(Ravindranath and Chandrasekhara, 1981). Pan et al. in 1999, administered 100 mg/kg

of curcumin I.P to mice to investigate the pharmacokinetics of curcumin. After one

hour of administration, curcumin level in spleen, liver, and kidney were 26.1, 26.9,

and 7.5 μg/g, respectively, and a minute trace levels (0.41 mg/g) were detected in the

brain (Pan et al., 1999).

Studies of curcumin’s pharmacokinetics in humans yielded more or less similar

data, a peak plasma level of 0.41–1.75 µM have been obtained after administration of

4 – 8 g of curcumin orally in humans (Cheng et al., 2001). After several experiments,

Perkins, 2002 concluded that humans need a daily dose of 1.6 g curcumin to produce

5

an effect (Perkins et al., 2002). Many groups have shown that the liver is the primary

site of curcumin metabolism, where it undergoes extensive reduction via alcohol

dehydrogenase, followed by conjugation (Garcea et al., 2004; Hoehle et al., 2006;

Wahlström and Blennow, 1978).

Almost all studies verified that unformulated curcumin has poor bioavailability in

animal models and humans. To improve curcumin bioavailability, different

formulations have been developed. For example, a nanocurcumin was developed to

enhance curcumin solubility in aqueous solution. Cheng et al. 2013, prepared a

nanoparticle form of curcumin that yielded a higher plasma concentration and higher

AUC by six times. Moreover, mean residence time in the brain was longer in a mice

model (Cheng et al., 2013).

In another study, Polylactic-co-glycolic acid (PLGA) is one of the forms of

formulated curcumin, was employed and was reported to enhance curcumin

bioavailability by 5.6 folds and also extended curcumin half-life. This was due to

improvement of water solubility of the compound -which is known to be highly

lipophilic as mentioned previously-, more induction of intestinal juices facilitating

ingestion, higher permeability enhancing absorption, and elongating residence time in

the intestine which allowed for more absorption (Xie et al., 2011)

Liposomal curcumin is another formulated curcumin a form of drug carrier which

helps to increase the solubility of the compound. Liposome-encapsulated curcumin

(LEC) increased curcumin bioavailability by facilitating cellular uptake and increasing

its absorption. Different forms have been generated, silica-coated flexible liposomes

loaded with curcumin (CUR-SLs) and curcumin-loaded flexible liposomes (CUR-

FLs), were found to enhance curcumin bioavailability by 7.76- and 2.35 folds higher,

respectively, compared to unformulated curcumin (Li et al., 2012).

6

Cyclodextrin (CD), is a form of cyclic oligosaccharides, which encapsulates and

facilitate its cellular uptake, bioavailability, and elongates its half-life (Prasad et al.,

2014).

CD encapsulated curcumin improves curcumin permeability 1.8 fold across skin

in animal model compared to unformulated curcumin (Rachmawati et al., 2013).

Curcumin bioavailability can be enhanced up to 2000% in humans and to 154% in

rats, by administrating piperine (a component derived from pepper and a known

inhibitor of hepatic and intestinal glucuronidation) along with curcumin. Concomitant

piperine administration with curcumin significantly decreased elimination and half-

life clearance of curcumin (Anand et al., 2007; Shoba et al., 1998).

1.1.3 Molecular Targets of Curcumin

Based on the extensive pieces of evidence from both in-vitro and in-vivo studies,

several molecular targets of curcumin have been identified. Curcumin interacts with

transcription factors, e.g., nuclear factor-kB (NFκB), and signal transducer and

activator of transcription (STAT) proteins (Shishodia et al., 2007), growth factors and

their receptors, e.g. epidermal growth factor receptors and HER2 (Chen et al., 2006;

Soung and Chung, 2011), cytokines, e.g., interleukin 1b (IL-1b), interleukin 6 (IL-6)

(Cho et al., 2007), enzymes, e.g., hemoxygenase-1 (HO-1) (McNally et al., 2007), and

genes regulating cell proliferation and apoptosis (Aoki et al., 2007). This ability of

curcumin to modulate or interact with multiple cell signaling pathways and proteins,

strongly indicates that this polyphenol is an effective multi-targeted compound (Figure

2) (Goel and Aggarwal, 2010; Hasima and Aggarwal, 2012; Rainey et al., 2015;

Ravindran et al., 2009). This conclusion is in line with several recently published

7

reports identifying curcumin as a potent epigenetic regulator (Kunnumakkara et al.,

2016).

Figure 2: Molecular targets of curcumin.

Multiple cellular and molecular targets of curcumin has been identified, including:

transcription factors (STAT1, PPRPδ, FOXO,…), growth factors (IGF, TGF,

CDK2,…), inflammatory biomarkers (IL6, COX2, iNOS, NFκB,…), tumor suppressor

genes (P53, PTEN, Rb) protein kinases (MAPK, AKT, PKA, PKC,…), oncoproteins

(Fos, c-Myc, c-Met), and apoptotic genes (Bax, Bcl-2, caspase 8,…) (Hasima and

Aggarwal, 2012).

1.1.4 Biological Properties of Curcumin

Curcumin has been traditionally used in Asian countries as a dietary spice and as

a medical herb for several pathologies due to its anti-inflammatory (Ammon and Wahl,

1991; Brouet and Ohshima, 1995; Dikshit et al., 1995), antioxidant antimicrobial and

8

anticancer properties (Limtrakul et al., 1997; Rao et al., 1995), anti-arthritic (Deodhar

et al., 1980), hepatoprotective (Kiso et al., 1983), anti-thrombotic (Srivastava et al.,

1985), cardio-protective (Dikshit et al., 1995; Nirmala and Puvanakrishnan, 1996a;

Srivastava et al., 1985), and hypoglycemic properties (Arun and Nalini, 2002; Babu

and Srinivasan, 1995; Srinivasan, 1972). Moreover, it has been shown to exhibit a wide

range of pharmacological activities including the treatment of several

neurodegenerative diseases such as Alzheimer and Parkinson’s diseases (Figure 3). In

the next section, we will be discussing some of the biological properties of curcumin

(Zhou et al., 2011).

Figure 3: Therapeutic potential of curcumin.

Curcumin has been shown to exhibit a wide range of pharmacological activities in

central and peripheral body systems, through its anti-oxidant, anti-inflammatory and

neuroprotective properties (Zhou et al., 2011).

9

1.1.4.1 Anti-inflammatory Effects

Inflammation is a physiological process by which our body fight against infections

triggering host immune response. It is a complex interaction that aims at removing the

invading agent or damaged tissue. Over-activation of the immune system and

inflammatory responses may cause further tissue damage (Joe et al., 2004).

Inflammation plays a major role in a number of pathological conditions including:

neurodegenerative, autoimmune, cardiovascular, endocrine, and neoplastic diseases

(Brouet and Ohshima, 1995; Liu and Hong, 2003; Nosalski and Guzik, 2017).

Interaction with and modulation of the effects of various inflammatory mediators by

curcumin demonstrated its anti-inflammatory properties (Aggarwal and Harikumar,

2009; Jurenka, 2009). Curcumin can inhibit inflammatory cytokines, interleukins

(ILs), chemokines, as well as inflammatory enzymes, cycloxygenase-2 (COX-2),

inducible nitric oxide synthase (iNOS) and cyclinD1 (Creţu et al., 2012). Curcumin

has been shown to diminish GFAP level (Yu et al., 2010), suppress NF-kβ activity,

and reduce the levels of tumor necrosis factor- α (TNF-α) (Chen et al., 2014).

1.1.4.2 Anti-oxidant Effects

It is well known that oxidative stress plays a major role in acute, chronic, and

degenerative diseases. Oxidative stress results from imbalance between formation and

neutralization of reactive oxygen species (ROS) in our body, leading to generation of

free radicals and energy failure (Pham-Huy et al., 2008). Curcumin has a strong anti-

oxidant activity compared to vitamin C and E (Toda et al., 1985). The potent activity

of curcumin against pro-oxidants such as superoxide radicals, hydrogen peroxide and

nitric oxide radical, as well as enhancing anti-oxidant enzymes such as catalase,

superoxide dismutase (SOD), glutathione peroxidase (GPx) and heme oxygenase-1

10

(OH-1) results in a decrease in lipid peroxidation and subsequently organ damage

(Jeong et al., 2006; Reddy and Lokesh, 1994, 1992). Curcumin induced heme

oxygenase-1 and protected endothelial cells against oxidative stress (Motterlini et al.,

2000). Curcumin inhibition of free radical formation protected rat myocardial tissue

from isoprenaline (ISO)-induced ischemic injury (Manikandan et al., 2004; Nirmala

and Puvanakrishnan 1996a, 1996b). Curcumin provided anti-oxidant protection

comparable to vitamin E on renal cell lines by its inhibitory effect on lipid

peroxidation, cytolysis, and lipid degradation (Cohly et al., 1998).

The methoxy and phenolic groups on benzene rings and the β-diketone moiety in

the curcumin structure (Figure 4) are thought to be the cause of its anti-oxidant

properties (Sandur et al., 2007; Sreejayan and Rao, 1996).

Figure 4: Curcumin structural features.

Curcumin has three chemical entities in its structure: two aromatic ring systems

containing O-methoxy phenolic groups, connected by a seven carbon linker, consisting

of α, β-unsaturated diketone moiety [Modified from (Bagchi et al., 2015)].

Aromatic

ring

Aromatic

ring

11

1.1.4.3 Neuroprotective Effects

Our previous discussion has shown that both the anti-oxidant and anti-

inflammatory effects of curcumin together form the basis of beneficiary effects of

curcumin in several neurological diseases affecting the central as well as the peripheral

nervous system. Curcumin as a multi-targeted compound can serve as a

neuroprotective agent. Oral administration of (50, 100 and 200 mg/kg) curcumin

protected Swiss albino mice against rotenone-induced dysfunction in the

mitochondrial respiratory chain and conserved the mitochondrial enzyme complex

(Khatri and Juvekar, 2016). Anti-oxidant properties of curcumin improved the levels

of acetylcholine esterase enzyme in mice compared with negative control animals

which was reflected on motor behavioral assessments (Khatri and Juvekar, 2016).

Alzheimer’s disease is a neurodegenerative disorder and the most common cause of

dementia worldwide. The main pathological hallmark is the aggregation of Aβ

amyloid protein plaque formation, which has not been phagocytosed due to microglial

dysfunction. Curcumin has anti-protein aggregation properties (Darvesh et al., 2012),

and could inhibit plaque formation and accumulation, and activated microglial

phagocytic activity (Cole et al., 2007; Ono et al., 2004).

1.1.4.4 Anti-cancer Effects

In 1987, Kuttan and colleagues carried out the first clinical trial to investigate the

anti-cancer properties of curcumin. He included 62 patients having external cancerous

lesions and used an ointment containing ethanol turmeric extract. Patients who

received this treatment reported a significant improvement in their symptoms of pain,

itching, smell, and lesion size (Kuttan et al., 1987). Since this study, several other trials

have been conducted on different types of cancer insuring the dose dependent chemo-

12

preventive effect of curcumin in head and neck, breast, gastrointestinal (colon,

pancreatic, stomach, esophageal and oral carcinogenesis), and cervical cancers (Bayet-

Robert et al., 2010; Cao et al., 2016; Carroll et al., 2011; Cheng et al., 2001; Epelbaum

et al., 2010; Ghalaut et al., 2012; Kim et al., 2011). Curcumin was not tested as a single

anti-cancer agent only, but also as an adjuvant anti-tumor agent and to reduce adverse

effects of other chemotherapeutics (Belcaro et al., 2014; Garcea et al., 2005).

Curcumin can suppress carcinogenesis at different stages of promotion, angiogenesis,

and growth (Conney et al., 1991; Huang et al., 1992; Robinson et al., 2003).

1.1.5 Effects of Curcumin on Different Ion Channels and Receptors

Depending on the above discussion, several types of ligand-gated ion channels and

receptors have been suggested to be involved in mediating pharmacological actions of

curcumin. In this study, we are investigating the effect of curcumin application on the

functional properties of α7-nicotnic acetylcholine receptors mainly and other ligand

gated ion channels.

13

1.2 Acetylcholine Receptors

Acetylcholine (ACh) is one of the key neurotransmitters in the central and

peripheral nervous systems. It can bind and transmit signals through two types of

receptors, classified by their sensitivity to either muscarine or nicotine; muscarinic

acetylcholine receptors (mAChRs) and nicotinic acetylcholine receptors (nAChRs).

Muscarinic acetylcholine receptors are a family of G-protein-coupled receptors,

whereas nicotinic acetylcholine receptors are ligand-gated ion channels (LGIC) as

shown in Figure 5 (Hurst et al., 2013). Muscarinic receptors are involved in several

physiological functions namely heart rate, force of contraction of smooth muscle and

act as the main end-receptor stimulated by ACh. Their signals are relatively slow and

evolve over seconds to minutes. On the other hand, nicotinic receptors, respond to

endogenous ACh in muscle, autonomic ganglia, and the brain, and mediate fast

synaptic transmission in a millisecond time frame.

Figure 5: The activated forms of the acetylcholine receptor classes.

Nicotinic ligand-gated ion channels and muscarinic G-protein coupled receptors. For

each receptor, the non-overlapping binding sites: orthosteric and allosteric (De Smet

et al., 2014).

14

The central role of nicotinic receptors in converting chemical stimuli into electrical

signals has involved them broadly in a broad range of physiological functions

including muscle contraction, brain development, cognitive function, learning and

memory, arousal, reward, motor control, analgesia, synaptic plasticity as well as

pathological disorders including Alzheimer's disease, Parkinson's disease, epilepsy

and schizophrenia (Jensen et al., 2005; Lindstrom, 2003; Mineur and Picciotto, 2008;

Posadas et al., 2013). These receptors are the target of pharmacologically administered

nicotine.

1.2.1 Nicotinic Acetylcholine Receptors

Early in the twentieth century, nicotine became a fundamental molecule in the basic

science of pharmacology. In 1905, Langley reported that body muscles contract via a

“receptive substance” in muscles. The identification of the muscle nicotinic

acetylcholine receptors paved the way for the discovery of neurotransmitter receptors

(Langley, 1905). But it was not until 1970s when neuronal nAChRs, were identified

(Changeux et al., 1970; Miledi and Potter, 1971). A decade later, the extended family

of nicotinic receptor has been identified (Patrick et al., 1983).

Nicotinic acetylcholine receptors (nAChRs) were the first of all neurotransmitters

to be identified biochemically and functionally (Lindstrom, 2003). nAChRs are

members of a structurally related family of ligand gated ion channels that also include

receptors for neurotransmitters such as 5-hydroxytryptamine (5-HT), g-aminobutyric

acid (GABA), and glycine (Albuquerque et al., 2009; Hendrickson et al., 2013).

Initially, this class of receptors was named the Cys-loop family as all receptors contain

a conserved two disulfide-bonds cysteines separated by 13 amino acids in their

extracellular amino terminus (Figure 6A). Recent discovery of these receptors in

15

prokaryotic cells but lacking the character of Cys-loop led to the change the in name

from Cys-loop family to pentameric Ligand-gated ion channels (pLGIC) (Tasneem et

al., 2005).

Figure 6: Neuronal nicotinic acetylcholine structure.

(A) Each nAChR subunit contains four transmembrane domains (M1-M4), an

extracellular NH2- and COOH-terminal, and a prominent M3-M4 intracellular loop of

variable length. (B) Five subunits co-assemble to form a functional subunit. (C)

Homomeric receptors consist of α subunits. (D) Majority of nAChRs are heteromeric

and consist of a combination of α and β subunits. Multiple α subunits may co-assemble

with multiple β subunits in the pentameric nAChR complex (illustrated here by

α6β3β2). ACh binding sites are represented as red triangles [Modified from:

(Hendrickson et al., 2013)].

All nAChR subunits have an: 1) extracellular domain (ECD) approximately ̴ 200

amino acid long and hydrophilic in nature, 2) four transmembrane domains (TMD)

which are hydrophobic in nature (M1 – M4), 3) the intracellular domain (ICD) which

varies in length between different subunits, and lastly 4) an extracellular carboxy

terminal (Figure 6A) (Albuquerque et al., 2009). In general, the ECD is the site of

agonist (ACh) binding, TMD contains the allosteric binding site and is responsible for

Cys-loop

16

the ion pore, permeability and selectivity (especially M2 which is conserved

throughout LGIC), and ICD controls channel conductance (Albuquerque et al., 2009;

Changeux, 2010; Jones et al., 2010; King et al., 2015; Paulo et al., 2009).

Nicotinic acetylcholine receptors have a pentameric structure consisting of five

transmembrane subunits around a central water-filled pore selective for cation (Figure

6B). To date, 16 distinct subunits of nAchRs have been identified in the human

proteome. Subunits are divided into two subgroups, the α and β subunits of which 5

nAChR subunits that are expressed in muscle (α1, β1, γ, δ, and ε) and 11 nAChR

subunits are expressed in nervous tissue (α2-7*, α9, α10, β2-4). This study mainly

focuses on neuronal types of nAChRs. Each nAChR can be either a homomeric,

formed by five identical subunits or heteromeric receptor that result from the

combination of different subunits (Figures 6C & D). The nomenclature for the genes

that encode the nAChR subunits is CHRNxy where CHRN stands for cholinergic

receptor, nicotinic, and xy represents the subunit. For example, CHRNB4 is the gene

for the β4 subunit. The α7 neuronal nicotinic receptor gene, CHRNA7 is located on

the long arm of Chromosome 15, is widely expressed in both the brain (Sinkus et al.,

2015). CHRNA7 was first identified in chicken, α7 subunit immediately attracted

much interest of physiologists and geneticists, since it forms functional homomeric

receptors and has unique features in terms of its 1) genomic structure, 2) localization

and function with high calcium permeability (PCa/PNa≈10), 3) rapid activation and

desensitization by agonist (millisecond scale) (Bertrand et al., 1992; Couturier et al.,

1990), and 4) selective inhibition by α-bungarotoxin (α-Btx) and methyllycaconitine

(MLA) (Couturier et al., 1990; Séguéla et al., 1993; Turek et al., 1995). Because of its

simple organizational structure, the α7 subunit can be used to study structure–function

relationships. For example, mutation of a single amino acid in the channel domain will

17

cause the whole receptor complex to be modified, which provides a better

understanding of receptor function (Hurst et al., 2013). Despite its homomeric

arrangement, α7-nAChR can assemble in a heteromeric form with other subunits; α7β2

heteromeric receptors (Liu et al., 2012, 2009; Moretti et al., 2014; Thomsen et al.,

2015; Zoli et al., 2015).

Depending on the presence, abundance, and timing of ACh binding, nAChRs exist

in different states and undergo spontaneous conformational transitions: closed at rest,

open pore, and desensitized (Figure 7) (Hurst et al., 2013). Prolonged exposure to low

doses of ACh, nicotine, or a nicotinic agonist substantially will lead to desensitization,

stabilizing the receptor in a closed state, unresponsive to further agonist stimulation.

Figure 7: Proposed mechanism of activation and desensitization.

Channel transition between three main conformational states by the binding of agonist:

(A) closed, (B) open, and (C) desensitized, where the channel is having high binding

affinity to agonist, but impermeable to ions [Modified from (Nys et al., 2013)].

A

C B

18

Orthosteric is the term used to describe the binding site for the natural ligand, and

classical agonists are therefore referred to as orthosteric agonists. However, a group of

compounds that lack agonist activity on nAChRs and act via a distinct transmembrane

binding site are described as allosteric modulators, (allo- from the Greek meaning

"other") (Figures 5 & 8) (Flor and Acher, 2012). Allosteric modulators can modulate

the activity of the channel. They are of three types: 1) positive allosteric modulators

(PAMs), that potentiate the activity of the channel but only in the presence of an

agonist with minimal level of desensitization; 2) negative allosteric modulators

(NAMs), inhibiting channel activity upon binding, or acting as open-channel blocker;

3) silent allosteric modulators (SAMs), having no effect on orthosteric activity but

blocking allosteric modulation (Figure 8) (Corradi and Bouzat, 2016).

19

Figure 8: Types of Allosteric modulators.

(A) The allosteric ligands modulate the activity of the channel by binding to a

topographically distinct binding site from the orthosteric site and modulate the affinity

(red) and/or efficacy (green) of the orthosteric ligand (Nishikawa et al., 1983).

(B) The effect of different allosteric modulators on the functional response of the

agonist represented by the concentration-response curve of the agonist (solid black).

PAMs enhances orthosteric agonist affinity and/or efficacy (solid red, blue, and green),

while NAM inhibit the activity of the channel by lowing orthosteric agonist affinity

and/or efficacy (dashed red and green) (Kinon et al., 2015).

Based on their macroscopic effect, PAMs can be classified into ‘type I’ or ‘type II’

PAMs, depending on their effects on receptor desensitization. Type I increases the

current amplitude but do not modify the channel kinetics significantly, while type II

profoundly changes the kinetics of the currents by prolonging single channel opening

time (Figure 9) (Clementi et al., 2000; Lewis et al., 2017). When compared with

Allosteric

ligand

Orthosteric

ligand

20

regular α7-nAChR agonists, PAMs have emerged as an important pharmacological

target because they: 1) have greater structural diversity compared to orthosteric site

which is highly conserved in nAChRs (Yang et al., 2012); 2) allow more flexible

structural form and final effects; 3 have an extra neuroprotective activities, as

activation of α7-nAChR can be inactivated by desensitization, some α7-PAMs has the

ability to desensitize the receptor back to conducting state (Kalappa et al., 2013; Sun

et al., 2013; Uteshev, 2014). Moreover, it has been suggested that neuronal injury

activates cholinergic system, and the presence of PAM will reduce the level of agonist

stimulation required for its neuroprotective effect (Uteshev, 2014).

Figure 9: Molecular activation routes of the α7-nAChRs.

PAM type I and II compounds (grey lines) produce differential enhancement of the

inward currents generated by nicotinic agonists (black line). (A) PAM type I

profoundly increase the current amplitude, (B) PAM type II significantly change the

kinetics of the current by prolonging the single channel opening time.

A B

21

1.2.1.1 Distribution of Nicotinic Acetylcholine Receptors

The current existing data regarding the distribution on nAChRs suggest that it is

relatively conservative in all vertebrate species. The distribution of nAChRs is not

restricted to well-defined brain cholinergic pathways. The structure and localization of

the different nAChR subtypes have been investigated using a number of

complementary techniques, including in situ hybridization and PCR for specific

subunit RNAs, immune-precipitation for protein subunits, imaging by

autoradiography, PET and SPECT, and functional electrophysiological or

neurotransmitter release assays (Mineur and Picciotto, 2008). However, despite the

incomplete nature of the studies performed to date, it is possible to draw a map of

nAChR distribution in the human brain as shown in Figure 10, with the α7 and β2*

being the most widely distributed throughout the mammalian brain (the asterisk denote

the possibility of different subunits) (Millar and Gotti, 2009). The regional distribution

of nAChRs subtypes has been described in the temporal cortex, cerebellum, striatum

and basal forebrain and have been localized in a variety of brain structures, in particular

the thalamus, cortex and the striatum (Zoli et al., 2015).

Grady et al. examined the mouse brain using in situ hybridization to characterize

the mRNA expression pattern of nAChRs (Grady et al., 2007). Grady’s laboratory

demonstrated that the ventral tegmental area (VTA) and substantia nigra (SN)

expressed variable concentrations of α 3-7, β2, and β3. Also, they reported the presence

of heteromeric nAChRs subunits; α4α6β2β3, α6β2β3, α6β2, α4β2, and α4α5β2, on

dopaminergic terminals of mouse striatum (Grady et al., 2007; Le Novere et al., 1996).

Several studies have demonstrated a decline of specific nAChRs in Parkinson’s disease

(Guan et al., 2002).

22

Figure 10: Distribution of nicotinic acetylcholine receptors human brain.

Distribution of different subtypes of nAChRs in human brain by means of quantitative

immunoprecipitation studies using radiolabeled (3H-Epibatine or 125I-αBungarotoxin)

nAChRs obtained from post-mortem brains (Zoli et al., 2015).

1.2.1.2 Alteration of Nicotinic Acetylcholine Receptors Expression

Alteration of cholinergic neurotransmission by either genetic dysregulation or

cholinergic denervation has been demonstrated in various studies. Several studies have

correlated a decline of specific nAChRs to certain pathological conditions like,

Alzheimer’s disease (Clementi et al., 2000; Nordberg, 1992; Warpman and Nordberg,

1995), Parkinson’s disease (Guan et al., 2002; Quik et al., 2012), schizophrenia

(Marcus et al., 2016; Timofeeva and Levin, 2011), autism (Bacchelli et al., 2015;

Olincy et al., 2016), epilepsy (Steinlein et al., 1995; Weiland et al., 1996), and

neuropathic pain (Marubio et al., 2003, 1999; Sullivan et al., 1994; Umana et al.,

23

2013). Here, we will focus on the proposed mechanism of neuroprotective role of

neuronal α7-nAChRs in Parkinson’s disease (Figure 11).

Figure 11: Proposed mechanism of α7-nAChRs in Parkinson’s disease.

Activation of α7-nAChRs has a neuroprotective effect on dopaminergic neurons and

astrocytes via its anti-inflammatory and anti-apoptotic activities (Jurado-Coronel et

al., 2016).

Astrocytes Nicotine

24

1.3 Parkinson’s Disease

The basal ganglia are a core component in the pathogenesis of Parkinson’s disease.

The basal ganglia are a group of subcortical nuclei located near the base of the brain

including, the caudate, putamen, (both together form the corpus striatum or

neostriatum), the ventral stiatum, globus pallidus with its external and internal

segments (GPe, GPi, respectively), the subthalamic nucleus (STN), and the substantia

nigra pars reticulata (SNpr) and pars compacta (SNpr) (Figure 12).

Dysfunction of the basal ganglia results in a wide spectrum of movement

disorders that varies from hypokinetic disorders (e.g.; Parkinson's disease) to

hyperkinetic disorders (e.g.; Huntington's disease). The main focus of this research

work is the hypokinetic disorder namely, Parkinson’s disease.

Figure 12: Structures of the basal ganglia.

Coronal section of the brain illustrating major structures of basal ganglia. Substantia

nigra (gray) and its innervation to striatum. The putamen (purple) and caudate nucleus

(green) together forms the striatum. The The globus pallidus (blue) with its two parts

externus (GPe) and internus (GPi). The subthalamic nucleus (red). The thalamus

(orange) and the cortex (Aum and Tierney, 2018).

25

1.3.1 Background

Parkinson’s disease (PD) is the second most common neurodegenerative disease

after Alzheimer's disease (AD). Parkinson’s disease was first described by an English

physician and surgeon, James Parkinson in his Essay on the Shaking Palsy in 1817,

which was called later Parkinson’s disease (PD) by Jean-Marie Charcot (Parkinson,

2002). PD is an age-related disorder. The prevalence of the disease increases with

advancing age. The prevalence is around 1% over the age of 60 and 0.3% of all ages

in industrialized countries (de Lau and Breteler, 2006).

1.3.2 Pathophysiology

Parkinson is a slowly progressive multisystem disorder rather than just a disease

involving massive neuropathological alterations in the brain. Pathologically, the

hallmark of the disease is the phosphorylation of alpha synuclein protein and formation

of proteinaceous inclusions, Lewy bodies (LB) in neurons (Figure 13) and Lewy

neurites (LN) in axons and dendrites as well as degeneration of dopaminergic

nigrostriatal neurons (Braak et al., 1994; Del Tredici and Braak, 2016).

Other central nervous system (CNS) neurotransmitter systems are also affected to

varying degrees including cholinergic, GABA-ergic, glutamatergic, tryptaminergic,

noradrenergic and adrenergic nerve cells that may show similar damage in their

cytoskeletons (Braak and Braak, 2000). Mechanistically, some environmental insults

and/or gene mutations contribute to the degenerative changes observed in Parkinson’s

disease, causing mitochondrial dysfunction, oxidative stress, modifications in protein

handling, adaptations in immune-modulators, as well as alterations in other molecular

and cellular functions (Figure 14) (Franco-Iborra et al., 2016; Olanow and McNaught,

2011; Schapira and Jenner, 2011).

26

Figure 13: Lewy body in affected dopaminergic neurons.

Photomicrographs of various regions of substantia nigra in Parkinson’s patient show

deposition of Lewy bodies and Lewy neuritis at two different magnifications. The

upper panels (A & B) demonstrate are magnified 20 times to show the alpha-synuclein

aggregates forming Lewy bodies (red arrows). The lower panels (C & D) demonstrate

a 60-times magnification to show strand-like Lewy neurites (green arrows) and

rounded Lewy bodies of various sizes (red arrows) [Modified from (Rajan, 2012)].

D C

A B

27

Figure 14: Pathophysiology of Parkinson’s disease.

Parkinson’s disease is a multifactorial disorder, several factors have been involved in

dopaminergic neuron degeneration process: (1) Genetic mutation results in protein

misfolding and oxidative stress. (2) Exposure to environmental toxins causes

mitochondrial dysfunction and increase ROS formation. (3) Neuroinflammation and

chronic activation of microglia causes neuronal degeneration by releasing pro-

inflammatory mediators [Modified from (Blesa et al., 2015)].

1.3.3 Diagnosis

For identification and characterization of this anatomical, structural, and

neurotransmitter systems dysfunction; Braak and his collaborators has grouped PD

into six stages. Stage 1-2: where only medulla oblongata/pontine tegmentum and

olfactory bulb/anterior olfactory nucleus are affected by inclusion bodies with no

clinical symptoms. Stage 3-4: symptoms may start to appear as inclusion bodies invade

28

the substantia nigra and other nuclei of the midbrain and forebrain. Stage 5-6: end

stage of the disease as the neocortex is affected with a wide range of clinical

manifestation (Braak et al., 2004, 2003). The clinical symptoms of PD can be

categorized into motor and non-motor symptoms as shown in Table 2.

Table 2: Motor and non-motor symptoms of Parkinson’s disease

Motor symptoms Reference

Limb rigidity

Cogwheel phenomenon

Shuffling gait lack

Arm swing while walking.

Expressionless face (hypomimia)

Micrographia

Limb tremor

Resting pill-rolling

Loss of balance and falls

Freezing of movements

Postural instability

Speech disturbances

Swallowing problems

Dribbling of saliva

Dystonia

Postural deformities

(Jankovic, 2008)

(Virmani et al., 2015)

(Virmani et al., 2015)

(Williams et al., 2006)

(Perez-Lloret et al., 2012)

(Kalf et al., 2011)

(Kalf et al., 2012)

(Tolosa and Compta, 2006)

(Doherty et al., 2011)

Non-Motor symptoms Reference

Orthostatic hypotension

Constipation

Excessive sweating

Urinary control disturbance

Sleep disturbances

Visual hallucinations and illusions

Cognitive impairment and

Dementia

Anhedonia

Depression and anxiety

Loss of smell and taste

Limb pain

(Lahrmann et al., 2006)

(Jost, 2003)

(Hirayama, 2006)

(Jost, 2003)

(Monderer and Thorpy, 2009)

(Onofrj et al., 2007)

(Williams-Gray et al., 2006)

(Pont-Sunyer et al., 2015)

(Reijnders et al., 2008)

(Doty et al., 1988)

(Williams and Lees, 2009)

29

1.3.3.1 Motor Symptoms

Dopamine (DA) is neurotransmitter involved in coordination of movement. When

sixty to eighty percent of dopamine producing cells in the substantia nigra are

defunctionalized the extra pyramidal system loses the ability to effectively promote

movement and the motor symptoms of Parkinson's disease begin to appear (Chung et

al., 2001). UK PD Brain Bank has established specific criteria for diagnosis of

Parkinson’s disease and presented it in three steps (Table 2). Step one which is based

mainly on the patient’s motor symptoms include bradykinesia, defined as delay in

initiation of voluntary movement associated with progressive reduction in patient’s

speed and amplitude of repetitive actions (Antonini et al., 2013; Berardelli et al., 2001;

Goetz et al., 2007). It is one of the main and core symptoms that if found, in association

with one of the following additional symptoms i.e., resting tremor, muscular rigidity,

or postural instability- are a criterion for the diagnosis of PD (Hughes et al., 1992).

Step two; is to exclude any other cause for the motor symptoms such as parkinsonian

syndrome. Step three; establishes three supportive measures. Namely, unilateral onset

of symptoms, persistent asymmetry of clinical symptoms, induction of dyskinesia by

the dopaminergic treatment, and good response to levodopa treatment.

1.3.3.2 Non-Motor Symptoms

Clinical heterogeneity of Parkinson’s disease reflects the multisystem involvement

in the pathophysiology of the disease. Therefore, the International Movement

Disorders Society included a range of Non-motor symptoms (NMS) to the diagnostic

criteria of Parkinson’s disease (Postuma et al., 2015). It has been reported that NMS

starts to appear as early as ten years before the diagnosis of PD, outlining a prodromal

or an asymptomatic stage of the disease. There is a wide range of symptoms (Table 3),

30

most commonly: cognitive impairment, anxiety, depression, constipation, and sleep

disturbance. PD patients suffer more from NMS than motor symptoms with advancing

disease.

Table 3: UK Parkinson’s disease society Brain Bank clinical diagnosing criteria

(Hughes et al., 1992)

Step 1. Diagnosis of Parkinsonian Syndrome

Bradykinesia

• At least one of the following

o Muscular rigidity

o 4-6 Hz rest tremor

o postural instability not caused by primary visual, vestibular, cerebellar, or

proprioceptive dysfunction

Step 2. Exclusion criteria for Parkinson’s disease

• History of repeated strokes with stepwise progression of parkinsonian features

• History of repeated head injury

• History of definite encephalitis

• Oculogyric crises

• Neuroleptic treatment at onset of symptoms

• More than one affected relative

• Sustained remission

• Strictly unilateral features after 3 years

• Supranuclear gaze palsy

• Cerebellar signs

• Early severe autonomic involvement

• Early severe dementia with disturbances of memory, language, and praxis

• Babinski sign

• Presence of cerebral tumor or communication hydrocephalus on imaging study

• Negative response to large doses of levodopa in absence of malabsorption

• MPTP exposure

Step 3. Supportive prospective positive criteria for Parkinson’s disease

• Unilateral onset

• Rest tremor present

• Progressive disorder

• Persistent asymmetry affecting side of onset most

• Excellent response (70-100%) to levodopa

• Severe levodopa-induced chorea

• Levodopa response for 5 years or more

• Clinical course of ten years or more

31

1.3.4 Treatment

The etiology of PD is multifactorial. To date, there is no drug that cures or stops

disease progression. Being mainly a dysfunction in dopaminergic system in the brain,

Levodopa or L-dopa (L-3,4-dihydroxyphenylalanine) was introduced in 1960s as a

prodrug of dopamine enhancing intracerebral dopamine concentration. Since its

approval by the FDA in 1970, L-dopa has been the gold standard treatment for

Parkinson’s disease. Though, after several months to years of treatment with L-dopa,

patients develop the adverse effects of dyskinesias (Brotchie and Jenner, 2011; Huot

et al., 2013; Iravani et al., 2012). This is known as L-dopa-induced dyskinesias (LIDs)

and it will be discussed in greater details later. With the limitation of L-dopa use, other

strategies have been implemented to enhance dopamine release, such as; dopamine

agonist, monoamine oxidase type B inhibitors (MAO), catechol-O-methyl transferase

inhibitors (COMTIs), anticholinergic, beta-blocker, antipsychotic, and antiviral (Table

3 & Figure 15) (Connolly and Lang, 2014; Gazewood et al., 2013a). Surgical

intervention became an option with deep brain stimulation (DBS) in selected PD

patients (Benabid et al., 2009; Morgante et al., 2007; Rizzone et al., 2014).

32

Table 4: Treatment options of Parkinson’s disease

Drug group Mechanism of Action Indication Reference

Levodopa

Levodopa-carbidopa

Bind to and activate

dopamine post synaptic

receptors, to overcome

dopamine depletion

All motor

symptoms

(Hisahara

and

Shimohama,

2011)

Dopamine agonist

Bromocriptine

Pramipexole

Ropinirole

Rotigotine

Bind to and activate

dopamine receptors, to

overcome dopamine

depletion

All motor

symptoms

(Hisahara

and

Shimohama,

2011)

Monoamine

Oxidase B

inhibitors (MAO)

Selegiline

Rasagiline

Inhibit dopamine breakdown

by MAO-B enzyme, to

increase the amount of

available dopamine

Early mild

symptoms

and motor

fluctuations

(Grosset et

al., 2010)

Catechol-O-Methyl

Transferase

Inhibitors

(COMTIs)

Entacapone

Tolcapone

Prevent L-dopa breakdown

by COMT enzyme, to

increase the amount of

available dopamine

Motor

fluctuations

(Grosset et

al., 2010)

Anticholinergic

Benztropine

Trihexyphenidyl

Bind to and block cholinergic

receptors

Tremor (Gazewood

et al.,

2013b)

Beta-Blocker

Propranolol

Bind to and block β2

receptors, help in

symptomatic treatment of

tremor

Tremor (Ferreira et

al., 2013)

Antipsychotic

Clozapine

Bind to and block 5-HT2A

receptors

Tremor and

dyskinesia

(Ferreira et

al., 2013)

Antiviral

Amantadine

Bind to and block NMDA

glutamate (excitatory)

receptors, to reduce motor

complications associated

with L-DOPA

Gait

dysfunction

and

dyskinesia

(Wolf et al.,

2010)

33

Figure 15: Schematic presentation of pathophysiology and treatment of PD.

(A) Coronal and sagittal section of human brain. (B) The gold-standard treatment of PD with L-dopa can be enhanced by co-administration of

COMTIs, to prevent L-dopa degradation, and administration of a dopamine decarboxylase inhibitor (DDCIs); carbidopa to prevent peripheral

conversion of L-dopa to its active form dopamine and thus maximizing the amount of the drug that can pass BBB and reach striatum. The striatum

is densely innervated by corticostriatal glutamatergic afferents, dopaminergic afferents, cholinergic interneurons, and GABAergic medium spiny

neurons. (C) This heavy connection is the site of action of most of the anti-parkinsonian medications: L-dopa, dopamine agonists, MAOBIs, anti-

cholinergic, and anti-viral [Modified from (Connolly and Lang, 2014)].

A

C

B

34

1.3.5 Prognosis

Parkinson’s disease is a complex disorder, and with advancing age it is expected

that disease progression will vary between patients (Poewe, 2006). NMS precedes the

motor symptoms and is usually mild and unilateral. Later, it progresses to the

contralateral side but still remains very responsive to treatment, in what is called the

honeymoon period. However, with time treatment efficacy starts to decline and

patients’ symptoms and disability worsen, affecting their quality of life with the need

of home care and frequent hospital admissions (Low et al., 2015; Parashos et al., 2002).

Based on several studies, life expectancy of PD patients ranges from 6.9 to 14.3 years

(Macleod et al., 2014). Thus, the need for development of strategies to stop, slow

down, or preferably reverse the neurodegenerative process is mandatory. So, the next

question is how an interaction at nAChRs level may lead to an overall functional effect

such as protection against nigrostriatal damage. Three mechanisms suggested in recent

studies will be discussed in following sections.

1.4 The Neuroprotective Role of Nicotine and Nicotinic Receptors Against

Nigrostriatal Damage

Cigarette smoking is a well-known health hazard and a risk factor of serious

chronic disorders, including cardiovascular disorders, lung disease, and cancers.

However, unexpectedly, cigarette use appears to confer beneficial effects in

Parkinson’s disease. The initial evidence that nicotine which acts on nAChRs, may be

useful as a therapy for Parkinson’s disease stemmed from the results of various

epidemiological findings in the early 1960s (Allam et al., 2004; Elbaz and Moisan,

2008; Gorell et al., 1999; Noyce et al., 2012; Tanner, 2010; Wirdefeldt et al., 2011).

Nicotine is a highly lipophilic compound and the active addictive ingredient of

35

tobacco. After smoking, chewing, or sniffing, nicotine rapidly crosses the blood-brain

barrier and binds to nicotinic receptors. Nicotine is likely to mediate neuroprotective

effects, as many studies have shown a reduced risk of PD in current and former

smokers (Fahn, 2010; Nicoletti et al., 2010; Tanner, 2010). This apparent

neuroprotection against Parkinson’s disease is correlated with smoking duration,

intensity, and recentness, and is reduced with smoking cessation. Moreover, the

incidence of Parkinson’s disease within twin pairs was less in the twin that smoked

compared to the nonsmoker (Tanner et al., 2002), suggesting that the neuroprotective

effects of tobacco are independent of genetic background. Furthermore, clinical trials

with nicotine patches showed an attenuation of PD symptoms in PD patients

(Fagerström et al., 1994). In addition, recent studies showed that nicotine

administration reduces dyskinesia which is a major side effect of L-dopa used as the

primary treatment for Parkinson’s disease (Bordia et al., 2008; Huot et al., 2013;

Iravani et al., 2012; Quik et al., 2007). All these observations raised the possibility that

nAChR stimulation may be useful in Parkinson’s disease management (Bordia et al.,

2015; Huot et al., 2013; Iravani et al., 2012).

1.4.1 Dopaminergic and Cholinergic Systems Correlation and Dopamine Release

Dopamine inputs to the striatum arise from midbrain dopamine neurons located in

the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc), which

innervates the ventral striatum (nucleus accumbens (NAc)) and dorsal striatum

(caudate–putamen (CPu)) (Gerfen et al., 1987; Voorn et al., 2004). The striatum is

innervated by a high density of axonal varicosities of dopaminergic nerves forming

dopaminergic synapses (Descarries and Mechawar, 2000; Pickel et al., 1981).

36

The cholinergic striatal interneurons are the primary source of cholinergic input to

the striatum, containing both muscarinic and nicotinic receptors (Zhou et al., 2002,

2001). Nicotinic acetylcholine receptors are principal modulators of neuronal

excitability throughout the central nervous system. Presynaptic nAChRs influences the

release of neurotransmitters, while the postsynaptic nAChRs participate in fast

postsynaptic neurotransmission as an excitatory input in the hippocampus, and

subcortical areas including the ventral tegmental area (Dani, 2001; Mameli-Engvall et

al., 2006; Mansvelder et al., 2002; Pyakurel et al., 2018).

Functional evidence for the specificity of the cholinergic innervation to the VTA

and the SN came from studies of Blaha and Winn (Blaha et al., 1996; Blaha and Winn,

1993). Blaha and Winn measured dopamine efflux following nicotinic agonist

administration before and after performing a lesion in the VTA. In both cases, a

potentiation of DA efflux from the VTA or SN was noticed as a functional evidence

of cholinergic innervation to this area. Using electrical stimulation of the VTA, Forster

and Blaha demonstrated that DA efflux in the striatum is mediated through nicotinic

and glutamatergic receptors in the SN (Forster and Blaha, 2003). Later in 2006, Quik

and colleagues demonstrated that the levels of striatal tyrosine hydroxylase (TH),

dopamine transporter, vesicular monoamine transporter, dopamine and nicotinic

receptors were greater in nicotine-treated MPTP-lesioned parkinsonian animals than

in lesioned animals not receiving nicotine (Figure 16) (Quik et al., 2006).

37

Figure 16: Effect of nicotine treatment on MPTP- lesioned primates.

Increase in levels of tyrosine hydroxylase (A), dopamine transporter (B), vesicular

monoamine transporter (C), and striatal dopamine (D), in nicotine-treated MPTP-

lesioned primates than in lesioned animals not receiving nicotine [Modified from

(Quik et al., 2006)].

A

B

C

D

38

In 2009, studies of Quarta supported the previous results by using α7-nicotinic

receptor null mutant mice and compared the release of [3H]dopamine from mouse

striatal slices. Quarta measured [3H]dopamine release after administration of (1 μM)

Nicotine in the absence (black bar) or presence (light gray; αCTxMII, white; DHβE)

of β2* and α6β2* nicotinic receptor blockers respectively. α7 nicotinic receptors were

enhanced by 1 mM of choline (selective α7-nAChR agonist). Inhibition of

[3H]dopamine release in response to nicotine was similar in both groups of animals

due to blocking of different subunits of nicotinic receptors. Conversely, choline failed

to stimulate dopamine release in α7 null mutant mice, indicating that α7-nAChR plays

a major role in dopamine release (Figure 17). Along with other results, Quarta’s study

supported the important role of heteromeric as well as homomeric nicotinic receptor

subtypes in dopamine release (Quarta et al., 2009).

Figure 17: Striatal [3H]dopamine release of wild type and α7-nicotinic receptor null

mutant mice.

Nicotine (1 μM) was applied in the absence (black bar) or presence of αCTxMII (200

nM, light grey bars) or DHβE (10 μM, open bars); antagonists were applied 5 min

before nicotine and remained throughout the stimulation. α7-nAChRs were stimulated

by application of choline (1 mM; Cho; dark grey bars) (Quarta et al., 2009).

39

Recently, Pyakurel et al. (2018) tested the effects of various nicotinic receptor

agonists; acetylcholine, nicotine, and neonicotinoid on dopamine release using

Drosophila melanogaster strains (Figure 18A). Measurement of dopamine release

following acetylcholine stimulation (Figure 18B) using fast-scan cyclic

voltammogram indicated changes in dopamine over time (Figure 18C). This current

response was stable after subsequent stimulations. Interestingly, addition of dopamine

synthesis inhibitor; 3-iodotyrosine; 3-IT, significantly decreased Acetylcholine

stimulated-dopamine release (Figure 18D).

Figure 18: Stimulation of dopamine release in in Drosophila melanogaster ventral

nerve cord (VNC).

(A) Electrode and stimulating pipet in VNC of Drosophila. (B) Measurement of

acetylcholine-stimulated dopamine release by fast scan cyclic voltammetry (FSCV).

(C) Increasing the amount of acetylcholine caused an increase evoked dopamine

concentration. (D) Acetylcholine-stimulated dopamine release was significantly

inhibited by dopamine synthesis inhibitor, 3-iodotyrosine; 3-IT [Modified from

(Pyakurel et al., 2018)].

D C

A B

40

Moreover, Pyakurel confirmed that dopamine release is mainly mediated by

nicotinic not by muscarinic acetylcholine receptors. After incubation with two

different selective nicotinic receptor antagonists; (α-bungarotoxin and DHβE) (Figures

19A & C) the current response was lowered significantly, indicating lesser dopamine

release. In contrast, upon incubation with a muscarinic antagonist; 1 µM of atropine,

there was no significant change (Figure 19B) (Pyakurel et al., 2018).

Figure 19: Data of Acetylcholine stimulated dopamine release before and after bathing

with different nicotinic and muscarinic antagonists.

(A) Effect of 1 µM α-bungarotoxin (nicotinic antagonist) suppressed dopamine

release. (B) Effect of 1 µM of DHβE (β2/β4 antagonist) on dopamine release. (C) Effect

of 1 µM of atropine (muscarinic antagonist) on acetylcholine stimulated dopamine

release. (D) Histogram presentation of the data. (Pyakurel et al., 2018).

41

1.4.2 Immune Modulation via Nicotinic Receptors

Activation of the immune system in the CNS takes place in various neurological

disorders such as in; stroke, neurodegenerative diseases, spinal cord injury, multiple

sclerosis, and brain injury (Long-Smith et al., 2009; Napoli and Neumann, 2010;

Prokop et al., 2013; Yenari et al., 2010). In the CNS, the innate immune system is

represented by a type of macrophage, the microglia, a member of the

reticuloendothelial system and a resident immune cell in CNS. Under normal

physiological conditions, microglia are inactive, with a small cell body and highly

ramified branching processes. In response to injury or pathogen invasion, microglia

transform into active phagocytic microglia helping with the clearance of aggregated

proteins and cell debris (Stence et al., 2001). Chronic activation of microglia leads to

noxious effects on neurons by the release of pro-inflammatory molecules and, thus,

contributing to the pathophysiology of neurodegenerative diseases (Figure 20A)

(Gehrmann et al., 1995; Liu and Hong, 2003).

Figure 20: The role of microglia in health and disease.

(A) Unchallenged microglia perform functions in the healthy CNS including

phagocytosis of normal apoptotic debris, secretion of trophic factors (IGF-1, TGFβ),

and synaptic maintenance. (B) Challenged microglia respond to CNS damage

secreting pro-inflammatory factors (TNFα, IL-1β and ROS). When these mediators

persist unchecked, neurodegeneration can ensue (Derecki et al., 2013).

A B

42

Current advances in molecular biology have provided evidence that

neuroinflammation plays an important role in the pathogenesis of PD (Chung et al.,

2010; Moore et al., 2005). It has been suggested that immune reaction in the form of

glial activation and inflammatory processes may also participate in the cascade of

events leading to neuronal degeneration in PD. Activated microglia express various

cell-surface receptors, leading to increased levels of cytokines such as tumor necrosis

factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ in the substantia nigra of

PD patients (Figure 20B) (Kreutzberg, 1996; Nagatsu and Sawada, 2005). These

contribute to the development of chronic inflammation of the brain, leading to

neuronal dysfunction and death in the form of neurodegenerative disorders such as PD

(Chung et al., 2010; Orr et al., 2002).

Figure 21: Staining of Primary human macrophages with fluorescein isothiocyanate

(FITC)-labelled α-bungarotoxin (α -Bgt, 1.5 mgml21).

(A) Binding of α- Bungarotoxin to nicotinic receptors on the surface of macrophages

stained with (FITC)-labelled α-bungarotoxin. (B) 500 µmol of Nicotine was added

before the addition of α -bungarotoxin. (C & D) Higher magnification revealing

receptor clusters. Magnifications: a, b, X50; c, X200; d, X450 (Wang et al., 2003).

A B

C D

43

Neuronal nAChRs have been shown to regulate inflammation, in particular via the

α7-nAChR activation in microglia, this is known as ‘cholinergic anti-inflammatory

pathway’. In his letter to Nature journal, Wang first established the relationship

between the cholinergic and immune systems (Figure 21) (Wang et al., 2003).

A year later, Shytle and collaborators demonstrated the expression of α7-nAChRs

on microglia (Figure 22A) and their results suggested that α7-nAChRs play important

roles in the neuroinflammatory processes (Figure 22B) (Shytle et al., 2004). Consistent

with these findings, it has been demonstrated that nicotine has a neuroprotective effect

on dopaminergic neurons via an anti-inflammatory mechanism mediated by the

modulation of microglial activation (Park et al., 2007). Both Shytle and Park

documented that nicotine treatment decreased microglial activation, with significant

reduction of the bacterial cell wall endotoxin LPS-induced TNF- α release (Park et al.,

2007; Shytle et al., 2004).

44

Figure 22: RT–PCR analysis of α7-nAChRs expression on microglia using N9 and

primary cultured microglial cells.

(A) Expression of α7-nAChRs on microglia cell live. (B) Anti-inflammatory effect of

Nicotine or ACh on LPS-induced TNF-α release. This effect is abolished after addition

of α-Bgt. (C, D) The anti-inflammatory effect of Nicotine or ACh is dose dependent

[Modified from (Shytle et al., 2004)].

In following studies, α7-nAChRs has been also identified on astrocytes (Liu et al.,

2015). Activation of α7-nAChRs can inhibit H2O2-induced astrocyte apoptosis in a

concentration dependent manner (Figure 23A). This effect is abolished after

administration of a selective α7-nAChRs antagonist, methyllycaconitine (MLA)

(Figure 23B). Several protective mechanisms have been suggested including;

A

C

B

D

45

maintenance of the Bax/Bcl-2 balance, and inhibition of cleaved caspase-9 activity via

α7-nAChR activation. Nicotine prevents H2O2-induced loss of mitochondrial

membrane potential (ΔΨm) (Figure 23B) (Liu et al., 2015).

Figure 23: Nicotine inhibits H2O2-induced astrocyte apoptosis through protection of

mitochondrial membrane potential.

(A) Activation of α7-nAChRs on astrocytes by nicotine inhibits H2O2-induced

apoptosis. Addition of MLA abolish this protective effect. (B) Nicotine inhibits H2O2-

induced loss of mitochondrial membrane potential (ΔΨm). Addition of MLA abolish

this protective effect (Liu et al., 2015).

A

B

46

1.4.3 Effect of Nicotinic Receptors on L-Dopa Induced Dyskinesia

L-dopa-induced dyskinesia (LID) which occurs in majority of patients on prolonged

L-dopa treatment (Ahlskog and Muenter, 2001), can be very debilitating and impairs

patient’s quality of life. Indeed, LID represent one of the major drawbacks of L-dopa

treatment (Calabresi et al., 2008; Carta et al., 2008; Fahn, 2008; Fox et al., 2008;

Jenner, 2008).

The pathophysiological mechanism behind LID is not fully understood, but it is

primarily due to the altered handling of levodopa presynaptically as well as dopamine

receptors hypersensitization postsynaptically (Bastide et al., 2015).

Several pharmacological approaches to treat LID have been implicated. For

example the use of amantadine, a nonselective NMDA receptor antagonist has been

suggested (Blanchet et al., 1996) but its effect is limited, short-lived and associated

with many adverse effects (Sawada et al., 2010; Thomas et al., 2004; Verhagen

Metman et al., 1998)Reduction of L-dopa daily dose has also been considered as a line

of LID treatment, but this might lead to the re-emergence of PD symptoms (Goetz et

al., 2005; Tambasco et al., 2012). The surgical approach has been also suggested for

the treatment of LID, namely, deep brain stimulation (DBS). Deep brain stimulation is

based on targeting the nuclei of selective deep brain structures such as; the internal

segment of the globus pallidus (GPi), subthalamic nucleus (STN), and thalamus by

high-frequencies electrical stimuli. This is based on interruption and/or modulation of

the neuronal signaling of the structures within the basal ganglia (Brown and Eusebio,

2008; Obeso and Lanciego, 2011). Though it is an effective method of treatment, it

needs careful selection and screening of patients with dyskinesia with quality of life

47

severely affected, with on–off fluctuations, and treatment-resistant tremor. It is an

invasive procedure with considerable side effects (Okun, 2012).

The demand for safe and non-invasive approaches in treating LID is crucial. Many

laboratories are targeting different neurotransmitter systems in the brain, such as; the

serotonergic, glutamatergic, GABAergic, adrenergic, cannabinoid, opioid, adenosine

and other neurotransmitter systems to test the effect of pharmacological agents on

different neurotransmitter systems. Any one of these has the potential to improve

dyskinesias, in fact some laboratories have shown promising results (Blandini and

Armentero, 2012; Brotchie et al., 2005; Cenci and Lundblad, 2007, 2006; Fox et al.,

2009; Linazasoro et al., 2008; Olanow et al., 2006; Quik et al., 2008; Samadi et al.,

2006; Sgambato-Faure and Cenci, 2012).

One of the proposed strategies against LID is to target neuronal nicotinic

acetylcholine receptors. Accumulating evidence suggests that nicotine and nAChR can

reduce the abnormal involuntary movements or dyskinesias complicating L-dopa

treatment (Hickey and Stacy, 2013; Kerr, 2010; Quik et al., 2014, 2012). Pioneering

studies were done on MPTP-induced Parkinson in monkeys to investigate the effect of

long-term nicotine treatment against nigrostriatal damage in non-human primates

where they exhibited parkinsonian motor symptoms very similar to those in

Parkinson’s disease patients and develop abnormal involuntary movements after L-

dopa treatment analogous to those in L-dopa-treated Parkinson’s disease patients. The

results demonstrated the effectiveness of nicotine treatment in PD model (Quik et al.,

2006). This was followed by number of studies by Quik and his colleagues where they

could demonstrate that nicotinic administration to L-dopa-treated animal models led

to ~60% decrease in LIDs (Figure 24). Nicotine reduced LIDs -whether given orally

48

via the drinking water, by systemic injection or by minipump- showing the effect is

independent of rout of administration (Mihalak et al., 2006; Ween et al., 2010).

Figure 24: Nicotine administration reduces L-dopa-induced dyskinetic-like

movements in rats and monkeys.

(A) Unilateral 6-OHDA injectted rat, nicotine treatment reduced AIM score

significantly compared to vehicle group (Tanuja Bordia et al., 2008). (B) MPTP-

lesioned monkeys receiving oral nicotine treatment. Nicotine significantly reduced

dyskinesia compared to vehicle group (Bordia et al., 2008; Quik et al., 2007).

This reduction is more reflective with prolonged nicotine treatment with no

development of tolerance, indicating that long term molecular and cellular changes

underlie the nicotine-mediated decline in LIDs (Bordia et al., 2015, 2008; Quik et al.,

2013, 2007; Zhang et al., 2013). Neuroprotective effect of nicotine is achieved by

stimulation of various subtypes of nAChRs, mainly α7 and α4β2*/α6β2* nAChRs.

This was evidenced by using α7(−/−) and α4(−/−) knockout models (Quik et al., 2013).

LID Patients after a period also suffer from “on-off” fluctuations due to a progressive

reduction in their dopamine storing capacity. The term “on-time” refers to periods of

the day where Parkinsonian symptoms are under adequate control and the opposite is

true. The effect of TC-8831, an α6β2*/ α4β2* nAChR agonist on LID has been

evaluated. In addition, the duration and quality of ON-time (‘good’ ON-time: time

49

without disabling dyskinesia, ‘bad’ on-time: time with disabling dyskinesia) was

studied and compared to amantadine. TC-8831caused a significant reduction in the

duration of ‘bad’ ON-time (62%) as well as severity of LID (reduced chorea and

dystonia) with minimal effect on L-DOPA anti-parkinsonian benefits, in a dose

dependent manner. Parallel to TC-88331, amantadine reduced ‘bad’ ON-time by up to

61% but total ON-time has dropped by up to 23% (Figure 25) (Johnston et al., 2013).

50

Figure 25: Effect of TC-8831 and amantadine in combination with L-DOPA in MPTP-

lesioned monkeys.

(A) Effect of TC-8831 in combination with L-DOPA on duration and quality of ON-

time. TC-8831 treatment significantly reduced the duration of ‘bad’ ON-time at 0.03,

0.1, and 0.3 mg/kg compared to vehicle alone. (B) Effect of amantadine in combination

with L-DOPA on duration and quality of ON-time. Amantadine at 3 mg/kg, afforded

a significant reduction in duration of ‘bad’ ON-time and an increase in the duration of

‘good’ ON-time, compared to vehicle alone [Modified from (Johnston et al., 2013)].

A B

51

Quik and his colleagues, have tested several nAChR agonists compounds ABT-

089, ABT-894 (α6β2*/ α4β2* nAChR partial and full agonists respectively), and ABT-

107 (α7-nAChR agonist) in monkey model of Parkinson’s disease. All agents yielded

up to 60% reduction in LID in monkeys (Zhang et al., 2014, 2015). A newly developed

α7-nAChR agonist ABT-126, has proved to be effective in treating LID in moderate

to severe nigrostriatal damage in MPTP-treated squirrel monkeys. The anti-dyskinetic

effect of ABT-126 was dose dependent (Figure 26) (Zhang et al., 2014, 2015). ABT-

126 is safe and well tolerated by patients and currently has reached phase 2 clinical

trials for Alzheimer's disease (Gault et al., 2015).

Figure 26: Effect of ABT-126 on LID in MPTP treated monkeys .

(A) Various doses of ABT-126 were used (0.03, 0.1, 0.3 and 1mg/kg). (B) ABT-126

significantly reduced LID in a dose dependent manner compared to vehicle alone

[Modified from (Zhang et al., 2015)].

52

1.4.4 Molecular Neuroprotective Mechanisms of Nicotinic Acetylcholine

Receptors

Epidemiological and experimental evidence indicate that nicotine is protective for

the vulnerable dopamine neurons in Parkinson disease. The molecular mechanism

involves alterations in calcium (Ca2+) signaling, although calcium independent

nAChR-mediated mechanisms (cytosolic Ca2+) have also been reported (Dajas-

Bailador and Wonnacott, 2004; Picciotto and Zoli, 2008; Shimohama, 2009; Ward et

al., 2008).

Stimulation of nAChR mediates several intracellular changes, namely protein

kinases such as; protein kinase A (PKA), extracellular signal-regulated mitogen-

activated protein kinase (ERK/MAPK), calcium-calmodulin-dependent protein kinase

(CaM) and phosphatidylinositol 3-kinase (PI3K)/Akt-or protein kinase B-dependent

signaling (Toulorge et al., 2011). Modulation of protein kinases are all calcium

dependent. On the other hand, calcium independent modulation involves modifications

in the JAK2 (Janus kinase 2)/PI3K and/or JAK2/STAT3 (signal transducer and

activator of transcription 3) pathways (Hosur and Loring, 2011; Kawamata and

Shimohama, 2011). These pathways are also known as survival pathways leading to

cell survival by modulating different downstream signaling cascades such as; caspase

activity (3, 8 and 9), cell survival proteins such as Bcl-2 (B-cell lymphoma 2) and Bcl-

x, NFκB, CREB (cAMP response element-binding), and other molecular components

(Figure 27). Also it has been reported that nicotine-induced changes in basic fibroblast

growth factor-2 (FGF-2), brain-derived neurotrophic factor (BDNF) and nerve growth

factor (NGF) in brain dopaminergic and other regions attenuate neuronal damage

(Belluardo et al., 2000; Formaggio et al., 2010; Massey et al., 2006; Zhou et al., 2004).

In addition to neuroprotective role of nicotine- nAChRs stimulation, other mechanisms

53

are involved in its neuroprotective effect of nicotine, including: reduction in

mitochondrial complex 1 activity, inhibition of reactive oxygen species generation,

oxidative or anti-oxidative potential and radical scavenging properties (Cormier et al.,

2003; Ferger et al., 1998; Newman et al., 2002; Xie et al., 2005). Overall, all these data

suggest the involvement of multiple molecular transduction mechanisms in nicotinic

receptor-mediated neuroprotection under various pathological conditions.

54

Figure 27 : α7-nicotinic acetylcholine-mediated molecular signaling mechanism.

α7-nAChR neuroprotective mechanisms through Ca2+-dependent and non-

Ca2+dependent mechanisms, modulating an intracellular cascade of events via

activation of several protein kinases, leading to modification in immune activity,

suppression of apoptosis, alteration of synaptic plasticity and eventually cell survival

[Modified from (Quik, Perez et al., 2012)].

JAK2

PI3K

PKB

STAT3

CaMK

PI3K PKA

PKA

cAMP

ERK p-AKT

Ca2+

α7 and/or β2

nAChR

ACh, Nicotine

Ca2+

CREB ↑Bcl-2 ↓Caspase ↓NF-kB

Ca2+-dependent Ca2+-independent

55

1.5 Animal Models of Parkinson’s Disease

The use of animal models has contributed significantly to a better understanding

of disease process and made a significant breakthrough in development of new

therapeutic strategies specially in LID and surgery. An ideal animal model of PD

should include both pathological and clinical manifestation (motor and non-motor

symptoms), involving central and peripheral nervous system, dopaminergic and non-

dopaminergic neurons. Unfortunately, none of the available models includes the full

picture of PD, still they can replicate many of the disease characteristics. Experimental

models can be categorized into two main classes: toxic and genetic.

1.5.1 The Neurotoxin Model

Many pharmacological and toxic agents have been implicated in primates as well

as in rodents and other mammals. Though neurotoxic models considered to be the best

for testing nigrostriatal dopaminergic degeneration, some drawbacks need to be

mentioned: the rapidly progressive dopaminergic degeneration (within days – weeks)

compared to the disease progression in human taking years to develop, most of the

animal models lack the pathological hallmark; Lewy bodies, and motor/behavioral

abnormalities (Blesa and Przedborski, 2014).

1.5.1.1 Classical Model: 6-Hydroxydopamine

6-Hydroxydopamine (6-OHDA) is a catecholaminergic neurotoxin that

selectively damages dopaminergic and noradrenergic neurons (Luthman et al., 1989).

This model of “Chemical Denervation” was first established by Ungerstedt in 1968

and proved to knock out 60 – 90 % of tyrosine hydroxylase (TH)-positive neurons in

SNpc. 6-hydroxydopamine is a photosensitive compound and can be rapidly oxidized

56

if exposed to light, hence it should be protected from light exposure, also its dissolved

in ascorbic acid to protect it from oxidation (Bové and Perier, 2012). As 6-OHDA

cannot cross blood-brain barrier, it should be injected directly (mostly as unilateral

injection; hemiparkinsonian model) into the targeted area; medial forebrain bundle

(MFB), substantia nigra pars compacta (SNpc), striatum or intraventricular

administration (Blandini et al., 2008; Rodríguez Díaz et al., 2001).

Since it’s a hydroxylated analog of dopamine (Figure 28), 6-OHDA is taken up by

the neurons via dopamine transporters (DAT) and accumulate in the cytosol where it

gets oxidized leading to ROS formation and cytotoxicity due to oxidative stress (Figure

29) (Blum et al., 2001; Graham, 1978; Rangel-Barajas et al., 2015; Saner and Thoenen,

1971). Proteinaceous aggregates and Lewy-like inclusions are not produced in this

type of PD (Blesa and Przedborski, 2014).

Figure 28: Chemical structures of 6-hydroxydopamine (6-OHDA) and dopamine

(Bové and Perier, 2012)

57

For the following reasons, it was decided to use the animal model of PD in which

multiple injections of 6-OHDA into the striatum was employed: 1) Striatal nerve

terminals are more sensitive to 6-OHDA toxicity than the axon and cell bodies (Bruyn,

1983; Malmfors and Sachs, 1968). 2) Progressive and less extensive lesion are more

relevant to the pathophysiology of PD (Cannon and Greenamyre, 2010). 3) Striatal

injection produces also non-motor symptoms of the disease; cognitive, psychiatric, and

GI symptoms (Branchi et al., 2008; Tadaiesky et al., 2008). 4) Including wider and

larger area of striatum can increase the success rate (Tieu, 2011). 5) It provides a well-

characterized behavioral, biochemical, and pathological feature of the disease which

is correlated tightly with the number of injection sites (Rosenblad et al., 1999). 6) Any

model that induce a predictable lesion over a long period of time will provide a greater

opportunity for neuroprotective strategies to succeed, and striatum is therefore an ideal

target to test for neuroprotection (Kirik et al., 1998).

Following unilateral 6-OHDA injection, drug-induced rotation test (drug:

dopamine receptor agonist; apomorphine or dopamine releasing compound such as

amphetamine) are performed to assess the extent of motor impairment. Systemic

injection of any of these drugs will result in asymmetrical rotation. Number of turns

are counted to ipsilateral side in case of intraperitoneal injection of amphetamine or

contralateral side in case of subcutaneous injection of apomorphine (Dunnett and

Torres, 2011; Hefti et al., 1980b; Ungerstedt and Arbuthnott, 1970).

58

Figure 29: Molecular Mechanisms for different animal models of PD.

Schematic diagram demonstrates site of action of pharmacological agents or genetic

manipulations leading to nigrostriatal degeneration and striatal dopamine depletion.

The cell represents a substantia nigra dopaminergic neuron with its cell body in the

substantia nigra and its terminals in the striatum. Reserpine and Methamphetamine

deplete dopamine at the nerve terminals, resulting in striatal dopamine deficiency. 6-

OHDA can affect all catecholamine neurons; therefore, it is stereotactically targeted

into the substantia nigra, the nigrostriatal tract or the striatum. The neurotoxic effects

of 6-OHDA are believed to involve oxidative stress-related mechanisms. MPP+ the

active metabolite of MPTP, is selectively taken up by the dopaminergic neurons via

its affinity for the dopamine transporter (DAT). The mechanism of action of paraquat

is believed to involve oxidative stress; due to its structural similarity to MPP+, its toxic

effects could be via the mitochondria. Rotenone, a potent inhibitor of complex I, is

believed that rotenone-induced partial inhibition of complex I and the subsequent

oxidative stress renders dopaminergic cells selectively vulnerable to chronic low levels

of mitochondrial dysfunction. Direct administration of 3-NT into the striatum tests the

involvement of oxidative stress, specifically peroxynitrite, in nigrostriatal

dopaminergic degeneration. Mutations in the α-synuclein gene, linked to a small group

of familial PD cases, are suggested to play a role in neuronal degeneration and

increased protein aggregation (Sherer et al., 2003).

59

The site of 6-OHDA injection significantly affects the characteristics and extent of

neurodegeneration (Agid et al., 1973; Przedborski et al., 1995). Injection of 6-OHDA

into substantia nigra, cause a very rapid degeneration process of dopaminergic neurons

(up to 90% loss) to take place within hours (12 hours) producing a complete damage

in the nigrostriatal pathway, followed by loss of striatal terminals in 2 to 3 days (Faull

and Laverty, 1969; Jeon et al., 1995), this is similar to end-stage PD. In the medial

forebrain bundle (MFB) model, striatal terminals are degenerated before dopaminergic

neurons (80 - 90% loss) producing a near complete damage in the nigrostriatal pathway

within 3 to 4 days after injection which is equivalent to end-stage in PD. In the

consecutive 3 weeks, dopamine content is totally lost in the striatum. In comparison to

substantia nigra and medial forebrain bundle, 6-OHDA injection into striatum,

produces slow, (in 3 weeks’ time) progressive, and partial loss of dopaminergic

neurons and damage in the nigrostriatal pathway in a retrograde fashion which is more

relevant to PD (Przedborski et al., 1995; Sauer and Oertel, 1994). The percent of

dopaminergic cell death is dose-dependent and can vary from 30 to 75% depending on

number of injection sites. Up to four injections can be delivered into the striatum (Kirik

et al., 1998). This model is characterized by producing non-motor symptoms;

cognitive, psychiatric, and gastrointestinal dysfunction (Branchi et al., 2008; Cannon

and Greenamyre, 2010). Therefore, this model was employed in this study.

60

1.5.1.2 Gold Standard: MPTP

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model was discovered

accidentally in California 1983 in intravenous drug abusers using an analogue of the

synthetic opioid; meperidine which was contaminated with MPTP, patients showed

some movement abnormalities (bradykinesia) similar to PD (Davis et al., 1979;

Langston et al., 1983). L-dopa was successful in treating these patients and

confirmatory postmortem studies demonstrated the loss of nigrostriatal structures in

the brain of same patients (Davis et al., 1979; Langston et al., 1999). MPTP is a

lipophilic compound that can cross blood-brain barrier, once inside the brain it can be

taken up by the astrocytes and metabolized by MAO-B enzyme into its active toxic

form 1-methyl-4-phenylpyridinium MPP+ (Figure 30). The active metabolite MPP+

then can be taken up by the neighboring dopaminergic neurons via dopamine

transporters (DAT) and stored in the vesicles by vesicular monoamine Transporter

(VMAT) displacing and releasing dopamine from the storing vesicles (Figure 29).

Released dopamine intracellularly can form toxic compounds such as; DOPAL

(Panneton et al., 2010) where it can be exposed to superoxide radical (5-cysteinyl-DA)

and hydroxyl radical (6-OHDA). Still, MPP+ can be more damaging by inhibiting

complex I causing to mitochondrial electron chain inhibition leading to energy failure

and oxidative stress and dopaminergic cell death (Mizuno et al., 1987; Nicklas et al.,

1985). MPTP causes severe damage to nigrostriatal pathway with significant loss of

dopaminergic neurons in the striatum and SNpc (Dauer and Przedborski, 2003).

61

Figure 30: Chemical structures of MPTP and MPP+

(Bové and Perier, 2012)

MPTP is a toxic compound in a wide range of species including mice, monkeys,

sheep, dogs, guinea pigs, cats, but not rats which were found to be resistant to this of

toxicity (Bezard et al., 1998; Giovanni et al., 1994; Przedborski et al., 2001). Mouse

model is a practical and can provide a model of genetic mutation compared to the gold

standard-monkey model which needs highly trained lab workers (Giovanni et al.,

1994). MPTP model also lack the pathological hallmark of PD disease; Lewy bodies,

but in the other hand some reports have investigated the expression of α-synuclein

(Dauer et al., 2002; Halliday et al., 2009; Purisai et al., 2005; Shimoji et al., 2005; Vila

et al., 2000).

1.5.1.3 Rotenone

Rotenone is a natural compound found in plants that belong to family of

Leguminosa (Tieu, 2011). It has been used mainly in farming as a pesticide. Rotenon

model was developed for the first time by Greenamyre and his colleagues where the

administered rotenone chronically using low-dose treatment in rats (Betarbet et al.,

2000; Cannon et al., 2009; Sherer et al., 2003). Due its high lipophilicity, it crosses

blood-brain barrier and enter almost all cells independent of any transporter. Similar

to MPTP cytotoxic mechanism, rotenone also inhibits complex I with the

consequential oxidative stress and neuronal cell death (Parker et al., 1989; Schapira et

62

al., 1989). Interestingly, chronic systemic administration of rotenone to rats replicates

all pathological features of PD, namely; proteinaceous aggregate, Lewy-like bodies,

α-synuclein, behavioral abnormalities, and oxidative stress (Greenamyre et al., 2010;

Sherer et al., 2003). Rotenone can be administered by different routes of

administration, mainly intraperitoneal daily injection (Cannon et al., 2009),

subcutaneous, or intravenous (Fleming et al., 2004). Most recently, it has been tested

in mice via chronic intragastric rout or administration and direct stereotaxic injection

or infusion into the brain (Alam and Schmidt, 2004; Pan-Montojo et al., 2010). A

major drawback of this model is the high variability between different animals with

high mortality which may reach up to 50% (Blesa and Przedborski, 2014; Emborg,

2004). Moreover, trials to induce PD using rotenone in other species like mice or

monkeys were unsuccessful, and not even in humans (Blesa et al., 2012).

1.5.1.4 Paraquat

Paraquat (N,N’-dimethyl-4-4’-bipiridinium) is a divalent cation compound that has

a structure similarity with MPTP (Figure 29) (Snyder and D’Amato, 1985). Originally,

it was used as a herbicide that has been forbidden in European Union after 2007 (Bové

and Perier, 2012). Due to its hydrophilicity, paraquat uses neutral amino acid transport

mechanism to penetrate blood-brain barrier. Once inside the brain paraquat causes

cytotoxicity by inducing redox cycling with a cellular diaphorase in form of NADPH

oxidase and nitric oxide synthase leading ROS production such as superoxide radical,

hydrogen peroxide and the hydroxyl radical causing damage of cellular lipids,

proteins, DNA and RNA (Day et al., 1999). Injection of paraquat in mice causes dose-

and age-dependent nigral damage (Brooks et al., 1999; McCormack et al., 2002;

Thiruchelvam et al., 2003). Paraquat causes not only brain toxicity but multi-organ

63

toxicity; lung, liver, and kidney, which may result in death after acute exposure in

human (Grant et al., 1980; Hughes, 1988). Several reports established the presence of

α-synuclein aggregates and Lewy bodies in this model (Fernagut et al., 2007; Mak et

al., 2010; Manning-Bog et al., 2002).

Other neurotoxic models

Several other models gave have been used to induce nigrostriatal degeneration and

dopaminergic cell death. However, those models are not in common use.

1.5.1.5 Reserpine

Reserpine was one of the earliest pharmacological agents that have been used to induce

Parkinson’s disease in rodents, after the observation that it induces movement

abnormalities; akinesia, in injected animals (Carlsson et al., 1957). Remarkably, these

symptoms could be reversed by L-dopa treatment (Carlsson, 1959). Carlsson

demonstrated that reserpine depletes monoamines in general in the brains of injected

animals. Reserpine model has been reproduced also in other mammals such as; cats,

rabbits, guinea pigs, and monkeys (Bezard et al., 1998). Reserpine is not only

reversible but also non-specific; it mediates it’s action by hindering catecholamine

storage in synaptic vesicles through ATP- and magnesium mechanism (Bezard et al.,

1998), such mechanism has no direct relation to dopamine and not toxic to nigrostriatal

pathway. The aforementioned findings have weakened the use of this model.

1.5.1.6 α-Methyl-Para-Tyrosine

This reversible pharmacological agent is an enzyme inhibitor, it inhibits the

enzyme tyrosine hydroxylase in dopamine synthesis process, blocking dopamine

synthesis (Corrodi and Hanson, 1966; Spector et al., 1965). α-methyl-p-tyrosine has

64

the same weakness points of reserpine being reversible, non-specific, and not

triggering neurodegeneration in nigrostriatal pathway (Tieu, 2011).

1.5.1.7 Amphetamines

A pharmacological group of compounds that belongs to psychostimulant drugs,

has been found to be highly neurotoxic and producing behavioral and structural

alteration similar to PD (Cadet et al., 2007; Thrash et al., 2009). Amphetamine

derivatives such as: methamphetamine (METH) and 3,4-

methylenedioxymethamphetamine (MDMA), p-chloroamphetamine (PCA), and

fenfluramine are toxic to serotoninergic and dopaminergic terminals in the striatum,

nucleus accumbens, and frontal cortex (Hess et al., 1990; Krasnova and Cadet, 2009),

but SN and VTA neuronal cell bodies are affected only at higher doses (Sonsalla et al.,

1996; Trulson et al., 1985).

1.5.2 Genetic Models

Genetic animal models of PD have been designed on previously recognized targets

to better study the disease process and therapy (Bezard and Przedborski, 2011;

Meredith et al., 2008), noting that none of such genetic mutations are expressed in

humans. Moreover, most of the genetic models don’t exhibit the behavioral

phenotypes and neurodegeneration of PD (Dawson et al., 2010). For instance, many

studies have reported the presence of mitochondrial dysfunction (Exner et al., 2012;

Matsui et al., 2014; Morais et al., 2014), ubiquitin-proteasome dysfunction (Dantuma

and Bott, 2014), ROS formation (Gandhi et al., 2009; Joselin et al., 2012; Ottolini et

al., 2013). nevertheless, none of the studies could demonstrate significant changes in

65

dopaminergic neurons (Andres-Mateos et al., 2007; Goldberg et al., 2003; Hinkle et

al., 2012; Sanchez et al., 2014). Here are the commonly used transgenic models of PD:

1.5.2.1 α-synuclein

α-syn gene is the core component of Lewy bodies (LB), and a type of missense

mutations of α-syn. It is also called Park1. Up to date, two mutations in the gene

(A53T, A30P) identified and causes a dominantly-inherited form of familial PD

(Goedert et al., 2013; Krüger et al., 1998). This model generates a transgenic mice with

typical behavioral and motor phenotype, but with no effect on DA neurons (Abeliovich

et al., 2000; Thomas et al., 2011). Conversely, studies that have been done in

Drosophila with a mutant α-syn gene, showed a reduced TH expression with a

significant dopaminergic nigral cell loss, and inclusion bodies (Feany and Bender,

2000). With all these controversies, hence the role of α-syn gene is still to be

determined.

1.5.2.2 LRKK2

LRRK2 (leucine rich repeat kinase 2) gene is localized to membranes.

Mutation in LRKK2 causes an autosomal dominant inherited form of PD (Healy et al.,

2008). Several mutations have been identified (Rudenko and Cookson, 2014), but all

with no considerable disruption of SN dopaminergic neurons (Wang et al., 2008).

1.5.2.3 PINK1

PINK1 (phosphatase and tensin homolog- induced novel kinase 1) gene is

localized to the mitochondria, causing autosomal recessive PD, and it’s also called

PARK6 (Scarffe et al., 2014). In this form of mutation there is an age-dependent

66

moderate loss of dopaminergic neurons which is associated with overexpression of α-

syn (Oliveras-Salvá et al., 2014). This model shares a common phenotypes similarity

with those of Parkin KO and DJ-1 KO mice.

1.5.2.4 Parkin

Parkin is an autosomal recessive mutation, accounting for 50% of familial

cases of the disease and 20% of the young onset PD cases (Lücking et al., 2000;

Periquet et al., 2003). Parkin KO mice show reduced DA level with minimal or no

behavioral abnormalities (Itier et al., 2003; Kitada et al., 2009). One of this gene

mutations; Parkin-Q311X-DAT-BAC mice displays age-dependent motor impairment

along with loss of dopaminergic terminals in striatum and degeneration of SN cell

bodies (Lu et al., 2009).

1.5.2.5 DJ-1

DJ-1 mutation is an autosomal recessive and early-onset for the disease

(Puschmann, 2013). This form of genetic mutation results in motor and behavioral

abnormalities, reduced DA level, but no effect on dopaminergic neuronal cells in SN

(Goldberg et al., 2005; Kim et al., 2005).

67

1.6 Fundamental Methods of Assessing Structure and Function of Nigrostriatal

Pathway

Assessment of neuropathological process may vary from one study to another.

However, when using neurotoxic model, and planning to investigate

neurodegeneration or neuroprotection, like in our case- it is essential to study

nigrostriatal pathway integrity and function.

1.6.1 Dopaminergic Neurons in the Substantia Nigra Pars Compacta

Substantia nigra pars compacta (SNpc) is a very rich area in dopaminergic neurons,

its estimated that a mouse brain contains around 8000 ∼ 14,000 dopaminergic neurons,

and this is variable from one strain to another (Zaborszky and Vadasz, 2001). The

density of dopaminergic neurons is not distributed homogenously in SN being denser

in rostral region. In Figure 31, coronal section of SN from caudal to rostral regions

show variable distribution of the cells. Therefore, to sample the populations from all

regions of SN we should choose an unbiased quantification method ensuring sampling

of all regions at systemic section intervals. Currently, the gold standard method is to

use unbiased stereological cell counting with an optical fractionator system (West et

al., 1996, 1993, 1991).

68

Figure 31: Dopaminergic neuronal distribution in striatum and substantia nigra.

(I) Dopaminergic cell bodies reside in SNpc and their terminals project to striatum for

dopamine release. Coronal sections at SN from caudal (A) to rostral (H) showing

heterogenous distribution of dopaminergic neurons. Coronal sections at striatum from

caudal (J) to rostral (P) showing heterogenous distribution of dopaminergic terminals

(Tieu, 2011).

69

1.6.2 Dopaminergic Terminals in the Striatum

The density of dopaminergic terminals in the striatum correlates with the cell

bodies they originate from in the SN, hence, the distribution of striatal terminals would

vary moving from caudal to rostral (Bockaert et al., 1976; Widmann and Sperk, 1986).

Immunohistochemical reaction of TH activity is a good measure of quantification.

Another way of assessing dopamine terminals level is immunoblotting technique using

TH and DA markers.

1.6.3 Striatal Dopamine

All the above-mentioned forms of assessments are assessing the structural integrity

of nigrostriatal pathway, its important also to assess the function of this pathway. This

can be done by measuring striatal dopamine release and its reflection on motor activity

and behavior. Measuring of striatal dopamine release can be performed by using high-

performance liquid chromatography (HPLC) with an electrochemical detector.

1.6.4 Lewy Body Aggregates

Lewy bodies are considered the pathological hallmark in PD. There are several

methods for detection of this mis-/or unfolded protein aggregates. An easy straight

forward method is to use an antibody against α-synuclein (Beach et al., 2008).

1.6.5 Behavioral/Motor Assessment

As we mentioned earlier, dopamine depletion is reflected on animals’ behavior.

There are several behavioral tests to assess the locomotor activities of animal models,

such as; rotarod, drug-induced rotation, limb use asymmetry, forelimb placing test,

swim test, adhesive removal test, and pole test (Meredith and Kang, 2006).

70

Chapter 2: Aims and Objectives

The main goal of in this study was to investigate the effect of curcumin application

on the functional properties of human neuronal 7 nicotinic acetylcholine receptors

(nAChRs). This work was divided into two main parts:

2.1 In-vitro Electrophysiological Study

o In the first part of the study, human 7-nAChR were expressed homomerically in a

Xenopus oocyte expression system to investigate:

1. The effect of curcumin on the functional properties of 7-nAChRs.

2. Determination of the voltage-dependence of curcumin action on the α7-

nAChRs, and the role of endogenously expressed Ca2+ activated Chloride

channel and the role of pertussis toxin sensitive G proteins in mediating the

effect of curcumin on the α7-nAChR.

3. The effect of curcumin on other nAChRs subunits & ligand gated ion channels.

4. The effect of other curcumin analogues and metabolites on 7-nAChRs.

5. The binding affinity of curcumin and its derivatives with 7-nAChRs.

2.2 In-vivo Study

o To establish the neuroprotective effect of curcumin in animal model of Parkinson’s

disease by investigating:

1. Improvement of abnormal motor behavior of the animals.

2. Effect of curcumin on the survival of dopaminergic striatal nerve terminals.

3. Effect of curcumin on the survival of dopaminergic neurons in SNpc.

71

o To test whether the neuroprotective effect of curcumin in animal model of PD is

mediated through an α7-nAChRs.

72

Chapter 3: Materials and Methods

3.1 Electrophysiological In-vitro Study

Materials

3.1.1 Female Xenopus Oocytes

Mature female African clawed frogs (Xenopus laevis) were purchased from

Xenopus Express, Haute-Loire, France (Figure 32). They were housed in in a water-

tank (dimensions; 32 cm width, 130 cm length, and 66 cm height) filled with

dechlorinated water. The room temperature was maintained at 19-21°C with a 12/12-

hour light/dark cycle. Frogs were fed twice a week with food pellets, supplied by

Xenopus Express, France. Tank-water was changed twice a week. Animal care and

handling experiments conducted in this study were in accordance with institutional

guidelines and approved by the Animal Ethic Committee of the CMHS/UAEU.

Figure 32: An adult female Xenopus laevis (Professor Murat Oz’s laboratory)

73

3.1.2 Chemicals

Table 5: Chemicals required for the experiments

Compounds Formula weight Manufacturer/ CAS #

Curcumin 368.38 Santa Cruz/458-37-7

α-Bungarotoxins ~ 8500 Sigma / T-0195-

Acetylcholine Chloride 181.7 Sigma, USA/ A6625

5-Hydroxytryptamine 387.4 Sigma, USA / H-7752

Glycine 75.07 Sigma, USA / G7126

Sodium Chloride (NaCl) 58.44 Sigma, USA/ S-3014

HEPES 238.3 Sigma, USA/ H3375

Potassium Chloride (KCl) 74.55 Mallinckrodt, USA/ 6858

Magnesium Sulphate (MgSO4) 246.5 Sigma, USA/ M9391

Sodium Bicarbonate (NaHCO3) 84.01 Sigma, USA/ S6014

Magnesium Chloride (MgCl2) 95.22 Sigma, USA/ M8266

Calcium Chloride (CaCl2) 110.9 Sigma-Aldrich, USA/C-4901

Barium Chloride (BaCl2) 244.28 BDH, England/R-20-25 S:45

Sodium Hydroxide (NaOH) 40 Amresco, USA/

Lot.#214613010

Hydrogen Chloride (HCl) 37% Sigma-Aldrich, USA/

25.814.B

Gentamycin Sulphate salt

(mixture of 3 major components

designated as C1, C1a, and C2)

C1 = 477.6

C1a = 449.5

C2 = 463.6

Sigma-Aldrich, USA/ G1264

Theophylline 180.2 Sigma/ T-1633

Sodium Pyruvate 110.04 Sigma-Aldrich, Japan/P5280

Penicillin G 356.4 Sigma, USA/ P3032

Streptomycin sulfate 1457.4 Sigma-Aldrich, USA/ S9137

BAPTA 764.68 Sigma-Aldrich / A-1076

Benzocaine 165.2 Sigma, USA/ E-1501

Collagenase-A CLS-1 (from

Clostridium histolyticum)

Concentration

200 u/mg

Worthington, biochemical

corporation, NJ, USA /

LS004196

PKC-412 570.64 Tocris, Minneapolis, MN

Go-6983 442.51 Tocris, Minneapolis, MN

KT-5720 537.61 Tocris, Minneapolis, MN

KN-62 721.84 Tocris, Minneapolis, MN

74

3.1.3 Other Materials

Table 6: Other materials and devices used in the study

Device or Material Specifications Company of Purchase

Electrode Holder -- World Precision Instruments,

Sarasota, FL, USA

Magnetic stand and

Manipulators Catalog #7739 Narishige, Tokyo, Japan

Silver Wires -- World Precision Instruments,

Sarasota, FL, USA

Borosilicate Glass

tubing

for microelectrodes

Glass thin-walled

with filament 1.5 mm

Catalog #TW150F-4

World Precision Instruments,

Sarasota, FL, USA

Vertical Puller

Model 700D, heater

adjusted to 48 and

solenoid adjusted to 70 οC to get optimal

resistance of 1-2 MΩ

David Kopf Instruments,

Tujunga, CA, USA

Microfill filling

syringe --


Sarasota, FL, USA

Micro-4

Microsyringe

pump controller

Model UMC4-C World Precision Instruments,

Sarasota, FL, USA

Automatic Nano-liter

injector

Nanoject

Drummond Scientific

Company, Broomall, USA

RNAase free water in

1.8ml Eppendorf

tubes

Lot #M25/80502 Epicenter Biotechnologies

Madison, Wisconsin, USA

pH Meter Model 450 Corning pH meter, Albany,

NY, USA

Stirrer Rotomix type 50800,

Model #M50825

Barnstead/ thermolyne,

Dubuque, IA, USA

Picofuge Catalog #400550 Stratagene, Santa Clara, CA,

USA

Petri Dishes Catalog #127,60mm Sterillin, Newport, UK

Surgical Accessories Scissors, forceps,

scalpels


Sarasota, FL, USA

Surgical Sutures

Catgut Chrom, reverse

cutting 3/8 circle, USP

4/0, SMI

DemeTech Corporation,

Miami, Florida, USA

Dissecting

microscope Model GSZ

Bunton Instruments Co Inc,

Rockville, MD, USA

75

3.1.4 Experimental Setup

The experimental setup for electrophysiological recordings using the two electrode

voltage-clamp is shown in Figure 33 and 34. Two-electrode voltage clamp (TEVC)

technique was applied using a GeneClamp-500B amplifier (Axon Instruments,

Molecular Devices, Inc., Sunnyvale, CA, USA), as described previously (Ashoor et

al., 2013) recording setup included magnetic holding devices (Kanetec USA

Corporation, Bensenville, IL, USA), two manual micromanipulators (M33;

Märzhäuser, Wetzlar, Germany) and head-stages for voltage (HS-2A Headstage, Gain

1 MG, Axon Instruments, Molecular Devices, Inc., Sunnyvale, CA, USA) and current

(HS-2A Headstage, Gain 10MG) were attached to the manipulators. The two glass

electrodes were inserted in electrode holders and then connected to the head-stages.

Micromanipulators were used to control the electrodes and impale the oocyte.

The perfusion apparatus consisted of perfusion tubes and bottles containing

extracellular solutions connected to the recording bath by silicon tubing (Cole Parmer

Instrument Company, I.D. 1/16 inch, O.D. 1/8 inch and WALL1/32, Vernon Hills,

Illinois, USA). Flow rate of perfusion was set to 3 - 5 ml/minute. A multichannel

perfusion system was used for drug applications included tubing (C-Flex tubing, Cole-

Parmer Instrument Company, I.D. 1/32 inch, O.D. 3/32 inch and WALL1/32 inch,

Vernon Hills, Illinois, USA), 50 mL glass syringes, and coupling devices. The drug

application system was based on gravity flow by means of a micropipette that was set

at a distance of about 2-3 mm from the oocyte position in the perfusion

chamber/recording chamber (Warner Instruments LLC, Hamden, CT, UK) designed

for placing oocyte to be impaled with the microelectrodes (Figure 34). The perfusion

76

solution in the recording chamber was removed by using a glass suction tube

connected to an adjustable vacuum source and collected in the waste tank.

Figure 33: Two-electrode voltage-clamp (TEVC) recording set-up from Xenopus

oocytes.

The oocyte was placed in the recording chamber and continuously bathed with

physiological solution. The oocyte membrane was penetrated with two

microelectrodes one for voltage sensing and the other for current injection. Drugs and

compounds were applied using gravity-based multichannel application system, at the

time of drug application the perfusion was stopped. TEVC is achieved using

Genclamp500 amplifier interfaced to a PC computer equipped with

electrophysiological software for data acquisition (Professor Murat Oz’s laboratory).

77

Figure 34: The oocyte impaled with two microelectrodes.

Illustration of the plastic chamber. The oocyte was placed in the recording chamber

and perfused continuously during the experiment (Professor Murat Oz’s Laboratory).

A fiber optic light source was used for illumination of the recording chamber (Fiber

Lite, High Intensity Illuminator Series 180, Dolan-Jenner Industries Inc. Boxborough,

MA, USA). A Low-power stereo-dissection microscope was used for visual

observation of the recording chamber (Olympus, Tokyo, Japan, SZ-STB1, 100 AL0.5

X, WD186). Computer set up for data acquisition consisted of a Compaq computer,

(Compaq Corporation, Wynyard, UK) and analog-digital converter, BNC 2081

(National Instruments, Austin Texas, USA).

78

Methods

3.1.5 Preparation of Required Solutions

3.1.5.1 Modified Barth’s Solution

Modified Barth’s Solution (MBS) was used during oocytes isolation process. The

composition of MBS is shown in table below:

a) Calcium free MBS solution:

Table 7: Calcium free MBS solution composition

Compound Concentration

(mM)

1x

(Weight in grams)

10x

(Weight in grams)

NaCl 88 5.14 51.4

HEPES 10 2.38 23.8

NaHCO3 2.1 0.2 2

KCl 1 0.075 0.75

MgSO4 0.8 0.2 2

The above components were dissolved in distilled water to get total volume of 1 L and

the pH was adjusted to 7.5 using NaOH.

b) Calcium containing MBS solution:

Similar to previous composition, with the addition of 0.22 g and 2.2 g of CaCl2

(2mM) to make stock solutions of 1x and 10x respectively. All compounds were

dissolved in distilled water to get total volume of 1 L and the pH was adjusted to 7.5

3.1.5.2 Oocyte Storage Solution

The storage solution was prepared as following: 1) 100 ml of 10x Calcium

containing MBS solution; 2) 900 ml Distilled water; 3) Antibiotic materials shown in

(Table 8) were dissolved in the previous solution; 5) pH was adjusted to 7.5; 6) The

mixture was filtered and stored in a sterile container.

79

Table 8: Antibiotic materials

Compound Concentration Weight in grams

Penicillin G 10,00 U/L 0.02

Gentamycin Sulphate

salt 50mg/L 0.1

Streptomycin 10mg/L 0.01

Sodium pyruvate 2mM 0.22

Theophylline 0.5mM 0.09

3.1.5.3 Extracellular Solution

ND96 extracellular solution was used in the two-electrode voltage-clamp

technique to record ion currents mediated by the nAChRs, and normal Ringer’s

solution was used to record ion currents mediated by the glycine and 5-HT3 receptors.

The recipes for both ND96 and normal Ringer’s solution used were as follow:

a) ND96 solution:

Table 9: ND96 solution composition

Compound Content

(mM)

1x

(Weight in grams)

10x

(Weight in grams)

NaCl 96 5.61 56.1

KCl 2 0.15 1.5

MgCl2 1 0.10 1.0

HEPES 5 1.19 11.9

CaCl2 or

BaCl2

1.8

1.8

0.20

0.439

2.0

4.39

The above components (1x) are dissolved in Distilled Water to produce a total volume

of 1 L and the pH was adjusted to 7.5 using NaOH.

80

b) Normal Ringer’s solution:

Table 10: Normal Ringer’s solution composition

Compound Content

(mM)

1x

(Weight in grams)

10x

(Weight in grams)

NaCl 115 6.72 67.2

KCl 1 0.074 0.74

CaCl2 1.8 0.199 1.99

HEPES 10 2.38 23.8

The above components (1x) were dissolved in distilled water to get total volume of 1

L and the pH was adjusted to 7.4 using NaOH.

3.1.6 Drug Application

Stock solutions of the test compounds were prepared in ND96 solution when

recording ion currents mediated by the nAChRs (or in normal Ringer’s solution when

recording ion currents mediated by glycine and serotonin receptors) using the

following formula:

Weight (mg) = (MW) x (Volume (L)) x (concentration (mM))

Further dilutions were prepared using the Charles equation:

C1 x V1 = C2 x V2

Where,

C1 = concentration of stock solution

V1 = volume of stock solution to be used

C2 = desired concentration to be prepared

V2 = desired volume to be prepared

Stock solutions and required dilutions were prepared freshly before

starting the experiments.

81

3.1.7 Isolation and Maintenance of Oocyte from Xenopus Laevis

Xenopus laevis female frogs were anesthetized in 1L of 0.03% w/v benzocaine

solution, prepared by dissolving 300 mg of ethyl p-aminobenzoate in 15 mL of 70 %

ethanol and then adding that to 1L of cold tap water. The end point of anesthesia was

determined by failure to respond to noxious stimuli induced by pinching of the lower

limbs. Under described conditions, usually 5-10 minutes to achieve full anesthesia, the

anesthetized frog was placed on crushed ice covered with a wet paper towel to avoid

skin from drying out during surgery while maintaining low core body temperature

during surgery. A small incision of about 1.5 cm length was made through the

epidermal layer and in the inner muscular layer of the lower abdominal area slightly

to the left or right of the midline, and a similar cut. Using sterilized forceps, one to

two ovarian lobes (small clumps of oocytes) were removed and placed in a petri dish

containing Ca2+- free MBS (Figure 35).

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Figure 35: Steps of oocyte isolation and preparation.

A flow diagram of oocytes isolation steps. Female Xenopus frog was anesthetized, and

ovarian lobes were taken out and kept in Ca2+- free solution. Fine forceps were used

to separate oocyte clusters into small pieces, followed by enzymatic treatment using

collagenase enzyme. Finally, the collagenase solution washed away with Ca2+- free

solution and then with Ca2+-containing solution for several times. Mature healthy-

looking oocytes were collected for injection [Modified from (Nakagawa and Touhara,

2013)].

After removing the ovarian lobes, the muscular layer as well as the outer skin were

sutured with absorbable Catgut sutures. After surgery, the frog was kept in a container

filled with tap water with the head elevated (to prevent drowning) and the rest of the

body submerged, and closely monitored for recovery, based on free swimming

behavior. Recovery from the anesthesia usually take up to 1 hour. After 3-4 hours after

recovery, the frog was returned to the main frogs’ container. Each frog was utilized

for three to four surgeries with a gap of two to three months between each procedure.

83

3.1.8 Oocyte Preparation

The preparation of oocytes was performed according to procedures described earlier

(Oz et al., 2003). Briefly, using fine forceps, the inner ovarian epithelium, theca, and

follicular layers were removed as much as possible to produce smaller clusters of

oocytes (Figure 36).

The resulting oocyte clusters were then treated with collagenase A solution

(collagenase enzyme solution was prepared by dissolving 80 mg of Collagenase, type

A, in 25 ml of Ca2+ Free MBS solution). The oocyte clusters were incubated in a small

conical flask containing 12.5 ml collagenase solution with constant stirring (60-80

rotations/minute) at room temperature for 1.5 hour. After that, collagenase solution

was replaced with fresh collagenase solution 12.5 ml and kept in stirring for another

1.5 hour. Finally, oocytes were washed gently; 5 times using Ca2+ Free MBS, then 5

times with Ca2+ containing MBS solution. Subsequently, oocytes were transferred to

a petri dish filled with Ca2+ containing MBS for oocyte selection.

Only healthy-looking mature oocytes (stage V-VI) were selected under a dissecting

microscope (Bunton Instruments Co Inc., Model GSZ, Rockville, MD, U.S.A.). Stage

V and VI oocytes characterized by larger size with 1.0 mm - 1.2 mm diameter, rounded

shape, and clear dark brown animal pole and yellow vegetal pole divisions (Figures 36

& 37) were selected. The oocytes were subsequently maintained in MBS at 18 °C and

used within 5 - 7 days.

Throughout the electrophysiological experiments, we employed a Xenopus oocyte

expression system for the following reasons: 1) methods for harvesting and

maintaining oocytes are well established and straightforward. As a result, high

numbers of cells with desirable expression levels were routinely available. 2) the

84

oocytes are freshly derived from living frogs, thus they can be treated as primary cell

culture. 3) the two-electrode voltage clamp technique is a relatively less laborious and

a high-yield electrophysiological assay system. 4) the two-electrode voltage clamp

technique is stable for many hours, making it an ideal method for experiments

requiring long-recording times.

Figure 36: Frog’s ovarian lobe.

(A) An ovarian lobe, containing oocytes at different developmental stages. (B)

Separating oocytes by removal of epithelial and follicular layers manually with fine

forceps. Isolated stage V and VI oocyte of Xenopus laevis after collagenase treatment.

A

B

85

Figure 37: Stages of oocyte development.

Oocytes vary in size according to maturation stage. Oocyte size range from 100 – 1300

µM (Wozniak et al., 2018).

3.1.9 Synthesis of cRNA

The cDNA clone of human α7-nAChR was kindly provided by Dr. J. Lindstorm

(University of Pennsylvania, PA, U.S.A.). Capped cRNA transcripts were synthesized

in vitro using a mMESSAGE mMESSAGE kit (Ambion, Austin, TX, U.S.A.) and

analyzed on 1.2% formaldehyde agarose gel to check the size and quality of the

transcripts (Figure 38).

Human α7-nAChR mRNA was prepared by in vitro transcription and confirmed

by gel analysis. Restriction enzyme (Xbal) was used to digest the cDNA of human α7

nAChR, and it was cleaned by the Qiagen kit. Linearized plasmid cDNA was

transcribed in vitro by SP6 RNA polymerase to produce α7-nACh receptor RNA using

a mMESSAGE mMACHINE kit. This RNA was cleaned and purified by

phenol:chloroform extraction and ethanol precipitation. The quantity of RNA was

86

estimated by OD260 measurement and quality was assessed by agarose gel. Capping

enzyme and 2’-O-methyltransferase were used to add a cap and poly(A) tail to RNA,

and the resulting mRNA was suspended in DEPC-treated water. Once more, mRNA

was cleaned by phenol:chloroform extraction and ethanol precipitation, then run

against RNA in agarose gel to confirm the addition of 5’ cap and 3’ poly(A)tail (Figure

38).

Figure 38: Agarose gel analysis of mRNA

Lane 1: NEB ssRNA ladder with 9,7,5,3,2,1, and 0.5 kilo bases markers.

Lane 2: 2 μg RNA of 5-HT3A receptor.

Lane 3 and 4: 1.5 and 0.75 μg mRNA of 5-HT3A receptor, respectively.

Lane 5: 3μg RNA of α7-nAChR.

Lanes 6 and 7: 1.8 and 0.9 μg mRNA of α7-nAChR, respectively.

3.1.10 In-vitro cRNA Synthesis

The mMessage mMachine kit used to synthesize the capped cRNA transcripts was

purchased from Ambion (SP6 kit, Austin, TX, USA). The synthesized capped cRNA

transcripts were analyzed on 1.2% formaldehyde agarose gel to check the quality and

87

size of the transcripts. The cDNA clones of human α7-nAChR was provided by Dr. J.

Lindstorm (University of Pennsylvania, PAM, USA).

3.1.11 Microinjection of cRNA into Oocytes

The mRNA concentration of synthesized human α7-nAChRs and other subunits

used in this study are shown in (Table 11). They were stored as 1μl aliquots in freezer

at -80o C. Once the oocytes were prepared and sorted, only one RNA aliquot was

transferred in an ice bucket to the laboratory. To maintain a RNase free environment

while working with cRNA, the tube was centrifuged using a microcentrifuge at 1200

rpm for 1- 3 min, and then by using a sterile pipette and RNase/DNase free pipette tips

(Denville Scientific Inc., Metuchen, NJ, USA), and resulting cRNA pellet was diluted

to a final concentration of 10 ng/50 nl in RNase-/DNase- free water. Only 3 to 4 μL of

diluted cRNA was used for each batch of oocytes, and the remaining cRNA was

returned to the freezer. A vertical puller (Model 700 D, David Kopf Instruments

Tujunga, CA, USA) was used for making glass microelectrodes with fine needle

shaped tip from autoclaved glass capillaries (World Precision Instruments, Sarasota,

FL, USA). The tip of each glass microelectrode was broken by applying gentle

pressure using a fine pair of forceps (Fine Science Tools Inc., Vancouver, Canada)

under a dissecting microscope (Bunton Instruments Co., Rockville, MD, USA).

The glass needle was backfilled with mineral oil (Sigma, St. Louis, MO, USA) using

a 1 ml glass syringe. Then the glass needle was fitted into a microdispenser connected

to the micromanipulator (Figures 39 & 40).

88

Table 11: The initial concentration of all subunits

Receptor Subunit Main stock conc. (µg/µl)

nACh receptors

α7 3.7

α3 2.43

α4 2.15

β2 1.66

β4 1.71

Glycine receptor

α1 2.18

α2 1.678

α3 1.595

5-HT3 receptor A 2.1

Subsequently, 3 to 4 μL of diluted cRNA or distilled water was dropped on the

center of a mineral oil drop placed on parafilm (American National Can Co.,

Greenwich, CT, USA). By focusing on the drop under the dissecting microscope, the

tip of the glass needle was placed at the center of the droplet, and the aqueous phase

was carefully withdrawn into the glass needle using the withdrawal option on the

microdispenser controller. After loading the sample, the sorted oocytes were arranged

in a U-shaped pattern in a small petri dish, containing MBS, with mesh-bottom to hold

the oocytes in place during the microinjection procedure. Each oocyte was impaled by

the glass needle and gently injected with 50 nL of cRNA solution or distilled water

using the microinjector (driven by a Micro-4, micro syringe pump controller).

89

Figure 39: Microelectrode set-up for cRNA injection.

Schematic presentation of microinjector set-up. The glass microelectrode backfilled

with cRNA solution for oocyte injection (Bianchi, 2006; Nakagawa and Touhara,

2013).

Figure 40: cRNA injection set-up (Professor Murat Oz’s Laboratory)

Low-power stereo micro-scope

Volume control

Microinjecto

90

Following the injection, the oocytes were stored in 25 ml petri dishes filled with

oocyte storage solution and incubated at 18ºC. In initial experiments, the oocytes were

divided into two groups; oocytes that were injected with cRNA encoding the human

α7-nAChR, and oocytes that were injected with distilled water (control). Xenopus

oocytes were used in the electrophysiology study 2-3 days after microinjection to allow

maximal expression of receptors. Healthy oocytes were transferred to fresh dishes with

new storage solution daily. A bottle of storage solution was stored in the incubator

with the oocytes. The oocytes were used for about 7-10 days.

3.1.12 Two Electrode Voltage Clamp

The voltage clamp technique is a method that allows ion flow across the cell

membrane to be measured as an electric current while the transmembrane potential is

held constant (clamped) with a feedback amplifier. Functional properties of ion

channels expressed in Xenopus oocytes can be studied effectively using the two-

microelectrode voltage clamp (TEVC). In TEVC, two intracellular microelectrodes

were employed, a voltage sensor electrode and a current injection electrode to maintain

the transmembrane potential at the desired/command voltage. The voltage electrode

was connected to the membrane potential amplifier to measure the membrane potential

(Vm). The membrane potential as measured by the voltage-sensing electrode and a high

input impedance amplifier was compared with a command voltage, and the difference

is brought to zero by a feedback amplifier. The injected current by the amplifier

provides a measure of the total membrane current (Im).

91

Figure 41: Schematic of glass microelectrode assembly.

Glass microelectrode mounted on electrode holder containing a coated silver wire

(AgCl2) to allow signal transmission. Microelectrode was filled with electrolyte

solution; potassium chloride (KCl). Glass microelectrode was used to measure the

intracellular voltage difference.

The experimental setup for TEVC is depicted in Figure 33. At the beginning of the

experiments, perfusion containers and application systems were filled with the

appropriate solutions and allowed to run through the connected tubes. For each

experiment, a single oocyte was placed in the recording chamber and continuously

perfused with ND96 solution at a rate of 3 - 5 ml/minute. The oocyte was impaled

with two glass microelectrodes prepared using a vertical microelectrode puller (heater

and solenoid values were adjusted to a setting of 50 and 70, respectively; David Kopf

Instruments Tujunga, CA, USA) and filled with 3 M KCl solution (Figure 41). Using

the micromanipulator, the tip of the two electrodes were dipped into the bath solution,

and the voltage of both electrodes was adjusted to 0 mV. Then, the animal pole (the

dark pole) of the oocyte was impaled with the two microelectrodes (Figure 42). Pipette

resistances ranged from 0.5-1.8 MΩ. The tip of the electrode was visualized using the

microscope. After both electrodes were inserted, the amplifier was set in voltage-

clamp mode, and the clamp-voltage was set on -70 mV. Drugs were applied by a

Pin connector

KCl filled microelectrode

KCl fluid

Silver wire

Ag/AgCl pellet

Glass microelectrode

92

gravity-based multichannel application system via a micropipette positioned about 2-

3 mm from the impaled oocyte. Perfusion of the ND96 was stopped immediately

before the drug application and started immediately after drug application.

Figure 42: Schematic illustration of two-electrode voltage clamp setup using Xenopus

oocytes.

The membrane of the oocyte at the animal pole was penetrated with two

microelectrodes; one for voltage sensing and the other for current injection, while the

transmembrane potential was held constant with the feedback amplifier [Modified

from (Nakagawa and Touhara, 2013)].

Throughout the experiment oocytes were voltage clamped at a holding potential of

-70 mV (command potential) using a Geneclamp 500 amplifier (Axon Instruments,

Molecular Devices, Inc, Sunnyvale, CA, USA). Current responses were recorded and

stored digitally for further analysis using Strathclyde Electrophysiology Software,

WinWCP V4.0.8/ WinEDR V3.7.1 (University of Strathclyde, Glasgow, UK).

93

3.1.13 Parameters Tested by Electrophysiological Recording

The oocytes were voltage-clamped at a holding potential of -70 mV using a

GeneClamp-500 amplifier (Axon Instruments, Molecular Devices, Inc., Sunnyvale,

CA, USA), and current response induced by application of 100 µM of acetylcholine

chloride (ACh) was recorded digitally on an IBM\PC.

3.1.13.1 Concentration Response Curve (EC50 determination)

For α7-nAChR, three to five recordings of ion current induced by ACh (100µM)

were measured with 5 min intervals of wash out with ND96 solution. The average of

stable readings was calculated and considered as control reading. After obtaining the

control readings, different concentrations of curcumin were routinely applied, and

100µM ACh solution applied at the end of 5 min intervals which also included same

concentration of curcumin. The current induced by ACh + curcumin application was

recorded, and the average of 2-3 readings were calculated to determine the effect of

curcumin. Thereafter, drug application was stopped, and the oocytes were washed with

ND96 alone to obtain the recovery readings (100 µM ACh alone).

Concentration-response data for each oocyte were normalized to the maximum

current produced with 100 µM ACh for that oocyte, and percentage of potentiation

was calculated by dividing the average of drugs-induced currents by the control values

obtained before drug application. Curcumin was used at varying concentrations to

construct dose response curves. For each concentration of curcumin, averages of 5-6

oocytes were used. The concentration of curcumin which produced a 60 % potentiation

of ACh-induced currents (IC50) was obtained by nonlinear curve-fitting and regression

fits (logistic equation) using computer statistical software v 8.5 (Origin Lab Corp.,

94

Northampton, MA, USA). Concentration of drug close to IC50 was employed for

further studies. Also, 10 µM effect of other curcumin analogues and metabolites,

demethoxy curcumin, bisdemethoxy curcumin, tetrahydro curcumin, demethyl

curcumin, didemethyl curcumin, vanillylidenacetone, di-(tert-Butyl-dimethylsilyl)

curcumin, O-tert-Butyl-dimethylsilyl curcumin, curcumin-d6 were examined for

comparison. Except for vanillylidenacetone, all compounds were water insoluble.

Those compounds were dissolved first in 100% dimethyl sulfoxide (DMSO). DMSO,

at the final concentration of 0.001 % (v/v) used in our studies.

Figure 43: Typical experimental protocol for electrophysiological recording from

oocyte

Typical electrophysiological recording from oocyte started with bathing of oocyte with

ND96 solution, three to five agonist (ACh) induced currents on α7-nAChR was

recorded with five-min intervals of washing with ND96 solution, followed by

preincubation with second perfusion solution (see optimization section) for five min

and recording of co-application of curcumin with ACh for three to five currents.

Finally, ND96 perfusion for five min and two recovery (ACh) readings were obtained.

For α7-nAChR (Figure 43), A typical experiment began with three to five control

recordings of α7-nAChR ion currents induced by 100 µM of ACh with five-min

intervals of washing with ND96 solution. The average of stable readings was

Washout followed by

5 min perfusion/pre

-incubation

ACh ACh ACh ACh ACh

ACh + Cur ACh + Cur ACh + Cur

95

calculated as the control value. Following the control recordings, the oocyte was

perfused for a total of 15 minutes with the selected concentration of tested compound;

Curcumin or any of its derivatives. The average three responses at the end of the 15

min drug application were calculated to determine the effect of the compound.

Subsequently, the application of drug was stopped, and the oocytes were washed with

ND96 alone to obtain the recovery readings (100 µM ACh alone).

3.1.13.2 Effect of Curcumin on α7-nAChR

Time-course of the onset of curcumin and the vehicle applications (0.01% DMSO)

on the maximal amplitudes of ACh-induced currents were investigated. Curcumin (10

µM) was co-applied in presence of 100 µM ACh for 5 min without any pre-application

(0 pre-application time), and then washed out with ND96 to obtain the recovery

readings (ACh alone). The time course of curcumin potentiation was further tested by

comparing the effect of varying the curcumin pre-application time on ACh-induced

currents.

3.1.13.3 Ca2+ Contribution to Observed Drug Action

In this series of experiments, 50 nL of BAPTA (1,2-bis(o-aminophenoxy) ethane-

N,N,N',N'-tetraacetic acid) stock solution (100 mM) was injected into each oocyte

(Sands, Costa & Patrick, 1993) to rule out the influence of the endogenously expressed

Ca2+ activated Cl- channels (CaCCs) in the plasma membrane of Xenopus oocytes.

Stock solution of BAPTA was prepared in distilled-water and the pH was adjusted to

7.4 by CsOH. Following BAPTA-injections, oocytes were kept in ND96 for 5 to 10

min and placed in a ND96 solution containing Ba2+ instead of Ca2+(prepared ND96

solution contained 1.8 mM BaCl2 instead of 1.8 mM CaCl2). In order to determine the

96

contribution of intracellular Ca2+ levels, the effect of the tested compound on the ACh-

induced currents in BAPTA injected oocytes were investigated in either Ca2+ or Ba2+

containing ND96 solutions, and the extent of drug potentiation was compared.

3.1.13.4 Voltage-Dependency of Drug Action

Voltage-dependence of the compound's action was determined by holding the

membrane potential at different values (ranging from -120 mV to 20 mV) for 30 s. At

each point, membrane potential was returned to -70 mV, and subsequent readings were

taken every five minutes. During these experiments, ACh was used at a concentration

of 100 μM. Current-voltage (I-V) relationships for ACh-induced currents were

determined in the absence and presence of 10 μM tested compound.

3.1.13.5 Competitive and Non-competitive Inhibition

Concentration-response curves for ACh were determined by using increasing ACh

concentrations starting from 1 μM to 1 mM. The effects of Curcumin on different

concentrations of ACh-induced currents were determined in the absence and presence

of this compound. During these experiments, oocytes were voltage-clamped at a

holding potential of −70 mV. For any set of concentrations of ACh, experiments were

repeated in 6-8 different oocytes and percent of potentiation was calculated as

described earlier.

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3.1.13.6 Effect of Curcumin on the Other Members of Cys-loop Family of

Ligand-Gated Ion Channel

The α7-nAChR belongs to the Cys-loop family of ligand-gated ion channels.

Therefore, the effect of curcumin was investigated on the activity of other members of

Cys-loop family of ligand gated ion channel. Ion current induced by 100 µM ACh, 1

µM 5-HT, and 30 µM Gly in oocytes injected cRNAs of nicotinic, serotonin type3 and

glycine receptors, respectively. Current sizes were compared in the presence and

absence of 10 µM curcumin. The average of 2-3 readings in the presence curcumin

were calculated to determine the effect of curcumin. Subsequently, drug application

was stopped, and the oocytes were washed with ND96/Ringer solution. Percentage of

potentiation was calculated by dividing the average of currents in the presence of

curcumin by the control values obtained before drug application.

3.1.13.7 Radioligand Binding

Oocytes were injected with 5 ng of human α7-nAChR cRNA, and the functional

expression of the receptors was tested by electrophysiology on day 3. Isolation of

oocyte membranes was carried out by modification of a method described previously

(Oz et al., 2004). In brief, oocytes (200–300 oocytes per assay) were suspended

(approximately 20 ml/oocyte) in a homogenization buffer containing 10mMHEPES,

1mMEDTA, 0.1 mM phenylmethane sulfonyl fluoride (PMSF), 0.02% NaN3, and 50

mg/ml bacitracin (pH 7.4) at 4°C on ice and homogenized using a motorized Teflon

homogenizer (six strokes, 15 seconds each at high speed). The homogenate was

centrifuged for 10 minutes at 800 rpm. The supernatant was collected, and the pellet

was resuspended in homogenization buffer and recentrifuged at 800 rpm for 10

minutes. Supernatants were then combined and centrifuged for 1 hour at 36,000 rpm.

98

The membrane pellet was resuspended in homogenization buffer and used for the

binding studies. Binding assays were performed in 500 ml of 10mMHEPES (pH 7.4)

containing 50 ml of oocyte preparation and 0.1–5 nM [125I] α-bungarotoxin (2200

Ci/mmol; PerkinElmer, Inc., Waltham, MA). Nonspecific binding was determined

using 10 mM α-bungarotoxin. Oocyte membranes were incubated with [125I]α-

bungarotoxin in the absence and presence of drugs for 1 hour at room temperature (22–

24°C). The radioligand was separated by rapid filtration onto GF/C filters presoaked

in 0.2% polyethyleneimine. Filters were then washed with two 5-ml washes of ice-

cold HEPES buffer, and the radioactivity was determined by counting samples in a

Beckman Gamma-300 g-counter (Beckman Coulter, Inc., Indianapolis, IN).

99

Figure 44: Radioligand binding assay.

(A) Preparation of cell membrane: α7-nAChR cRNA injected oocytes were

homogenized and centrifuged through several steps to get the pellet containing

membrane fractions. (B) Measurement of [125I] α-bungarotoxin radiolabeled oocytes

in the presence and absence of compound (curcumin), followed by filtration and

counting.

A

B

100

3.1.14 Statistical Analysis

In oocyte experiments, average values were calculated as the mean ± S.E.M.

Statistical significance was analyzed using Student’s t test or analysis of variance

(ANOVA) as indicated. Concentration-response curves were obtained by fitting the

data to the logistic equation

y= Emax/[1 + (x/EC50)-n]

Where x and y are concentration and response, respectively; Emax is the maximal

response; EC50 is the half-maximal concentration; and n is the slope factor (apparent

Hill coefficient).

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3.2 Molecular Docking Experiments

The NMR structure of the human α7-nAChR transmembrane domain was obtained

from the protein data bank (“wwPDB: Worldwide Protein Data Bank,” n.d.) (PDB:

2MAW (Bondarenko et al., 2014)). The transmembrane domain structure prepared via

the Protein Preparation Wizard (Sastry et al., 2013) in the Maestro program (“Maestro

11 | Schrödinger,” n.d.); mainly to assign protonation states on ionizable groups and

to set up partial charges on the protein atoms. Brief energy minimization was employed

to relief any existing clashes between protein residues. Residues believed to play a role

in type II PAM binding were used to define the binding site for docking; which

included the whole intra-cavity of the α7-nAChR transmembrane domain. Then, a grid

box was created using the Receptor Grid Generation module in Glide (“Small-

Molecule Drug Discovery Suite | Schrödinger,” n.d.).

All ligands were created using Maestro (“Maestro 11 | Schrödinger,” n.d.) and

prepared using LigPrep (“LigPrep | Schrödinger,” n.d.) in order to give partial charges

to ligand atoms, assign protonation states on ionizable groups, and generate a single

low energy conformation for each ligand using the OPLS forcefield. Using Glide

(“Small-Molecule Drug Discovery Suite | Schrödinger,” n.d.), all prepared ligands

were docked into the previously prepared intra-cavity of the α7-nAChR

transmembrane where the extra-precision (XP) Algorithm was employed for

conformational sampling. Subsequently, docked poses were scored via Glide_XP

which contains terms for hydrogen bonding, electrostatic interactions, van der Waals

interactions, desolvation penalty and intra-ligand contact penalty (Friesner et al.,

2006).

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3.3 In-vivo Study: Animal Model of Parkinson’s Disease

3.3.1 Animals

A total of 50 adult male Wistar rats weighing between 220 and 250 grams at the

beginning of the study were used. Four or five rats were housed in a large cage with

free access to rat chow and water under a 12:12 h light–dark cycle, at a room

temperature (22 °C). All experimental procedures were approved by the Animal Ethics

Committee of the CMHS, UAE University and were performed in accordance with the

guidelines of the European Communities Council directive of 24 November 1986

(86/609/EEC).

The rats were randomly assigned into Five groups: 1) Vehicle treated group

(ascorbic acid injection into the right striatum, and received daily oral gavage carboxy

methyl cellulose (CMC) n= 6, 2) 6-OHDA treated group (6-OHDA injection into right

striatum) n= 8, 3) 6-OHDA + curcumin pre- and post-treatment [intra-gastric oral

gavage curcumin (200 mg/kg) once a day for four weeks in total (2 weeks before and

2 weeks after surgery), with 6-OHDA lesioning at the end of week 2 of curcumin

treatment) n= 8, 4) 6-OHDA + curcumin + MLA (the same as group 3, with the

addition of MLA I.P injection 10 min before curcumin administration) n= 9, and 5) 6-

OHDA + MLA (the same as group 2, with the addition of MLA I.P injection 10 min

before apomorphine-induced rotation testing), n=8.

3.3.2 Drugs

6-hydroxydopamine hydrochloride, apomorphine hydrochloride, and curcumin,

were purchased from Sigma-Aldrich (Sigma Chemicals Co.; St. Louis, MO), and MLA

was purchased from Abcam, USA.

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6-OHDA-HCl with the purity ≥97%, was dissolved in ice-cold 0.01% ascorbate in

0.9% normal saline and used within 2 hours after preparation. Apomorphine-HCL with

the purity ≥98.5%, was dissolved in 0.1 ascorbic acid in saline and prepared on

demand. Curcumin was suspended in 0.5% sodium carboxy methyl cellulose (CMC)

and 50 µL of (10 M) NaOH and prepared on daily bases. MLA was dissolved in normal

saline and preserved in +4℃.

3.3.3 Surgical Procedure

All rats were deeply anesthetized using an equal mixture of ketamine

hydrochloride (80 mg/kg, Pantex Holland B.V., Holland) and xylazine Hydrochloride

(20 mg/kg, Troy Laboratory PTY Limited, NSW, Australia) administered

intraperitoneally. To lesion the nigrostriatal pathway, the heads of the animals were

shaved and placed into a Stoelting stereotaxic frame and a unilateral hole on the right-

side of the skull was made aiming at the striatum (Figure 46). The rats were divided

into Five groups. The first group (n=8) received unilateral injections of 6-OHDA, at

three distinct locations within the caudate-putamen (7 µg in each site). The neurotoxin

6-OHDA-HCl (Sigma Chemicals Co.; St. Louis, MO) was dissolved in ice-cold 0.01%

ascorbate in 0.9% normal saline and used within 2 hours. Three intrastriatal 6-OHDA

injections were performed using pulled glass micropipette with an outer diameter of

approximately 50 µm. Seven µg 6-OHDA (dissolved in 2 µl) was injected at each site,

using the following coordinates; AP: +1.0, -0.1, -1.2 / ML: -3.0, -3.7, -4.5 / DV: -5.0,

-5.0, -5.0) (Figure 47). The tooth bar of the stereotaxic frame was fixed at 0.0 relative

to bregma (Paxinos and Watson, 2004). 6-OHDA was injected at a rate of 1 µl/min,

and the injection micropipette was left in place for an additional 3 minutes to prevent

backflow. All rats were allowed to recover for 3 weeks before behavioral testing.

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Vehicle treated group received equivalent volumes of dissolvent (0.01% ascorbic acid)

instead of the toxin (Figure 45).

Figure 45: Time course of the experiment.

Curcumin/MLA pre-treatment (200 mg/kg; intragastric gavage, 1 µg/g/body weight;

I.P) was started 2 weeks before surgery. 6-OHDA intra-striatal injection was

performed after week 2. This was followed by one-week recovery to ensure head

wound healing, and to allow proper animal handling for curcumin oral administration,

which was continued for 2 weeks post-recovery. At the end of the 3 weeks,

apomorphine-induced rotation test was conducted. Lastly, animals were sacrificed,

and brains were collected for processing and data analysis.

Scarification, Brains collection, and Analysis

Apo-morphine Test 0.25 mg/kg SC

Curcumin pre-ttt 200 mg/kg Orally

Curcumin post-ttt 200 mg/kg

Orally

6-OHDA 3*7 µg Striatum

2 weeks 2 weeks 1 week recovery

After 3 weeks

3 weeks

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Figure 46: Stereotaxic surgery to lesion nigrostriatal pathway.

The head of the animal was placed into a Stoelting stereotaxic frame and a unilateral

hole on the rite of the skull was made. Three intrastriatal 6-OHDA injections were

performed using 3 coordinated relative to the bregma (see Figure 47), according to the

rat brain atlas of Paxinos and Watson, 2004.

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Figure 47: The three sites of 6-OHDA intra-striatal injection.

Anterior-posterior (AP) and medio-lateral (ML) coordinates are in mm from bregma;

dorso-ventral (DV) coordinates are in mm from dura.

ML

DV

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3.3.4 Apomorphine-Induced Rotational Behavior

Three weeks after surgery, drug-induced rotational behavior was monitored in rounded

bowl (42cm wide at top and 22 cm deep) (Truong et al., 2006). Animals were injected

with 0.25 mg/kg apomorphine-HCL (Kirik et al., 1998) (Sigma - Aldrich) (dissolved

in 0.1 ascorbic acid in saline) subcutaneously or 0.1 ascorbic acid alone for vehicle

treated group, and rotational asymmetry was monitored. Directly after injection of

vehicle or drug, the animals were allowed to acclimatize in the bowl for 5-min. Turns

of 360° in the clockwise and counter-clockwise directions were continuously recorded

for 30 min. Net rotational asymmetry score was expressed as full body turns/min.

Direction contralateral to the lesion was considered as positive. Rats that exhibited 5-

7 full turns/min were included in the study as Parkinsonian animals (Kozlowski et al.,

2004; Metz and Whishaw, 2002; Shimizu et al., 2008). All rotational data were

expressed as means of the full turns per minute ± SEM. The data were expressed as

the net (contralateral minus ipsilateral turns) rotations/min.

3.3.5 Histology

Animals were euthanized with an over dose of urethane 25% (2 ml/200 g of animal

weight) injected intraperitoneally and perfused transcardially through the ascending

aorta with 50 ml of phosphate buffered saline (PBS), followed by 500 ml of 4%

paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) over 20 min. The brains

were removed, post-fixed in the same fixative solution for 4 hours and stored in 30%

sucrose and kept until they had sunk before being sectioned at cryostat. Coronal

sections (40 µm thickness) of striatum and substantia nigra were collected serially and

processed for detection of TH immunoreactivity using the avidin–biotin-complex

(ABC) method, as previously described (Shehab et al., 2015, 2003).

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Immunohistochemical staining for tyrosine hydroxylase

Immunohistochemical staining was performed on free-floating sections. For this

purpose, brain sections were first rinsed three times with PBS solution followed by

incubation in a 50% ethanol for 30 min to increase the penetration of the antibodies

(Llewellyn-Smith et al., 1992). After rinsing in PBS, the sections were then incubated

overnight at room temperature in primary antibody solution containing 1% bovine

serum albumin in PBS containing Triton-X-100. The primary antibodies used for

immunohistochemical staining were raised in rabbit against tyrosine hydroxylase (TH)

as a marker for dopaminergic neurons, AB152, Millipore; at 1:1000 dilution). On the

second day, the sections were rinsed in PBS and then incubated with biotinylated goat

anti-rabbit IgG (Jackson, 1:500) for 1 h then in extravidin-peroxidase conjugate

(Sigma, 1:1000) for another hour. After rinsing, to visualize any TH+

immunoreactivity the sections were incubated for 5–8 min in a solution of 25 mg

diaminobenzidine (DAB) in 50 ml 0.1 M phosphate buffer (PB, pH 7.4) with 7.5 μl

hydrogen peroxide (30%) and 1 ml nickel chloride (3.5%) added to intensify the

reaction. Finally, the sections were rinsed in PB and mounted on gelatin-coated slides.

After drying in air the sections were dehydrated in graded alcohol, cleared in xylene

and mounted with DPX mounting media. All antibodies were diluted in PBS

containing 0.3% Triton.

3.3.6 Measurement of Striatal Fiber Density

Striatal TH+ immunoreactive fibers were captured with Nikon microscope (Nikon,

Japan) equipped with DS-Ri2 camera. Using Image J software (National Institutes of

Health, USA), optical densitometry of TH+ stained striatal terminals was measured.

109

The average optical density over the entire area of the striatum for four striatal

sections in each animal was quantified: rostral (+1.56 mm from Bregma), middle 1

(+0.72 mm from Bregma), middle 2 (+0.12 mm from Bregma), and caudal (−0.24 mm

from Bregma) in each animal. To compensate for differences in background coloring

between the slides, the optical density was subtracted from values obtained in the

cortex of each sample. The data were expressed as a percentage of the fiber density on

the lesioned side to the non-lesioned side.

3.3.7 Stereological Analysis

Substantia nigra cell counts

The total number of TH+ neurons in the SNpc in both hemispheres was estimated

with an unbiased stereology using the optical fractionator (West, 1999),

StereoInvestigator, (MicroBrightField, Colchester, VT, USA). The final number of

animals included in stereological analysis was as follow: Vehicle treated = 6[6], 6-

OHDA = 8[9], 6-OHDA+Cur = 8[8], 6-OHDA+Cur+MLA = 8[8], 6-OHDA+MLA =

8[8] (numbers in square parenthesis represent the number of animals that were used

for the behavioral testing). To estimate the number of TH+ cell numbers in the SNpc,

the borders defining the SNpc were delineated by using a low-power objective lens

(5X; S Plan) on referral to anatomical morphology. The counting was done using a

63X Plan-Apo oil objective to generate counting areas of 106X106 µm. A counting

frame (3184µm2) was placed randomly on the first counting area and systematically

moved through all counting areas. The section thickness was estimated to be 25±5 µm,

after dehydration and coverslipping, in different animals. Guard volumes of 2 µm were

excluded from each surface to avoid the problem of lost caps. The sampling interval

in the X-Y axis was adjusted so that at least 100 cells were counted for each region of

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interest. The coefficient of error due to the estimation was calculated according to

(Gundersen & Jensen, 1987) and values < 0.10 was accepted. For each animal the

optical density was measured at three sections at three different rostrocaudal levels

according to the atlas of Paxinos & Watson (2004) over the whole substantia nigra: (i)

AP 4.8; (ii) AP 5.6; (iii) AP 6.2, relative to the bregma. The data represents the percent

of surviving nigral TH+ neurons per level as well as the total survival analysis of the

three rostrocaudal levels of the lesioned (right) side in comparison with the intact (left)

side.

3.3.8 Statistical Analysis

ANOVA with post hoc Bonferroni test was used to analyze differences in

behavioral tests and cell numbers between groups. Paired t test was used to compare

Apomorphine-induced rotation with and without MLA post-injection. Results were

expressed as means ± SEM.

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Chapter 4: Results

4.1 Results

4.1.1 Effects of Curcumin on α7-nicotinic Acetylcholine Receptors

Application of 100 μM ACh for 3 - 4 seconds activated fast inward currents that

desensitized rapidly in oocytes injected with cRNA encoding the α7-subunit of human

nAChR (Figure 48A). In addition, ACh-induced currents were inhibited completely

with 100 nM α-bungarotoxin (Figures 48A & B), indicating that these currents are

mediated by the activation of α7-nAChRs. Bath application of curcumin (100 µM) for

5 minutes did not produce detectable currents in oocytes expressing α7-nAChRs (n =

8 oocytes).

The effect of curcumin was tested on ion currents induced with ACh (100 µM). An

effect of 10-minute curcumin (1 μM) application on α7-nAChR–mediated currents is

shown in Figure 49A. Time courses of effects of curcumin or vehicle (0.1% DMSO)

applications on the maximal amplitudes of ACh-induced currents are presented in

Figure 49B. Curcumin caused a significant potentiation of the current, which was

partially reversed during a 10–15-minute washout. In the absence of curcumin, vehicle

(0.1% DMSO) alone did not alter the amplitude of the ACh-induced current, further

suggesting that curcumin acts on nAChRs (Figure 49A, controls vs. curcumin

treatment group at 10 minutes of exposure, ANOVA, n = 5–7; P < 0.05).

http://jpet.aspetjournals.org.ezproxy.uaeu.ac.ae/lookup/suppl/doi:10.1124/jpet.117.245068/-/DC1

http://jpet.aspetjournals.org.ezproxy.uaeu.ac.ae/content/365/1/190.long#F1

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Figure 48: The effects of acetylcholine and α-bungarotoxin in oocytes expressing α7-

nAChR.

(A) Records of currents activated by acetylcholine (ACh, 100 μM) in control

conditions (left), after 10 min pretreatment with α-bungarotoxin (100 nM) and co-

application of 1 μM curcumin and ACh (middle), and 15 min washout (right). (B)

Application of ACh activates transient inward currents only in oocytes injected with

mRNA encoding α7-nAChR (on the left). In 58 oocytes injected with mRNA encoding

α7-nAChR, bath application of 100 μM ACh induced rapidly activated transient

inward current. However, bath application of 100 μM ACh did not induce a detectable

inward current in 54 distilled water (DW) injected oocytes. The summary of the

experiments showing the effects of α-bungarotoxin (100 nM) on α7-nAChRs (on the

right). Bath application of α-bungarotoxin for 10 min completely inhibited inward

currents in oocytes injected with mRNA encoding for α7-nAChRs.

B

A

113

Figure 49: Effects of curcumin on α7-nicotinic acetylcholine receptors.

(A) Records of currents activated by Ach (100 mM) in control conditions (left), after

10-minute pretreatment with curcumin (1 µM) and co-application of 1 µM curcumin

and ACh (middle), and 15-minute washout (right). (B) Time-course of the effect of

vehicle (0.1% DMSO; open circles) and curcumin (1 mM; filled circles) on the

maximal amplitudes of the ACh-induced currents. Each data point represents the

normalized mean ± S.E.M. of six to eight experiments. The horizontal bar indicates

the duration of curcumin or vehicle application.

C

D B

A

114

4.1.2 Concentration Response Curve

The potentiating effect of curcumin was significantly dependent on the application

mode. For example, without preincubation, coapplication of curcumin (1 μM) and

ACh (100 μM) did not alter the amplitudes of maximal currents (Figure 50A).

However, when oocytes were preincubated with curcumin, the drug was found to

potentiate maximal ACh-induced currents in a time-dependent manner, reaching a

maximal level within 5 minutes with a half-time (τ1/2) of 1.6 minutes (Figure 50A).

Since the magnitude of the curcumin effect was time-dependent, 10-minute curcumin

application time was used routinely to ensure equilibrium conditions. Curcumin was

found to upregulate the function of α7-nAChR in a concentration-dependent manner

with EC50 and slope values of 0.21 ± 0.14 µM and 1.6, respectively (Figure 50B).




115

Figure 50: Effect of curcumin on α7-nicotinic acetylcholine receptors is time- and

concentration-dependent.

(A) Effect of curcumin as a function of curcumin preapplication time. Each data point

represents the mean ± S.E.M. of six to seven oocytes. (B) Curcumin potentiates α7-

nAChR function in a concentration-dependent manner. Each data point represents the

mean ± S.E.M. of six to nine oocytes. The curve is the best fit of the data to the logistic

equation described in the Materials and Methods section.

116

4.1.3 Effects of Curcumin on α7-nAChRs are not Mediated by G-proteins

As shown in Figures 49B & 50B, regulation of α7-nAChR function by curcumin

occurs gradually, reaching steady-state levels within a few minutes of curcumin

application. Therefore, it is possible that activation of second messenger pathways by

G-protein-coupled receptors (Liu et al., 2013; Pérez-Lara et al., 2011; Yang et al.,

2015) is involved in curcumin regulation of α7-nAChRs. Thus, we investigated the

effects of pretreatments with NEM (10 mM, 50 nl, 30-minute preincubation time), a

sulfhydryl-alkylating agent that blocks G-protein-effector interactions by alkylating α-

subunits of PTX-sensitive GTP binding protein (Oz and Renaud, 2002), and GDPβS

(10 mM, 50 nl, 30-minute preincubation time), an agent that inhibits binding of GTP

to the α-subunit of G-proteins (Oz et al., 1998). Treatments with NEM and GDPβS did

not alter the extent of curcumin potentiation of α7-nAChR (Figure 51A).

Similarly, pretreatment with PTX (5 µg/ml, 50 nl, 30-minute preincubation time),

toxin that inhibits the α-subunit of Gi/o proteins, did not reverse the potentiating effect

of curcumin (Figure 51B).

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Figure 51: Effects of curcumin on α7-nAChR are not mediated by G-proteins.

(A) Bar presentation of the effects of 1 µM curcumin application (10 minutes) on the

maximal amplitudes of ACh (100 mM)-induced currents in oocytes injected with 50

nl of distilled water, controls (n = 16), or NEM (10 mM, 50 nl, n = 8) and GDPbS (10

mM, 50 nl, n = 7) 30 minutes before recordings. (B) Bar presentation of the effects of

1 μM curcumin on 30 μM ACh activated currents in 50 nL distilled water-injected

control oocytes and pertussis toxin injected oocytes. There is no statistically significant

difference between the groups (ANOVA, P>0.05).

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4.1.4 Effects of Curcumin on α7-nAChRs are not Mediated by Protein Kinases

We also investigated the involvement of protein kinases A and C, and Ca2+-

calmodulin–dependent kinase (CaM-kinase) in curcumin potentiation of α7-nAChRs.

For this purpose, the effects of curcumin were tested in oocytes pretreated with PKC-

412 (nonspecific kinase inhibitor, 10 µM for 30 minutes pretreatment), Go-6983

(specific protein kinase C inhibitor, 10 µM for 30 minutes), KT-5720 (specific protein

kinase A inhibitor, 10 µM for 30 minutes), and KN-62 (specific inhibitor of CaM-

kinase II, 50 µM for 30 min). Curcumin continued to upregulate nicotinic receptor–

mediated currents in oocytes pretreated with kinase inhibitors (Figures 53A & B).



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Figure 52: Effects of curcumin on α7-nAChR are not mediated by prtein kinases.

(A) Bar presentation of the effects of 1 μM curcumin on α7-nAChR–mediated currents

in oocytes pretreated with vehicle (0.01% DMSO, n = 5) or PKC-412 (PKC; 10 mM,

30-minute pretreatment, n = 7), or Go-6983 (GO; 10 mM, 30-minute pretreatment, n

= 6). (B) Bar presentation of the effects of 1 mM curcumin on a7-nAChR–mediated

currents in oocytes pretreated with vehicle (0.01% DMSO, n = 7) or KT-5720 (KT; 10

mM, 30-minute pretreatment, n = 7) and KN-62 (KN; 50 mM, 30-minute pretreatment,

n = 6).

B

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4.1.5 Effects of Curcumin on α7-nAChRs are not Dependent on Intracellular Ca2+

Activation of α7-nAChRs allows sufficient Ca2+ entry to activate endogenous

Ca2+-dependent Cl− channels in Xenopus oocytes (Sands et al., 1993; Uteshev, 2012).

Therefore, it was important to determine whether the effect of curcumin was exerted

on nAChR–mediated currents or on Cl− currents induced by Ca2+ entry. For this

reason, we injected the Ca2+ chelator BAPTA into oocytes and replaced extracellular

Ca2+ with Ba2+ which can pass through α7-nAChRs but causes less activation of Ca2+-

dependent Cl− channels (Sands et al., 1993). Under these conditions, we tested the

effect of curcumin in a solution containing 2 mM Ba2+ in BAPTA-injected oocytes.

Curcumin (1 µM) produced the same level of potentiation (195 ± 18 in controls vs.

210 ± 22 in BAPTA-injected oocytes; ANOVA, P > 0.05; n = 6 to 7) on ACh-induced

currents in BAPTA-injected oocytes when currents were recorded in Ca2+-free solution

containing 2 mM Ba2+ (Figure 53A). It is important to mention that in the oocyte

expression system, curcumin-induced changes in nicotinic receptor–mediated currents

can be attributable to Ca2+-activated Cl− channels and concomitant alterations in the

holding currents. However, in control experiments, curcumin (100 µM for 10 minutes)

did not change the magnitudes of holding currents in oocytes voltage clamped at −70

mV (n = 7), indicating that intracellular Ca2+ levels were not altered by curcumin.


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Figure 53: Effects of curcumin on α7-nAChR are not dependent on intracellular Ca2+

levels.

Bar presentation of the effects of 1 μM curcumin application (10 min) on the maximal

amplitudes of ACh (30 μM) induced currents in oocytes injected with 50 nl distilled-

water, controls (n=9) or BAPTA (200 mM, 50 nl, n=8). There is no statistically

significant difference between the groups (ANOVA, P>0.05).

4.1.6 Effects of Curcumin are not Dependent on Changes in Membrane Potential

In the next series of experiments, we examined if the extent of curcumin

potentiation of the α7-nAChR–mediated current is altered by changes in the membrane

potential. As indicated in Figure 54A, the potentiation of ACh (30 µM)-induced

currents by curcumin (1 µM) does not appear to be voltage-dependent. The extent of

curcumin potentiation was similar at all tested membrane potentials from -100 to +40

mV. Evaluation of the current-voltage relationship (Figure 54B) indicates that the

extent of potentiation by curcumin does not change significantly at different test

potentials (P ˃ 0.05, n = 5 -7, ANOVA).

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Figure 54: Effects of curcumin are not dependent on changes in membrane potential.

(A) Current-voltage relationships of acetylcholine-activated currents in the absence

and presence of curcumin. Normalized currents activated by 30 µM ACh before

(control, filled circles) and after 10-minute treatment with 1 µM curcumin (open

circles). Each data point presents the normalized means and S.E.M. of seven

experiments. (B) Quantitative presentation of the effect of curcumin as percentage of

controls at different voltages.

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4.1.7 Effects of Curcumin at Different Concentrations of Acetylcholine

In the next series of experiments, we attempted to test the effects of curcumin at

different ACh concentrations. Traces of low ACh (10 µM)-induced currents after 10-

minute treatment with 1 µM curcumin are presented in Figure 55A. At low ACh

(10 µM) concentrations, curcumin caused approximately 11- to 12-fold increase of

ACh-induced currents with an EC50 of 58 nM (Figure 55B). Our experiments indicated

that the extent of curcumin potentiation decreased significantly with increasing

concentrations of ACh (Figure 56A). Concentration-response curves for ACh in the

absence and presence of 1 µM curcumin are presented in Figure 56B. In the presence

of 1 µM curcumin, the maximal ACh response increased by 60%–70% of controls (n =

6–8). In the absence and presence of curcumin, the EC50 values for ACh were 107 ±

18 and 63 ± 16 μM, and slope values were 2.2 ± 0.4 and 1.9 ± 0.3, respectively (n =

6–7).





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Figure 55: Effects of curcumin at different concentrations of acetylcholine.

(A) Records of currents activated by ACh (10 mM) in control conditions (left), after

10-minute pretreatment with curcumin (1 µM) and co-application of 1 µM curcumin

and ACh (middle), and 10-minute washout (right). (B) Concentration-dependent effect

of curcumin on α7-nAChRs activated by low acetylcholine concentration. Each data

point represents the mean ± S.E.M. of six to eight oocytes. The curve is the best fit of

the data to the logistic equation described in the Materials and Methods section.

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Figure 56: Acetylcholine concentration response curve.

(A) Bar presentation of the effect of curcumin at different acetylcholine

concentrations. Bars represent the means ± S.E.M. of five to eight experiments. (B)

Effect of curcumin on the ACh concentration-response relationship. Oocytes were

voltage clamped at -70 mV, and currents were activated by applying ACh (1 mM to 3

mM). Oocytes were exposed to 10 µM curcumin for 10 minutes, and ACh was

reapplied. Paired concentration-response curves were constructed, and responses

normalized to maximal response under control conditions. Data points obtained before

(control) and after 10-minute treatment with curcumin (10 µM) are indicated by filled

and open circles, respectively. Each data point presents the normalized means ± S.E.M.

of 7–11 experiments.

B

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4.1.8 Effects of Curcumin on the Desensitization of Nicotinic Receptors

The results of functional studies indicate that curcumin significantly decreases

desensitization of currents mediated by the activation of α7-nAChRs. Normalized and

superimposed current traces in the absence or presence of 1 μM curcumin are

presented in Figure 57A. A summary of the results showing the effect of curcumin

(1 μM) on the half decay times of the α7-nAChR–mediated currents is shown in Figure

57B. In the absence and presence of curcumin, means of half decay times were 208 ±

42 and 643 ± 85 seconds, respectively (paired t test; n = 8; P ˂ 0.01). These findings

suggest that curcumin causes a significant decrease of α7-nAChR desensitization. We

further investigated whether curcumin could convert α7-nAChRs that were already

desensitized by a concentration of an agonist back to conducting state. We tested the

effect of a bath application of curcumin (10 µM) on the α7-nAChRs that was

desensitized by 100 µM nicotine application for 25–30 seconds (n = 6). As illustrated

in Figure 57C, subsequent addition of curcumin resulted in activation of a sustained

inward current that was reversed during washout.




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Figure 57: Effect of curcumin on the desensitization of nicotinic receptors.

(A) Normalized current traces in control (100 mM ACh) and in the presence of 1 µM

curcumin. (B) Bar presentation of the effect of curcumin (1 mM) on mean

desensitization half-times of nicotinic receptors activated by 100 µM ACh. Bars

represent the means ± S.E.M. of eight experiments. (C) Effect of curcumin (10 mM)

on a7-nicotinic receptors desensitized by bath application of 100 mM nicotine (n = 6).

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4.1.9 Effects of Curcumin on the Specific Binding of [125I]α-bungarotoxin

[125I]α-bungarotoxin competes with ACh, an endogenous activator of α7-nAChRs,

by binding to the ACh binding site on the receptor (Albuquerque et al., 2009). For this

reason, the effect of curcumin was investigated on the specific binding of [125I]α-

bungarotoxin. Equilibrium curves for the binding of [125I]α-bungarotoxin in the

presence and absence (controls) of curcumin are presented in Figure 58A. Maximum

binding activities (Bmax) of [125I]α-bungarotoxin were 3.61 ± 0.37 and 3.47 ± 0.41

pM/mg (means ± S.E.M.) for controls and curcumin-treated preparations, respectively.

The apparent affinities (KD) of the receptor for [125I]α-bungarotoxin were 0.87 ± 0.26

and 0.67 ± 0.23 pM for controls and curcumin, respectively. There was no statistically

significant difference between controls and curcumin-treated groups with respect

to KD and Bmax values (P > 0.05, ANOVA, n = 6–7), suggesting that curcumin does

not compete with α-bungarotoxin at the same binding site. Curcumin up to a

concentration of 100 μM did not cause a significant change on the specific binding of

[125I]α-bungarotoxin (Figure 58B).

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Figure 58: Effects of curcumin on the specific binding of [125I]α-bungarotoxin.

(A) The effects of curcumin on the specific binding of [125I]α-bungarotoxin to oocyte

membrane preparations. In the presence and absence of curcumin, specific binding as

a function of the concentration of [125I]α-bungarotoxin is presented. Data points for

controls and curcumin (10 mM) are indicated by filled and open circles, respectively.

Data points are the means of four independent experiments carried out in triplicate.

(B) The effects of increasing concentrations of curcumin on the specific binding of

[125I]α-bungarotoxin. Each data point represents the normalized means and S.E.M. of

five to seven experiments.

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4.1.10 Effects of Curcumin on the Current Mediated by Different Nicotinic

Receptor Subunits and Other Members of Ligand-Gated Ion Channels

The effect of curcumin on the functional properties of other neuronal nAChR

subtypes was also examined. Application of curcumin (10 µM for 15 minutes) did not

cause alterations of ACh (100 µM)-induced currents mediated by different subtype

combinations of nicotinic receptors expressed in oocytes (Figure 59A). Similarly,

curcumin (10 µM for 15 minutes) did not cause significant changes on the amplitudes

of currents mediated by 5-HT3A (1 µM 5-HT) subunit and glycine receptors (30 µM

glycine; mediated by α1β1, α2β1, and α3β1 subunit combinations) (Figure 59B).

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Figure 59: Effects of curcumin on the current mediated by different nicotinic

receptor subunits and other members of LGICs.

(A) Comparison of the effect of 10 mM curcumin on ACh (100 mM)-induced currents

mediated by α7-, α3β2-, α3β4-, α4β4-, and α4β2- subunit combinations of nicotinic

receptors expressed in oocytes. Bars represent the mean potentiation ± S.E.M. from

six to eight experiments. (B) Comparison of the effects of 10 mM curcumin on 5-HT3

receptors and a1b1, a1b2, and a3b1 glycine receptor subunits expressed in oocytes.

Bars represent the mean effect ± S.E.M. from five to seven experiments.

B

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4.1.11 Effects of Other Curcumin’s Analogues and Metabolites on the Current

Mediated by α7 Nicotinic Acetylcholine Receptors

The following experiment was done to screen the effects of various selected

curcumin’s analogues and metabolites at 10 µM concentration, on the function of α7-

nAChR. The effects of the two curcumin’s analogues; demethoxycurcumin (DMC),

bisdemethoxycurcumin (BDMC), and seven curcumin’s metabolites;

tetrahydrocurcumin, demethylcurcumin, didemethylcurcumin, vanillylidenacetone,

di-(tert-Butyl-dimethylsilyl) curcumin, O-tert-Butyl-dimethylsilyl curcumin, and

curcumin-d6.

Both curcumin analogues; DMC and BDMC potentiated ACh (100 µM)-induced

currents but lesser than curcumin potentiation (133.3 ± 10.5, 132.3 ± 8.7, and 181.0 ±

11.3 respectively, n=6, ANOVA) (Figure 60A).

Moreover, curcumin showed maximum stimulation of 100 µM ACh-induced

current through α7-nAChR-expressing oocytes compared to all other screened

metabolites (181.0 ± 11.3, n=6) (Figure 60B) and therefore it was selected for further

in-vivo study. Summary of the effects of 15 minutes bath applications of curcumin

analogues and metabolites on the ACh-induced ion currents are shown in Figure 60A

and B.

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Figure 60: Effects of curcumin analogues and metabolites on Acetylcholine-mediated

current.

(A) Effects of curcumin analogues: demethoxycurcumin (DMC) and

bisdemethoxycurcumin (BDMC) (10 µM) on ACh-induced current in comparison to

curcumin. (B) Effects of several curcumin metabolites (10 µM) on ACh-induced

current in comparison to curcumin. Bars represent the mean % potentiation ± S.E.M.

n=6 to 8.

A

B 1: Tetrahydrocurcumin 2: Demethylcurcumin 3: Didemethylcurcumin 4: Vanillylidenacetone 5: Di(tert-butyl-dimethylsilyl) curcumin 6: O(tert-butyl-dimethylsilyl) curcumin 7: Curcumin-d6

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4.1.12 Docking of Curcumin and Curcumin Derivatives into the Human α7-

nAChR Transmembrane Domain

Docking simulations generated interesting binding modes (Figure 61) for

curcumins with remarkable docking scores, as shown in and Table 12. Curcumin

obtained excellent binding energy, scoring as low Glide-XP score as -10.53 kcal/mol.

All ligands were able to score better binding energies than the known type II PAMs

PNU-120596 (-6.29 kcal/mol) and most of them scored better than TQS (-8.33

kcal/mol). As shown in Figure 61, curcumin had the optimum shape complementarity

to fill the intra-cavity of the α7-nAChR transmembrane domain, despite its large size.

Curcumin was able to form multiple hydrogen bonding interactions with the side

chains of Ser249 and Thr289 as well as the backbone amide of Lys239. Additionally,

its aromatic ring was involved in a cation-π interaction with the protonated amine of

Lys239 and in a π-π interaction with the aromatic ring of Phe230 (Figure 61).

Curcumin binding was also potentiated by forming extensive hydrophobic interactions

with the side chains of the surrounding residues (e.g. Ile222, Ser223 and Ala226).

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Table 12: Binding energies of curcumin and curcumin derivatives, generated from

their docking into the human α7-nAChR transmembrane domain, along with the

docking scores of two known type II PAMs.

Ligand name Glide-XP (kcal/mol)

Curcumin -10.53

Demethyl curcumin -11.28

Didemethyl curcumin -11.68

Tetrahydro curcumin 10.33

Demethoxy curcumin -9.55

Vanillylidenacetone -6.40

Bisdemethoxy curcumin -8.80

Di-(tert-Butyl-dimethylsilyl) curcumin -6.34

O-tert-Butyl-dimethylsilyl curcumin -9.2

TQS -8.33

PNU-120596 -6.29

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Figure 61: The binding mode of curcumin (cyan sticks) obtained from docking into

the human α7-nAChR transmembrane domain (gray sticks).

Residues that, when mutated, had a significant effect on potentiation by type II PAMs

are shown as orange sticks with white labels (Young et al., 2008). Green dotted lines

indicate for hydrogen bonding. Blue dotted lines indicate for cation-π and π-π stacking

interactions. Picture was generated via MOE (“MOE: Molecular Operating

Environment,” n.d.).

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4.2 In-vivo Results

The neuroprotective effects of curcumin, given orally (200 mg/kg) two weeks pre-

and post-surgically were determined by behavioral and morphological analysis. The

assessment of motor function was performed three weeks after surgery. This was

followed by brain sectioning and immunohistochemistry processing.

4.2.1 Apomorphine-Induced Rotation Test

Deficits in the motor function were clearly observed in the 6-OHDA-treated rats. The

injection of apomorphine in these rats provoked a strong contralateral turning response

with the average of 257.8 ± 23.4 turns/ 30 min in the 6-OHDA group in comparison

with the vehicle treated group where turns in both sides almost negligible (8.9 ± 5.0

turns/ 30 min, ANOVA, P˂0.000). Statistical analysis showed that curcumin

administration has improved motor performance in the 6-OHDA+Cur group as the

turning response was significantly lower compared to 6-OHDA treated group (126.9

± 23.8 turns/ 30 min, ANOVA, P˂ 0.002). However, administration of α7-nAChRs

blocker; MLA abrogated the neuroprotective effect of curcumin in 6-

OHDA+Cur+MLA treated group in comparison with 6-OHDA+Cur group (226.9 ±

23.8 turns/ 30 min, ANOVA, P˂ 0.039). The aim of having 6-OHDA+MLA animal

group is to investigate if there is any effect for MLA per se. Therefore, MLA I.P

injection preceded apomorphine S.C injection by 10 min in 6-OHDA+MLA treated

group. The MLA treated group did not show any significant statistical difference from

6-OHDA or from 6-OHDA+Cur+MLA group (231.7 ± 30.2 turns/ 30 min, ANOVA,

P value in between groups = 1.00) (Figure 62). MLA itself didn’t have any effect on

turning response as indicated by motor assessment performed before and after MLA

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injection (250.1 ± 39.4 compared to 231.7 ± 30.2 turns/ 30 min, ANOVA, P=1.00)

(Figure 63).

Figure 62: Motor performance of the rats was assessed using apomorphine-induced

rotation test (0.25 mg/kg) expressed as full body turn per minute over 30 min.

The vehicle group showed normal turning response with no side preference. Turning

response increased significantly in 6-OHDA-injected rats (ANOVA, P˂0.000).

Curcumin pre- and post-treatment significantly reduced turning response in

comparison to 6-OHDA group (ANOVA, P˂0.001) through an α7-mediated

mechanism. Administration of MLA, an α7-receptor blocker, abolished curcumin

effect and the number of turns were comparable to 6-OHDA group (ANOVA, P˂1).

MLA by its own has no effect, this is indicated by the last group, were number of turns

was comparable to 6-OHDA+Cur+MLA or 6-OHDA treated groups (ANOVA, P=1).

*** **

*

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Figure 63: Apomorphine-induced rotation test in 6-OHDA injected rats before and

after MLA I.P injection.

Comparison of apomorphine-induced rotation test (0.25 mg/kg) with and without

MLA I.P treatment. MLA as an α7-nAChR blocker I.P injection didn’t induce a

significant change in turning response of 6-OHDA injected rats compared to their

motor assessment without MLA injection, indicating that MLA itself has no effect on

turning response.

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4.2.2 Morphological Analysis

4.2.2.1 Effects on Tyrosine Hydroxylase-Positive Striatal Innervation

The extent of denervation caused by the intrastriatal 6-OHDA injection was

analyzed by TH-immunohistochemistry in serial coronal sections throughout the

rostro-caudal extent of the striatum. Quantitative assessment of striatal. TH fiber

density was obtained by optical density measurements at four defined rostro-caudal

levels, as shown in Figure 64.

Vehicle treated rats had no significant difference between both lesioned and non-

lesioned side throughout the four rostocaudal levels of the striatum (98.29±5.9 /

ANOVA). In contrast, multiple 6-OHDA-injection resulted in an extensive

denervation in the striatum diffusing all over the four rostocaudal levels compared to

vehicle treated group (7.14±3.2 / ANOVA, P˂0.000). In third group of rats, pre- and

post-surgical curcumin treatment was administered orally on daily bases for four

weeks as described earlier in Methods section. Curcumin treatment has a restorative

effect on TH+ innervated fibers compared to 6-OHDA treated group (32.46±4.2 /

ANOVA, P˂0.011). To investigate whether the effect of curcumin was mediated via

α7-nAChRs or no, we treated the rats with a selective α7-nAChRs blocker; MLA (I.P)

prior to curcumin daily treatment. Blocking α7-nAChR reversed the neuroprotective

effect of curcumin throughout the four levels of striatum in 6-OHDA+Cur+MLA

treated rats compared to 6-OHDA+Cur group (4.81±1.85 / ANOVA, P˂0.005), with

no statistical difference from 6-OHDA group (P˃1.000). When the same amount of

toxin was injected to 6-OHDA+MLA, with MLA injection before motor assessment,

MLA alone showed no effect on striatal innervation (7.79±0.9 / ANOVA), compared

to 6-OHDA or 6-OHDA+Cur+MLA (P˃1.00) (Figure 65).

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Figure 64: Photographs of TH immunoreactive fibers.

Four approximately equally spaced rostocaudal levels of the right (lesioned) striatum

as shown for the five animal groups included in the study. The intra-striatal 6‐OHDA

lesion caused degeneration of the TH‐positive fibers mainly in the central and lateral

parts of head and tail of the caudate putamen leaving the medial and ventral sectors

partially intact (compare A and B). Curcumin treatment protected TH‐positive fiber

innervation to a large extent. 6-OHDA+Cur+MLA treatment reversed the protective

effect curcumin. MLA treated rats alone showed no difference from 6‐OHDA or 6-

OHDA+Cur+MLA groups.

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Figure 65: Striatal TH-immunoreactive fiber density expressed as a percentage of the

fiber density on the lesioned side to the non-lesioned side.

The density was measured at four rostrocaudal levels of the striatum: rostral (+1.56

mm from Bregma), middle 1 (+0.72 mm from Bregma), middle 2 (+0.12 mm from

Bregma), and caudal (−0.24 mm from Bregma). The multiple intra-striatal 6-OHDA

injection caused an extensive damage to TH+ fibers throughout the four levels of

striatum compared to vehicle treated group (ANOVA, P˂0.000). The neuroprotective

effect of curcumin was markedly obvious in curcumin treated group through an α7-

receptor mediated mechanism, at all rostrocaudal levels of stratum; in comparison with

6-OHDA treated group (ANOVA, rostral P˂0.011). MLA, an α7-receptor blocker,

reversed the protective effect of curcumin in 6-OHDA+Cur+MLA group (ANOVA,

P˂0.000 at all levels). MLA antagonist had no effect by itself as indicated by the last

animal group; 6-OHDA+MLA showed no statistical difference from 6-

OHDA+Cur+MLA group or 6-OHDA treated rats (ANOVA, P˃1.000 at all level).

Values represent mean % of control side ± SEM.

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** **

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4.2.2.2 Effects on Tyrosine Hydroxylase-Positive Neurons in Substantia Nigra

The total number of SN TH+ neurons were assessed bilaterally (using the left un-

lesioned side as a control) in each animal by the unbiased stereological analysis using

the optical fractionator principle. The counted region included substantia nigra pars

compacta. The number of TH positive neurons in three different levels of SN (caudal,

middle, and rostral) were counted for each animal; to overcome several anatomical and

technical confounding factors such as; non-homogeneous distribution of dopaminergic

neurons, variability between animals, variability in the immunohistochemical staining

from one batch of specimens to another. The intra-striatal 6-OHDA injection caused a

substantial loss of SN cells with sparing of VTA TH+ neurons. There was no

significant difference bilaterally in vehicle treated group at the three levels; caudal,

middle, rostral, and total survival SN cell analysis [rostral: 100.3±6.2, middle:

94.0±6.6, caudal: 105.6±5.0, total: 99.7±2.5 / ANOVA]. However, on day 21 after 6-

OHDA lesioning, the number of the TH-positive neurons were markedly decreased on

the lesioned side compared with vehicle treated group at all rostrocaudal levels

[ANOVA, rostral: 11.3±2.9 (P˂0.000), middle: 10.2±2.0 (P˂0.000), caudal: 8.9±2.6

(P˂0.000), total: 9.9±1.9 (P˂0.000)]. Curcumin pre- and post-treatment had a

neuroprotective property and could significantly restore 6-OHDA damaging effect on

dopaminergic neurons in comparison with 6-OHDA treated animals at caudal, middle,

rostral and total survival SN cell count in 6-OHDA+Cur treated group [rostral: caudal;

[ANOVA, rostral: 30.2±5.1 (P˂0.003), middle: 39.2±6.8 (P˂0.000), caudal: 42.5±4.6

(P˂0.000), total: 32.0±4.7 (P˂0.000)]. In contrast, blocking of α7-nAChR with MLA

I.P injection prior to curcumin oral administration significantly reversed the

neuroprotective effect of curcumin at all the three levels in 6-OHDA+Cur+MLA

treated group [ANOVA, rostral: 6.7±0.9 (P˂0.000), middle: 12.3±2.6 (P˂0.000),

144

caudal: 12.9±1.3 (P˂0.000), total: 10.3±1.4 (P˂0.000)], supporting that curcumin

neuroprotective effect is mediated via α7-nAChR. The SN cell count in 6-

OHDA+MLA animal group were comparable to 6-OHDA+Cur+MLA or 6-OHDA

groups with no statistical difference between the three groups at all levels [ANOVA,

rostral: 6.7±1.9, middle: 7.0±1.2, caudal: 8.0±3.3, total: 7.1±2.0 (P˃1.0 at all levels)],

as it was not expected from this group to have any change at cellular level and the main

target of this group was to test the behavioral variation due to MLA administration.

Noting that, in all sections, the SN on the un-lesioned left side, both the

morphology and the number of TH-positive neurons remained unchanged (Figures 66

& 67).

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Figure 66: Photomicrographs of coronal sections of SN for TH immunohistochemistry.

Sections show three rostrocaudal levels from the SN on the lesion side. 6-OHDA

treated rats showed sever loss of TH-positive cells in the SN at all levels compared to

vehicle group (ANOVA, P˂0.000 at all levels). Curcumin treated rats showed

significant improvement in cell survival in comparison with 6-OHDA injected rats

(ANOVA, rostral P˂0.003, middle P˂0.000, and caudal P˂0.000). MLA, an α7-

receptor blocker, reversed the protective effect of curcumin in 6-OHDA+Cur+MLA

group (ANOVA, P˂0.000 at all levels). MLA antagonist had no effect by itself as

indicated by the last animal group; 6-OHDA+MLA showed no statistical difference

from 6-OHDA+Cur+MLA group or 6-OHDA treated rats (ANOVA, P˃1.0 at all

level).

146

A

****

** ****

B

****

**** ****

****

**** ****

C

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Figure 67: Stereological assessment of total numbers of TH-positive cell bodies in the

SN at all three levels; rostral, middle, and caudal.

The multiple intra-striatal 6-OHDA injection caused a dramatic loss of TH+ neurons

in the pars compacta compared to vehicle treated animals (ANOVA, rostral P˂0.000,

middle P˂0.000, caudal P˂0.000, and total P˂0.000). The neuroprotective effect of

curcumin was markedly obvious in curcumin treated group through an α7-receptor

mediated mechanism, at all rostrocaudal levels; rostral (A), middle (B), and caudal (C)

in comparison with 6-OHDA treated group (ANOVA, rostral P˂0.003, middle

P˂0.000, caudal P˂0.000, and total P˂0.000). MLA, an α7-receptor blocker, reversed

the protective effect of curcumin in 6-OHDA+Cur+MLA group (ANOVA, P˂0.000 at

all levels). MLA antagonist had no effect by itself as indicated by the last animal group;

6-OHDA+MLA showed no statistical difference from 6-OHDA+Cur+MLA group or

6-OHDA treated rats (ANOVA, P˃1.0 at all level (D)).

D

**** ****

****

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Chapter 5: Discussion

5.1 Discussion

In the present study, electrophysiological, in silico (computational), behavioral,

and morphological analyses were used to provide evidence that curcumin (i)

upregulates the function of human α7-nAChRs expressed in Xenopus oocytes and (ii)

reverses neurodegeneration in rat models of 6-OHDA of PD through an α7-nAChR

mechanism.

5.1.1 Effects of Curcumin on α7-Nicotinic Acetylcholine Receptor

Many researchers have extensively studied curcumin in recent years. However,

molecular and cellular targets mediating the pharmacological actions of curcumin

remain largely unknown. In this study, the effects of curcumin on the functional

properties of α7-nicotinic acetylcholine receptor have been investigated, using the two-

electrode voltage clamp technique. The effect of curcumin was tested on ion currents

induced with acetylcholine. Curcumin caused a significant potentiation of the current,

which was partially reversed during washout.

The potentiation effect of curcumin was significantly dependent on the mode of

application. For example, without preincubation, the co-application of curcumin and

ACh did not alter the amplitude of maximal currents. Whereas, preincubation of

curcumin caused an increase in the extent of α7-nAChRs potentiation. It is likely that

this preincubation time is crucial for oocyte preparation to adapt the effect of curcumin

on the expressed α7-nAChRs by the oocytes. This is because the preincubation timing

allows longer timeframe for the compound with the same experimental dose used,

eventually resulting in a more potent effect.

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The time course of the effect of curcumin on the maximal amplitudes of ACh-

induced currents was relatively slow which might indicate the possible interaction of

curcumin with the lipid membrane. The enhancement of α7-nAChR function by

curcumin is reversible and occurs in a time- and concentration-dependent manner.

Moreover, other curcumin derivatives have been screened and as expected, they

showed potentiation effects on α7-nAChRs, although to a lesser extent in comparison

to curcumin. Being products of curcumin themselves, curcumin metabolites had a

comparable potentiation effects on the receptor function.

The potentiation effect of curcumin was also obvious on other subtypes of nAChRs

and the other members of ligand gated ion channels; serotonin and glycine receptors.

Curcumin was found less effective on other nicotinic receptor subunit combinations

and other members of ligand-gated ion channels.

5.1.2 Effects of Curcumin on α7-Nicotinic Receptor are not Mediated by G-

proteins and Protein Kinases, and are not Dependent on Intracellular

Ca2+ Levels, and Membrane Potential

A relatively slow time course of curcumin effect and the results of earlier studies

on curcumin modulation of various second messenger pathways and kinases

(Mahmmoud, 2007; Takikawa et al., 2013) suggest that activation of G-protein-

coupled receptors (Liu et al., 2013; Pérez-Lara et al., 2011; Yang et al., 2015) and/or

kinase-mediated phosphorylation is involved in curcumin-induced upregulation of α7-

nAChRs (Talwar and Lynch, 2014; Zhang et al., 1995). However, neither treatments

with established kinase inhibitors nor pharmacological disruption of G-protein activity

reversed curcumin potentiation of α7-nAChRs, suggesting that curcumin acts directly

on ion channel-receptor complex. Furthermore, the enhancement of α7-nAChR

function by curcumin is not altered by changes in intracellular Ca2+ levels, or

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membrane potential. This was evidenced by the same level of curcumin potentiation

at all tested membrane potentials.

In Xenopus oocytes, activation of α7-nAChRs, due to their high Ca2+ permeability,

allows sufficient Ca2+ entry to activate endogenous Ca2+-dependent Cl− channels

(Hartzell et al., 2005; Sands et al., 1993). Ca2+-activated Cl− channels are highly

sensitive to intracellular Ca2+ levels [KD of Ca2+-activated Cl−channels for Ca2+ is less

than 1 µM; see review by (Hartzell et al., 2005)], and alterations in intracellular

Ca2+ levels would be reflected by changes in the holding current under voltage-clamp

conditions. Curcumin has been reported to alter Ca2+ homeostasis in various cell types

(Dyer et al., 2002; Ibrahim et al., 2011; Moustapha et al., 2015; Wang et al., 2012).

Therefore, the direct actions of curcumin on Ca2+-dependent Cl− channels may

contribute to the observed effects of curcumin on ACh-activated currents in this

expression system. However, in Xenopus oocytes injected with BAPTA and recorded

in a solution containing 2 mM Ba2+, co-application of curcumin did not cause

alterations in baseline or holding currents and continued to potentiate α7-nAChR–

mediated ion currents after the chelation of intracellular Ca2+ by BAPTA, suggesting

that Ca2+-dependent Cl− channels were not involved in curcumin potentiation of

nicotinic responses. In addition, the reversal potential in solutions containing Ba2+ was

not altered in the presence of curcumin, suggesting that the potentiation by curcumin

is not due to alterations in the Ca2+ permeability of the α7-nAChR-channel complex.

Negative results in BAPTA calcium chelating experiment; may advocate a second

review of experimental set-up including solutions. More importantly, it would have

been appropriate to test BAPTA effectiveness on any other well-established

intracellular Ca2+-dependent pathway as a positive control study before starting

oocytes’ experiments.

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5.1.3 Effects of Curcumin at Different Concentrations of Acetylcholine

Previous studies have demonstrated that curcumin acts on several integral

membrane proteins, including enzymes, transporters, and ion channels (Li et al., 2017;

X. Zhang et al., 2014), T-type Ca2+channels in bovine adrenal cells (Enyeart et al.,

2009); IC50 = 10–20 µM), L-type Ca2+ channels in hippocampal neurons (Liu et al.,

2013); IC50 ≈ 10 µM), TREK-1 K+ channels (Enyeart et al., 2008); IC50 = 0.9 µM),

Kv1.4 K+ channels (Liu et al., 2006) in bovine adrenal cells, Kv1.4. K+ channels (Lian

et al., 2013); IC50 = 4.2 µM) in human T-lymphocytes, ERG K+ channels (Hu et al.,

2012), IC50= 5.5 µM; (Choi et al., 2013), IC50 = 10.6 µM; (Banderali et al., 2011),

IC50 = 2 µM), and K+ channels in rabbit coronary arterial smooth muscle cells (Hong

et al., 2013); IC50 = 1.1 µM). In addition to voltage-dependent conductance, curcumin

has also been shown to act on transient-receptor potential receptors (Yeon et al., 2010;

Zhi et al., 2013). In this study, curcumin was applied in the concentration range of 1

nM to 100 µM, and it was found that it can enhance the effects of ACh on the function

of α7-nAChRs in a concentration-dependent manner, with EC50 values ranging from

58 nM to several micromolar. The concentration of curcumin in plasma and its ability

to pass the blood-brain barrier following oral and intravenous administration have been

studied previously (Anand et al., 2007). When curcumin was given orally at a dose of

2 g/kg to rats, a maximum serum concentration of 1.35 µg/ml ± 0.23 μg/mL was

attained at time 0.83 h (Shoba et al., 1998). Since curcumin is a highly lipophilic

compound with a logP (octanol–water partition coefficient) value of 3.3

(https://pubchem.ncbi.nlm.nih.gov/compound/curcumin#section=Top), its membrane

concentration is expected to be considerably higher than blood levels. Therefore, the

functional modulation of α7-nAChRs demonstrated in this study can be

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pharmacologically relevant. Because of curcumin’s lipophilicity, it would be

interesting to test the effects of curcumin with a different application such as

intracellular injection allowing direct interaction with its transmembrane binding site.

As mentioned earlier, the roles of G-proteins, kinases, and intracellular Ca2+ levels

in curcumin actions were excluded in our functional and pharmacological studies.

Further experiments indicated that the extent of curcumin potentiation decreased

significantly with increasing concentrations of ACh suggesting that curcumin acts

through a non-competitive mechanism of action.

5.1.4 Effects of Curcumin on the Specific Binding of [125I]α-bungarotoxin

By definition, an antagonist is a compound that upon binding to the receptor, have

no effect on their own but rather block the action of an endogenous or exogenous

agonist (Williams and Raddatz, 2006). There are two types of antagonist; competitive

antagonist where the antagonist and the agonist bind to the same active binding site,

so both are competing to bind to the same receptor. While non-competitive antagonist

binds to a different site other than the active site and block the action of the agonist

(Swinney, 2004).

Our results demonstrated the negative correlation between an increase in ACh

concentrations and the extent of curcumin potentiation, and this assumption was

supported by competition radioligand binding experiments. Notably, binding of α-

bungarotoxin, a competitive antagonist of ACh, was not altered in the presence of

curcumin, suggesting that curcumin does not interact with the ACh binding site in the

receptor.

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5.1.5 Effects of Curcumin on Desensitization of Nicotinic Receptors

It is likely that curcumin, a highly lipophilic agent, first dissolves into the lipid

membrane and then diffuses into a non-annular lipid space to potentiate the function

of the ion channel-receptor complex. Consistent with this idea, the effect of curcumin

on α7-nAChR reached a maximal level within 5–10 minutes of application, suggesting

that the binding site(s) for these allosteric modifiers is located inside the lipid

membrane and requires a relatively slow (in minutes) time course to modulate the

function of the receptor. It is likely that these hydrophobic agents affect the energy

requirements for gating-related conformational changes in ligand-gated ion channels

(Spivak et al., 2007).

α7-nAChRs recovery from desensitization is mainly dependent on agonist potency,

concentration, and duration of exposure. Therefore, for desensitization experiment we

have used a stronger and highly selective nicotinic agonist; nicotine with a dose of 100

µM and much longer exposure time (25 - 30 sec). Contrarily, ACh was used in all our

experiments. This is because ACh mimics the endogenously secreted agonist in its fast

receptor activation of both muscarinic and nicotinic, rapid degradation, and short

response duration.

The hallmark charactarestics of α7-nAChRs include its high permeability to Ca2+,

allowing huge amount of Ca2+ influx into the cell which may have a cytotoxic effect

(Guerra-Álvarez et al., 2015). However, the fast kinetics of α7-nAChRs exemplified

in fast desensitization and brief opening duration may be the reason behind avoidance

of cell toxicity.

It is plausible that curcumin acts as an allosteric modulator for various receptors

and ion channels at the lipid membrane, accounting for some of its pharmacological

154

actions in animal studies (Zhang et al., 2014). Allosteric modulators alter the

functional properties of ligand-gated ion channels by interacting with sites that are

topographically distinct from the ligand binding sites [see review by (Onaran and

Costa, 2009)]. Two different types of positive allosteric modulator (PAM) have been

postulated (Chatzidaki and Millar, 2015; Uteshev, 2014). Whereas type I enhances

agonist-induced currents without affecting macroscopic current kinetics, type II PAMs

delay desensitization and reactivate desensitized receptors. Further analysis of the

curcumin effect indicated that curcumin significantly (more than 3-fold) decreased

desensitization of the receptor and reactivate the already-desensitized nAChR to its

conducting state suggesting that curcumin acts as a type II PAM.

Rapid desensitization of homomeric α7-nAChRs can be tested under different

conditions, by assembling α7 subunit with different stoichiometries; namely β2,

forming functional heteromeric receptor subtype. It is expected that β2 subunit would

result in a slower receptor kinetics. α7β2 receptors have been detected in various brain

areas (Liu et al., 2012, 2009; Moretti et al., 2014; Thomsen et al., 2015; Zoli et al.,

2015). Interestingly, α7β2 has been shown to play a role in the neuropathy of

Alzheimer’s disease and is highly susceptible to inhibition by

the volatile anesthetic isoflurane (Liu et al., 2009; Mowrey et al., 2013).

5.1.6 Docking of Curcumin and Curcumin Derivatives into the Human α7-

nAChR Transmembrane Domain

All of these indications together, have guided us towards molecular docking

studies to identify the exact binding site of curcumin -as a type II positive allosteric

modulator- in relation to α7-nAChRs. The binding site of type II positive allosteric

modulators (PAMs) were proposed to be within the transmembrane domain of α7-

nAChRs (Gill et al., 2011; Young et al., 2008). Several pieces of evidence have

155

indicated that amino acids within TM1-TM3 are responsible for the potentiation effect

induced by PAMs. Out of these residues, mutations of two particular residues Ala225

(in TM1) or Met253 (in TM2) had the greatest influence on α7-nAChR potentiation

by the known type II PAM, PNU-120596 (Young et al., 2008). Corradi and Bouzat

(2016) claimed that PNU-120695 is the most efficacious type II PAM and, therefore

it has been used along with TQS as reference compounds in docking experiments.

Like other type II PAMs, the intra-cavity of transmembrane α7-nAChR domain

could be proposed as the binding site of curcumin and its derivatives. Fortunately,

Bondarenko et al. have recently discovered the structure of the human α7-nAChR

transmembrane domain, intra-cavity of which was used for curcumin docking

(Bondarenko et al., 2014).

Interestingly, all of the interacting residues of curcumin are located in TM1, TM2

and TM3 which are known for their importance for type II PAM binding. Also, many

of those residues, that when mutated play an important role in type II PAM activity,

appear to be in close proximity to curcumin; most importantly Ala226 which had direct

contacts with the ligand carbons. It is highly favorable that mutation at those specific

residues lining the cavity would affect curcumin interaction with the receptor.

Furthermore it has been postulated that this intracavity is a common PAM modulatory

site within the pLGIC superfamily, allowing wide variety of compounds to mediate an

allosteric effect (Corradi et al., 2011; Jayakumary et al., 2010; Nury et al., 2011;

Sauguet et al., 2014).

To sum up, curcumin and curcumin derivatives have what it takes to fit nicely into

the α7-nAChR transmembrane domain intra-cavity, the proposed binding site of type

II PAMs.

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5.1.7 Neuroprotective Properties of Curcumin in Parkinson’s Disease

Neurodegenerative diseases such as Alzheimer and Parkinson’s disease result from

loss of neurons as a sequel of their dysfunction. As discussed earlier, degenerative

changes in PD occur due to three main mechanisms; mitochondrial dysfunction,

oxidative stress, and modifications in protein handling, affecting all cellular functions

(Franco-Iborra et al., 2016; Olanow and McNaught, 2011; Schapira and Jenner, 2011).

A crucial unmet demand in the management of Parkinson’s disease is the discovery

of new approaches that could slow down, stop, or even reverse the process of

neurodegeneration. Several pieces of experimental evidence have revealed that the

cholinergic system is a potential pharmacological target for the treatment of PD (Guan

et al., 2002; Quik et al., 2012). Nicotine, a selective agonist of α7-nAChRs, enhances

the dopaminergic system in the striatum in animal models of PD (O’Neill et al., 2002;

Picciotto and Zoli, 2008; Quik and Wonnacott, 2011). The widespread distribution of

various subtypes of nicotinic receptors in the brain contribute significantly to nicotinic

receptor-mediated neuroprotection mechanisms. Several in-vitro studies using primary

cultures from different brain regions such as striatal, nigral, cortical, or neuronal cell

lines have demonstrated that pre-treatment with nAChR agonists have a

neuroprotective activity against toxic insults via α7 or α4β2* nAChRs mediated

mechanisms (Bordia et al., 2015; Gatto et al., 2004; O’Neill et al., 2002; Picciotto and

Zoli, 2008; Quik and Kulak, 2002; Roncarati et al., 2009; Ward et al., 2008; Yang et

al., 2017). Thus, drugs/compounds that can modulate/regulate α7-nAChRs may

possibly have a neuroprotective effect against 6-OHDA induced toxicity, which leads

to dopaminergic neuronal loss.

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Our in-vitro results demonstrated vividly that curcumin enhances the effects of

ACh on the function of α7-nAChRs in a concentration-dependent manner, and that

curcumin significantly decreases desensitization of the receptor leading the proposal

that it acts as a type II PAM. These results suggest that curcumin may play a key role

in brain regions affected by 6-OHDA in animal model of PD. We therefore tested this

effect after systemic in-vivo administration in rats. α7-nAChRs are widely distributed

throughout the brain in neuronal and non-neuronal immune cells, such as microglia

and astrocytes (Liu et al., 2015; Park et al., 2007; Shytle et al., 2004; Wang et al.,

2003). α7-nAChR expressed on immune cells are involved in the initiation,

maintenance, and resolution of inflammation, and modulate neuro-inflammatory

processes. In addition, α7-nAChRs are expressed on microglia regulating

inflammatory factors in the CNS (Bagdas et al., 2018).

A recent systematic literature review involving 13 studies of different PD animal

models, between the periods 2005 to 2014. All these studies except one, elaborated on

the anti-oxidant, anti-inflammatory, and anti-apoptotic properties of curcumin (Figure

68) and proved its neuroprotective activity and ability to improve neurological

functions in different animal models of PD (Wang et al., 2017).

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Figure 68: Neuroprotective mechanisms of curcumin in PD.

Curcumin marked efficacy in different animal models of Parkinson’s disease through

its anti-inflammatory, anti-oxidant, anti-apoptotic properties, and its ability to suppress

iron deposition in dopaminergic neurons (Wang et al., 2017).

In the present study we found that curcumin is a type II PAM of α7-nAChR. PAMs

are compounds that facilitate endogenous neurotransmission and/or enhance the

efficacy and potency of exogenous agonists, without directly stimulating the agonist

binding sites. Since α7-nACh receptor PAMs have been reported to be active in animal

models of Parkinson’s disease (Bagdas et al., 2015; Freitas et al., 2013b, 2013a; Munro

et al., 2012), in the next step, the effects of curcumin on motor function were assessed

in the 6-OHDA induced model of PD.

A variety of agents and toxins have been used to induce neurotoxicity which

involves the destruction of dopaminergic system in animal models of PD. These

include 6-OHDA, MPTP, rotenone, and many others. 6-OHDA is the most commonly

used rat model of PD (Cicchetti et al., 2009; Uversky, 2004). In this study, we found

that multiple intra-striatal injection of 6-OHDA successfully developed PD in the rat

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as evidenced by the loss of TH-immunoreactive cells, a marker of dopaminergic

neurons and motor dysfunction of the SNpc. The mortality rate of around 6.8 % in this

study which was calculated from the loss of one rat from 6-OHDA group and two rats

from 6-OHDA+Cur group was relatively low.

The drug-induced rotation test is the gold standard assessment of unilateral 6-

hydroxydopamine (6-OHDA) lesions. The direction of rotation differs according to

the drug used. Amphetamine, a dopamine releasing compound, induces striatal

dopamine release, and also inhibits its reuptake-, thus strongly activating DA release

in the intact side. Therefore, amphetamine induces animals to rotate in a direction

ipsilateral to the lesion side which has weak or no DA to be stimulated (Figure 69)

(Schwarting and Huston, 1996; Ungerstedt, 1971). In contrast, Apomorphine is a direct

DA agonist and has a postsynaptic mechanism of action. Because of apomorphine's

predominant postsynaptic mechanism of action, it stimulates post-synaptic D1 and D2

receptors preferentially in the lesioned side which become supersensitive to

apomorphine stimulation, hence, a dose of 0.25 mg/kg of apomorphine induces a

contralateral rotation in 6-OHDA treated animals (Figure 69) (Hefti et al., 1980a;

Hudson et al., 1993). Previous studies have shown that intra-striatal injection of 6-

OHDA causes retrograde degeneration of SN dopaminergic neurons, resulting in

depletion of striatal dopamine (Singh et al., 2003). Consistent with previous studies,

we found that 21 days after 6-OHDA treatment, rats displayed significant impairment

in the motor functions.

Apomorphine-induced rotation test was used to assess motor abnormalities

between different groups as it induces contralateral rotations only in rats with sever

DA depletion (≥ 75 – 95%) (Schwarting and Huston, 1996). This test is a reliable,

objective, and closely related to the degree of nigrostriatal dysfunction as well as DA

160

depletion (Schwarting et al., 1991). Vehicle treated rats rotated equally in both

directions and results were expressed as contralateral turns (subtracting number of

right turns from left turns). In comparison, 6-OHDA treated rats showed a significant

increase in number of turns contralaterally. Curcumin pre- and post-treatment

produced significant improvement in rotation response to 6-OHDA- injected rats and

decreased turning behavior significantly.

Figure 69: Drug-induced rotation test in rats that had 6-OHDA injection in the right

striatum.

(A & B) Systemic amphetamine injection results in ipsilateral rotation. (C & D)

Systemic apomorphine administration results in hypersensitivity of dopamine

receptors causing contralateral rotations (Dunnett and Torres, 2011).

A

B

C

D

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Based upon our findings that curcumin treatment significantly decreased turning

response in 6-OHDA treated rats, we investigated the neuroprotective effects of

curcumin against neuronal loss.

According to the study of Dadhaniya et al. (2011), the LD50 of the preparation in

rats as well as in mice was found to be greater than 2000 mg/kg body weight after 90

days treatment protocol. They concluded that no Observed-Adverse-Effect Level

(NOAEL) for the standardized novel curcumin preparation is determined as 750

mg/kg/day, (the highest dose tested) (Dadhaniya et al., 2011). To study the effect of

curcumin at the cellular level, a non-toxic dose of curcumin (200 mg/kg) was used.

Thus, our dose for the in-vivo study is considered safe. Indeed, it is equivalent to 2

g/day in human and very close to the recommended dose suggested by Perkins et al.

(2002).

The present study was designed to assess the ability of oral curcumin

administration to preserve the integrity of nigrostriatal dopaminergic system (cell

bodies, striatal terminals, and motor behavior). The total number of TH+ neurons in

the SNpc of both hemispheres was evaluated using an unbiased stereology technique.

In 6-OHDA+Cur treated group, curcumin significantly reduced the 6-OHDA-induced

degenerative effect on SNpc dopaminergic neuronal cells, indicating that curcumin has

a neuroprotective effect on DA neuronal damage by 6-OHDA. In the next step of the

study, the involvement of α7-nAChRs in curcumin neuroprotection was investigated.

The results demonstrated that neuroprotective effect of curcumin is mediated via α7-

nAChRs as demonstrated in 6-OHDA+Cur+MLA group. In these animals the number

of TH positive neurons in SN were comparable to 6-OHDA group with no significance

difference between both groups. The restorative effect of curcumin was distinctly

reflected on denervated TH+ DA striatal fibers, as curcumin significantly protected the

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striatal fibers from the 6-OHDA toxin through an α7-nAChRs mediated mechanism as

indicated by the abolished effect of curcumin after the use of MLA in 6-

OHDA+Cur+MLA group. Taken together, our data suggest that, curcumin improved

turning response, striatal fiber density, and SNpc TH-positive neuronal cells, are

considered as a good index for curcumin to have an anti-Parkinsonian effect.

α7-nAChR is considered to be a potential pharmacological target for several

neurological disorders, such as Parkinson’s disease, Alzheimer's disease, and

Schizophrenia. Currently, a number of α7-nAChR agonists and modulators are under

clinical trials (Yang et al., 2017). In case of Parkinson’s disease and nicotinic

receptors, several clinical trials have been conducted as shown in Table 13.

Keeping in view that many clinical trials in which α7-nAChR agonists were used

(e.g.: Tropisetron, EVP-6124) have been either suspended or terminated. This could

be attributed to the lack of high selectivity indicated by cross-activity with other LGIC,

(for example: 5-HT3 receptors) (Huang et al., 2014; Macor et al., 2001).

In comparison, α7-nAChR PAMs, presented very positive and promising results,

especially PNU-120695; a type II PAM which has proved to have a pro-cognitive

effect in rodents and non-human primates but failed in clinical trials for being too-

potent drug causing a very high calcium influx and potential cytotoxic effect (Callahan

et al., 2013; Ng et al., 2007).

As a basic rule in research: “safety remains the most important starting point and

efficacy becomes a matter of validation”, the need of safe and effective approach in

regards of α7-nAChR is mandatory. Curcumin, as demonstrated in our research to be

a type II PAM, is a natural compound with a high safety profile with no reported

toxicity (Lao et al., 2006) has undergone several clinical trials for treatment of

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neurodegenerative disorders (Table 14) (e.g.: Alzheimer’s disease, cognitive

impairment).

Overall, current findings of clinical trials on nicotinic receptors and Parkinson’s

disease or curcumin and neuro-degenerative disorders such as Parkinson’s disease are

very promising, but further pre-clinical studies and clinical trials are needed to improve

curcumin’s bioavailability and define its hidden targets of curcumin.

Our findings are in agreement with many other previous studies carried out, where

motor, cellular, and biochemical alterations in PD rats have been improved by

curcumin and its derivatives (Agrawal et al., 2012; Khuwaja et al., 2011; Singh and

Kumar, 2017; Song et al., 2016; Tripanichkul and Jaroensuppaperch, 2012, 2013;

Yang et al., 2014; Zbarsky et al., 2005), and our findings suggest a new mechanism

for curcumin-induced neuroprotection.

Figure 70 illustrates the possible mechanisms by which curcumin may act through

α7-nAChR to protect against the neurotoxic effects in an animal model of Parkinson’s

disease. The results of in-vitro, in silico, and in-vivo experiments of this study suggest

that increasing Ca2+ influx through curcumin α7-nAChR potentiation may have a

neuroprotective mechanism in neuronal and non-neuronal cells via various

intracellular mechanisms.

The lipid signaling cascade initiated by PKC, via phosphorylation of

phosphatidylinositol 3-kinase (PI3K/Akt), is credited with modulating the activities of

neuroprotective and apoptotic factors, such as Bcl-2 and caspases, respectively. In

addition to to their neuronal expression, α7-nAChRs are expressed on microglia and

astrocytes, playing a major role in immune response via “cholinergic anti-

inflammatory pathway”, activation of α7-nAChR and increase in IC Ca2+

concentration modulate Janus kinase 2 (JAK2) and/or signal transducer and activator

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of transcription 3 (STAT3), ending up with upregulation of protein kinase B (PKB)

leading to inhibition of nuclear factor-kB (NFκB). IC Ca2+ trigger protein kinase A

(PKA) and/or calcium-calmodulin-dependent protein kinase (CaMK) which in turn

trigger extracellular signal-regulated mitogen-activated protein kinase (ERK/MAPK)

pathway. ERK/MAPK signaling is a crucial event in cell survival pathway via

upregulation of the cellular transcription factor; cAMP response element-binding

(CREB), increasing gene expression of tyrosine hydroxylase, and enhancing dopamine

release. As an end result, maintaining cell viability through α7-nAChR activation

induce dopamine release from synaptic vesicles via Ca2+-dependent facilitation

mechanism.

Collectively, all or some of these factors may contribute to decrease apoptosis,

modify immune response, and alter synaptic plasticity enhancing neuronal protection

and survival (Hosur and Loring, 2011; Picciotto and Zoli, 2008; Quik and Kulak, 2002;

Ward et al., 2008).

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Figure 70: Hypothetical model of Ca2+- dependent cell survival mechanism.

Curcumin modulate α7-nAChR allosterically allowing more Ca2+ entry into the cell as

depicted from the electrophysiological recording. Increase in intracellular Ca2+

concentration will lead to a cascade of events in dopaminergic neurons (from left to

right): Facilitation of dopamine release from synaptic vesicles. Activation of ERK by

PKA and/or CaMK, upregulate CREB protein, increase tyrosine hydroxylase activity,

and activate dopamine release. JAK2/STAT3 signaling pathway leads to inhibition of

NF-kB translocation via PKB activation. Increase in IC Ca2+ attenuates inflammatory

response in immune cells activating protein kinase C, PKC appears to activate

downstream signaling PI3K/AKT pathways that promotes Nrf-2 translocation

resulting in modulation of cell survival proteins; Bcl-2 and caspase.

ACh ACh+ Cur

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Table 13: nAChRs and PAMs in clinical trials for treatment of PD (“Home - ClinicalTrials.gov,” n.d.)

Title Status Conditions Interventions Characteristics Sponsor/Collabroters

Evaluation of 5-

[123I]-A-85380 and

SPECT imaging in

Individuals With

Parkinson’s Disease

Completed Parkinson’s

Disease

Drug: [1231]-

IA-85380

Phase:

• Phase 2

Study Design:

• Intervention Model: Single

Group Assignment

• Masking: None (Open

Lable)

• Primary Purpose:

Diagnostic

Institute of

Neurodegenerative Disorders

United States Department of

Defense

Cholinergic Nicotinic

Receptors and

Cognition in PD


Disease

Phase:

Study Design:

• Observetional Model:

Cohort

• Time Perspective: Cross-

Sectional

University of Michigan

Michael J. Fox Foundation

for Parkinson’s Research

Varenicline

Treatment for

Excessive Daytime

Sleepiness in


Active, not

recruiting

Parkinson’s

Disease

Drug:

Varenicline

Drugc: Placebo

Phase:

• Phase 4

Study Design:

• Allocation: Randomized

• Intervention Model:

Crossover Assignment

• Masking: Double

(Participant, Investigator)

• Primary Purpose:

Treatment

VU University Medical

Center

Center for Human Drug

Research, Netherlands

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Table 13: nAChRs and PAMs in clinical trials for treatment of PD (“Home - ClinicalTrials.gov,” n.d.) (Continued)

Title Status Conditions Interventios Characteristics Sponsor/Collabroters

PET Study of the

Nicotinic Receptors

in Human

Terminated Healthy

Parkinson’s

Disease

Alzheimer’s

Disease

Epilepsy

Drug: Curcumin Phase:

Study Design:

• Time Prespective:

Prospective

Commissariat A L’ebergie

Atomique

Single Photon

Emission Computed

Tomography to

Study Receptors in



Disease

Drug: I-123-5-

IA85380

National Institute of

Neurological Disorders and

Stroke (NINDS)

National Institutes of Health

Clinical Center (CC)

A Genetic and

Perfusion Study of

Response to

Cognitive Enhancers

in Lewy Body Disease

Completed Lewy Body

Disease

Drug:

Cholinesterase

Inhibitors

(Rivastigmine,

Aricept,

Galantamine)

Study Design:

• Observetional Model:

Cohort

• Time Perspective:

Prospective

Sunnybrook Health Sciences

Center

168

Table 14: Curcumin in clinical trials for treatment of various neurodegenerative disorders (“Home - ClinicalTrials.gov,” n.d.)


A Pilot Study of

Curcumin and

Ginkgo for Treating

Alzheimer’s Disease

Completed Alzheimer’s

Disease

Drug: Placebo

and ginkgo

extract

Drug:

Curcumin and

ginkgo extract

Phase:

• Phase 1

• Phase 2

Study Design:


• Intervention Model: Parallel

Assignment

• Masking: Double

• Primary Purpose: Treatment

Chinese University of

Hong Kong

BUPA Foundation

Kwong Wah hospital

Curcumin and Yoga

Therapy for Those at

Risk for Alzheimer’s

Disease

Recruiting Mild

Cognitive

Impairment

Drug:

Curcumin

Behavioral:

aerobic yoga

Behavioral:

non-aerobic

yoga

Dietary

Supplement:

Placebo

Phase:

• Phase 2

Study Design:


• Intervention Model: Factorial

Assignment

• Masking: Quadruple (Participant,

Care provider, Investigator, Outcome

Assessors)

• Primary Purpose: Prevention

VA Office of Reasech

and Development

heather.d’[email protected]

169

Table 14: Curcumin in clinical trials for treatment of various neurodegenerative disorders (“Home - ClinicalTrials.gov,” n.d.) (Continued)


Efficacy and Safety

of Curcumin

Formulation in

Alzheimer’s

Disease

Unknown

status

Alzheimer’s

Disease

Dietary

Supplement:

Curcumin

Formulation

Dietary

Supplement:

Placebo

Phase:

• Phase 1

Study Design:


• Intervention Model: Single Group

Assignment

• Masking: Double


Jaslok Hospital and

Research Center

Pharmanza Herbal Pvt

Ltd.

Verdure Sciences

University of California,

Los Angeles

A Randomized,

Double-blind,

Placebo controlled

Trial of Curcumin

in Leber’s

Hereditary Optic

Neuropathy

(LHON)

Completed Optic

Atrophy,

Hereditary,

Leber’s

Drug:

Curcumin

Phase:

• Phase 3

Study Design:


• Intervention Model: Prallel Assignment

• Masking: Double


Mahidol University

Curcumin in

Pateints With Mild

to Moderate

Alzheimer’s

Disease

Completed Alzheimer’s

Disease

Dietary

Supplement:

Curcumin C3

Complex

Phase:

• Phase 2

Study Design:


• Intervention Model: Parallel

Assignment

• Masking: Double


John Douglas French

Foundation

Institute for the Study of

Aging (ISOA)

National Institute on

Aging (NIA)

170

Chapter 6: Conclusions

To our knowledge, this is the first study to establish that:

• Curcumin selectively enhances the effect of ACh on the function of α7-

nAChRs in a concentration dependent manner.

• The effects of curcumin on α7-nicotinic receptor are not mediated by G-

proteins and protein kinases, and are not dependent on intracellular Ca2+ levels,

and membrane potential.

• Curcumin does not interact with ACh binding to receptors.

• Curcumin significantly decreased desensitization of the receptor and

reactivates the already-desensitized nAChR back to conducting state.

• Curcumin and curcumin derivatives fit nicely into the α7-nAChR

transmembrane domain intra-cavity, the proposed binding site of type II

PAMs.

• Curcumin pre- and post-treatment significantly improved motor function in the

6-OHDA induced model of PD.

• Curcumin pre- and post-treatment attenuated the toxic effects of 6-OHDA on

the reduction level of striatal dopamine.

• Curcumin treatment protected dopaminergic neuronal cells in SNpc from 6-

OHDA toxicity, suggesting that curcumin produced significant restoration of

dopamine in the nigrostriatal pathway.

171

Limitations and Future Directions

1- One of the limitations in this study was the lack of positive control in BAPTA

and protein kinase experiments. Testing BAPTA effectiveness on any well-

established intracellular Ca2+-dependent pathway, as a positive control study

before starting oocytes’ experiments, would have provided further evidence

that Ca2+-activated Cl- channels plays no role in curcumin α7-nAChR

activation, and the same applies for protein kinase experiments.

2- Curcumin solubility: DMSO was used as curcumin dissolvent in in-vitro study

with a final concentration of 0.001%. However, in in-vivo study higher doses

of curcumin and DMSO injected intraperitoneally were required. This method

resulted in a high mortality rate. Therefore, it was decided to use different

dissolvent (CMC) and different route of administration (intragastric).

3- Our results from molecular docking experiment demonstrated that curcumin

performed better than PNU-120695 which is considered as a reference

compound for PAM type II. Therefore, it would be very informative to test it

in-vivo and perform a comparative study in PD animal model.

4- Parkinson’s disease is neurodegenerative disorder, where inflammation and

oxidative stress plays a major role in the pathophysiology of the disease. Thus,

biochemical analysis and western blotting of anti-inflammatory, and anti-

oxidant biomarkers would have provided valuable information and should be

the next step.

172

References

Abeliovich, A., Schmitz, Y., Fariñas, I., Choi-Lundberg, D., Ho, W.H., Castillo, P.E.,

Shinsky, N., Verdugo, J.M., Armanini, M., Ryan, A., Hynes, M., Phillips, H.,

Sulzer, D., Rosenthal, A., 2000. Mice lacking alpha-synuclein display

functional deficits in the nigrostriatal dopamine system. Neuron 25, 239–252.

Aggarwal, B.B., Harikumar, K.B., 2009. Potential therapeutic effects of curcumin,

the anti-inflammatory agent, against neurodegenerative, cardiovascular,

pulmonary, metabolic, autoimmune and neoplastic diseases. Int. J. Biochem.

Cell Biol. 41, 40–59. https://doi.org/10.1016/j.biocel.2008.06.010

Aggarwal, B.B., Sundaram, C., Malani, N., Ichikawa, H., 2007. Curcumin: the Indian

solid gold. Adv. Exp. Med. Biol. 595, 1–75. https://doi.org/10.1007/978-0-

387-46401-5_1

Aggarwal, B.B., Sung, B., 2009. Pharmacological basis for the role of curcumin in

chronic diseases: an age-old spice with modern targets. Trends Pharmacol.

Sci. 30, 85–94. https://doi.org/10.1016/j.tips.2008.11.002

Agid, Y., Javoy, F., Glowinski, J., Bouvet, D., Sotelo, C., 1973. Injection of 6-

hydroxydopamine into the substantia nigra of the rat. II. Diffusion and

specificity. Brain Res. 58, 291–301.

Agrawal, S.S., Gullaiya, S., Dubey, V., Singh, V., Kumar, A., Nagar, A., Tiwari, P.,

2012. Neurodegenerative Shielding by Curcumin and Its Derivatives on Brain

Lesions Induced by 6-OHDA Model of Parkinson’s Disease in Albino Wistar

Rats. Cardiovasc. Psychiatry Neurol. 2012, 942981.

https://doi.org/10.1155/2012/942981

Ahlskog, J.E., Muenter, M.D., 2001. Frequency of levodopa-related dyskinesias and

motor fluctuations as estimated from the cumulative literature. Mov Disord

16, 448–58.

Alam, M., Schmidt, W.J., 2004. L-DOPA reverses the hypokinetic behaviour and

rigidity in rotenone-treated rats. Behav. Brain Res. 153, 439–446.

https://doi.org/10.1016/j.bbr.2003.12.021

Albuquerque, E. X., Pereira, E.F., Alkondon, M., Rogers, S.W., 2009. Mammalian

nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89,

73–120. https://doi.org/10.1152/physrev.00015.2008

Allam, M.F., Campbell, M.J., Hofman, A., Del Castillo, A.S., Fernandez-Crehuet

Navajas, R., 2004. Smoking and Parkinson’s disease: systematic review of

173

prospective studies. Mov Disord 19, 614–21.

https://doi.org/10.1002/mds.20029

Ammon, H.P., Wahl, M.A., 1991. Pharmacology of Curcuma longa. Planta Med. 57,

1–7. https://doi.org/10.1055/s-2006-960004

Anand, P., Kunnumakkara, A.B., Newman, R.A., Aggarwal, B.B., 2007.

Bioavailability of curcumin: problems and promises. Mol. Pharm. 4, 807–

818. https://doi.org/10.1021/mp700113r

Anand, P., Thomas, S.G., Kunnumakkara, A.B., Sundaram, C., Harikumar, K.B.,

Sung, B., Tharakan, S.T., Misra, K., Priyadarsini, I.K., Rajasekharan, K.N.,

Aggarwal, B.B., 2008. Biological activities of curcumin and its analogues

(Congeners) made by man and Mother Nature. Biochem. Pharmacol. 76,

1590–1611. https://doi.org/10.1016/j.bcp.2008.08.008

Andres-Mateos, E., Perier, C., Zhang, L., Blanchard-Fillion, B., Greco, T.M.,

Thomas, B., Ko, H.S., Sasaki, M., Ischiropoulos, H., Przedborski, S.,

Dawson, T.M., Dawson, V.L., 2007. DJ-1 gene deletion reveals that DJ-1 is

an atypical peroxiredoxin-like peroxidase. Proc. Natl. Acad. Sci. U. S. A.

104, 14807–14812. https://doi.org/10.1073/pnas.0703219104

Antonini, A., Abbruzzese, G., Ferini-Strambi, L., Tilley, B., Huang, J., Stebbins,

G.T., Goetz, C.G., Barone, P., MDS-UPDRS Italian Validation Study Group,

Bandettini di Poggio, M., Fabbrini, G., Di Stasio, F., Tinazzi, M., Bovi, T.,

Ramat, S., Meoni, S., Pezzoli, G., Canesi, M., Martinelli, P., Maria Scaglione,

C.L., Rossi, A., Tambasco, N., Santangelo, G., Picillo, M., Morgante, L.,

Morgante, F., Quatrale, R., Sensi, M., Pilleri, M., Biundo, R., Nordera, G.,

Caria, A., Pacchetti, C., Zangaglia, R., Lopiano, L., Zibetti, M., Zappia, M.,

Nicoletti, A., Quattrone, A., Salsone, M., Cossu, G., Murgia, D., Albanese,

A., Del Sorbo, F., 2013. Validation of the Italian version of the Movement

Disorder Society--Unified Parkinson’s Disease Rating Scale. Neurol. Sci.

Off. J. Ital. Neurol. Soc. Ital. Soc. Clin. Neurophysiol. 34, 683–687.

https://doi.org/10.1007/s10072-012-1112-z

Aoki, H., Takada, Y., Kondo, S., Sawaya, R., Aggarwal, B.B., Kondo, Y., 2007.

Evidence that curcumin suppresses the growth of malignant gliomas in vitro

and in vivo through induction of autophagy: role of Akt and extracellular

signal-regulated kinase signaling pathways. Mol. Pharmacol. 72, 29–39.

https://doi.org/10.1124/mol.106.033167

Arun, N., Nalini, N., 2002. Efficacy of turmeric on blood sugar and polyol pathway

in diabetic albino rats. Plant Foods Hum. Nutr. Dordr. Neth. 57, 41–52.

174

Ashoor, A., Nordman, J.C., Veltri, D., Yang, K.-H.S., Al Kury, L., Shuba, Y.,

Mahgoub, M., Howarth, F.C., Sadek, B., Shehu, A., Kabbani, N., Oz, M.,

2013. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

PloS One 8, e67674. https://doi.org/10.1371/journal.pone.0067674

Aum, D.J., Tierney, T.S., 2018. Deep brain stimulation: foundations and future

trends. Front. Biosci. Landmark Ed. 23, 162–182.

Babu, P.S., Srinivasan, K., 1995. Influence of dietary curcumin and cholesterol on

the progression of experimentally induced diabetes in albino rat. Mol. Cell.

Biochem. 152, 13–21.

Bacchelli, E., Battaglia, A., Cameli, C., Lomartire, S., Tancredi, R., Thomson, S.,

Sutcliffe, J.S., Maestrini, E., 2015. Analysis of CHRNA7 rare variants in

autism spectrum disorder susceptibility. Am. J. Med. Genet. A. 167A, 715–

723. https://doi.org/10.1002/ajmg.a.36847

Bagchi, D., Chaudhuri, S., Sardar, S., Choudhury, S., Polley, N., Lemmens, P., Pal,

S.K., 2015. Modulation of stability and functionality of a phyto-antioxidant

by weakly interacting metal ions: curcumin in aqueous solution. RSC Adv. 5,

102516–102524. https://doi.org/10.1039/C5RA21593E

Bagdas, D., Gurun, M.S., Flood, P., Papke, R.L., Damaj, M.I., 2018. New Insights on

Neuronal Nicotinic Acetylcholine Receptors as Targets for Pain and

Inflammation: A Focus on α7 nAChRs. Curr. Neuropharmacol. 16, 415–425.

https://doi.org/10.2174/1570159X15666170818102108

Bagdas, D., Targowska-Duda, K.M., López, J.J., Perez, E.G., Arias, H.R., Damaj,

M.I., 2015. The Antinociceptive and Antiinflammatory Properties of 3-furan-

2-yl-N-p-tolyl-acrylamide, a Positive Allosteric Modulator of α7 Nicotinic

Acetylcholine Receptors in Mice. Anesth. Analg. 121, 1369–1377.

https://doi.org/10.1213/ANE.0000000000000902

Banderali, U., Belke, D., Singh, A., Jayanthan, A., Giles, W.R., Narendran, A., 2011.

Curcumin blocks Kv11.1 (erg) potassium current and slows proliferation in

the infant acute monocytic leukemia cell line THP-1. Cell. Physiol. Biochem.

Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 28, 1169–1180.

https://doi.org/10.1159/000335850

Bastide, M.F., Meissner, W.G., Picconi, B., Fasano, S., Fernagut, P.-O., Feyder, M.,

Francardo, V., Alcacer, C., Ding, Y., Brambilla, R., Fisone, G., Jon Stoessl,

A., Bourdenx, M., Engeln, M., Navailles, S., De Deurwaerdère, P., Ko,

W.K.D., Simola, N., Morelli, M., Groc, L., Rodriguez, M.-C., Gurevich,

E.V., Quik, M., Morari, M., Mellone, M., Gardoni, F., Tronci, E., Guehl, D.,

Tison, F., Crossman, A.R., Kang, U.J., Steece-Collier, K., Fox, S., Carta, M.,

175

Angela Cenci, M., Bézard, E., 2015. Pathophysiology of L-dopa-induced

motor and non-motor complications in Parkinson’s disease. Prog. Neurobiol.

132, 96–168. https://doi.org/10.1016/j.pneurobio.2015.07.002

Bayet-Robert, M., Kwiatkowski, F., Leheurteur, M., Gachon, F., Planchat, E., Abrial,

C., Mouret-Reynier, M.-A., Durando, X., Barthomeuf, C., Chollet, P., 2010.

Phase I dose escalation trial of docetaxel plus curcumin in patients with

advanced and metastatic breast cancer. Cancer Biol. Ther. 9, 8–14.

Beach, T.G., White, C.L., Hamilton, R.L., Duda, J.E., Iwatsubo, T., Dickson, D.W.,

Leverenz, J.B., Roncaroli, F., Buttini, M., Hladik, C.L., Sue, L.I., Noorigian,

J.V., Adler, C.H., 2008. Evaluation of alpha-synuclein immunohistochemical

methods used by invited experts. Acta Neuropathol. (Berl.) 116, 277–288.

https://doi.org/10.1007/s00401-008-0409-8

Belcaro, G., Hosoi, M., Pellegrini, L., Appendino, G., Ippolito, E., Ricci, A., Ledda,

A., Dugall, M., Cesarone, M.R., Maione, C., Ciammaichella, G., Genovesi,

D., Togni, S., 2014. A controlled study of a lecithinized delivery system of

curcumin (Meriva®) to alleviate the adverse effects of cancer treatment.

Phytother. Res. PTR 28, 444–450. https://doi.org/10.1002/ptr.5014

Belluardo, N., Mudo, G., Blum, M., Fuxe, K., 2000. Central nicotinic receptors,

neurotrophic factors and neuroprotection. Behav Brain Res 113, 21–34.

Benabid, A.L., Chabardes, S., Mitrofanis, J., Pollak, P., 2009. Deep brain stimulation

of the subthalamic nucleus for the treatment of Parkinson’s disease. Lancet

Neurol. 8, 67–81. https://doi.org/10.1016/S1474-4422(08)70291-6

Berardelli, A., Rothwell, J.C., Thompson, P.D., Hallett, M., 2001. Pathophysiology

of bradykinesia in Parkinson’s disease. Brain J. Neurol. 124, 2131–2146.

Bertrand, D., Bertrand, S., Ballivet, M., 1992. Pharmacological properties of the

homomeric alpha 7 receptor. Neurosci. Lett. 146, 87–90.

Betarbet, R., Sherer, T.B., MacKenzie, G., Garcia-Osuna, M., Panov, A.V.,

Greenamyre, J.T., 2000. Chronic systemic pesticide exposure reproduces

features of Parkinson’s disease. Nat. Neurosci. 3, 1301–1306.

https://doi.org/10.1038/81834

Bezard, E., Imbert, C., Gross, C.E., 1998. Experimental models of Parkinson’s

disease: from the static to the dynamic. Rev. Neurosci. 9, 71–90.

Bezard, E., Przedborski, S., 2011. A tale on animal models of Parkinson’s disease.

Mov. Disord. Off. J. Mov. Disord. Soc. 26, 993–1002.


176

Bianchi, L., 2006. Heterologous expression of C. elegans ion channels in Xenopus

oocytes. WormBook. https://doi.org/10.1895/wormbook.1.117.1

Blaha, C.D., Allen, L.F., Das, S., Inglis, W.L., Latimer, M.P., Vincent, S.R., Winn,

P., 1996. Modulation of dopamine efflux in the nucleus accumbens after

cholinergic stimulation of the ventral tegmental area in intact,

pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental

nucleus-lesioned rats. J Neurosci 16, 714–22.

Blaha, C.D., Winn, P., 1993. Modulation of dopamine efflux in the striatum

following cholinergic stimulation of the substantia nigra in intact and

pedunculopontine tegmental nucleus-lesioned rats. J Neurosci 13, 1035–44.

Blanchet, P.J., Metman, L.V., Mouradian, M.M., Chase, T.N., 1996. Acute

pharmacologic blockade of dyskinesias in Parkinson’s disease. Mov Disord

11, 580–1. https://doi.org/10.1002/mds.870110516

Blandini, F., Armentero, M.-T., 2012. New pharmacological avenues for the

treatment of L-DOPA-induced dyskinesias in Parkinson’s disease: targeting

glutamate and adenosine receptors. Expert Opin. Investig. Drugs 21, 153–

168. https://doi.org/10.1517/13543784.2012.651457

Blandini, F., Armentero, M.-T., Martignoni, E., 2008. The 6-hydroxydopamine

model: news from the past. Parkinsonism Relat. Disord. 14 Suppl 2, S124-

129. https://doi.org/10.1016/j.parkreldis.2008.04.015

Blesa, J., Phani, S., Jackson-Lewis, V., Przedborski, S., 2012. Classic and new

animal models of Parkinson’s disease. J. Biomed. Biotechnol. 2012, 845618.

https://doi.org/10.1155/2012/845618

Blesa, J., Przedborski, S., 2014. Parkinson’s disease: animal models and

dopaminergic cell vulnerability. Front. Neuroanat. 8, 155.

https://doi.org/10.3389/fnana.2014.00155

Blesa, J., Trigo-Damas, I., Quiroga-Varela, A., Jackson-Lewis, V.R., 2015.

Oxidative stress and Parkinson’s disease. Front. Neuroanat. 9.


Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R., Verna,

J.M., 2001. Molecular pathways involved in the neurotoxicity of 6-OHDA,

dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s

disease. Prog. Neurobiol. 65, 135–172.

Bockaert, J., Premont, J., Glowinski, J., Thierry, A.M., Tassin, J.P., 1976.

Topographical distribution of dopaminergic innervation and of dopaminergic

177

receptors in the rat striatum. II. Distribution and characteristics of dopamine

adenylate cyclase--interaction of d-LSD with dopaminergic receptors. Brain

Res. 107, 301–315.

Bondarenko, V., Mowrey, D.D., Tillman, T.S., Seyoum, E., Xu, Y., Tang, P., 2014.

NMR structures of the human α7 nAChR transmembrane domain and

associated anesthetic binding sites. Biochim. Biophys. Acta 1838, 1389–

1395. https://doi.org/10.1016/j.bbamem.2013.12.018

Bordia, T., Campos, C., Huang, L., Quik, M., 2008. Continuous and intermittent

nicotine treatment reduces L-3,4-dihydroxyphenylalanine (L-DOPA)-induced

dyskinesias in a rat model of Parkinson’s disease. J Pharmacol Exp Ther 327,

239–47. https://doi.org/10.1124/jpet.108.140897

Bordia, T., McGregor, M., Papke, R.L., Decker, M.W., McIntosh, J.M., Quik, M.,

2015. The alpha7 nicotinic receptor agonist ABT-107 protects against

nigrostriatal damage in rats with unilateral 6-hydroxydopamine lesions. Exp

Neurol 263, 277–84. https://doi.org/10.1016/j.expneurol.2014.09.015

Bové, J., Perier, C., 2012. Neurotoxin-based models of Parkinson’s disease.

Neuroscience 211, 51–76. https://doi.org/10.1016/j.neuroscience.2011.10.057

Braak, H., Braak, E., 2000. Pathoanatomy of Parkinson’s disease. J. Neurol. 247

Suppl 2, II3-10. https://doi.org/10.1007/PL00007758

Braak, H., Braak, E., Yilmazer, D., de Vos, R.A., Jansen, E.N., Bohl, J., Jellinger, K.,

1994. Amygdala pathology in Parkinson’s disease. Acta Neuropathol. (Berl.)

88, 493–500.

Braak, H., Del Tredici, K., Rüb, U., de Vos, R.A.I., Jansen Steur, E.N.H., Braak, E.,

2003. Staging of brain pathology related to sporadic Parkinson’s disease.

Neurobiol. Aging 24, 197–211.

Braak, H., Ghebremedhin, E., Rüb, U., Bratzke, H., Del Tredici, K., 2004. Stages in

the development of Parkinson’s disease-related pathology. Cell Tissue Res.

318, 121–134. https://doi.org/10.1007/s00441-004-0956-9

Branchi, I., D’Andrea, I., Armida, M., Cassano, T., Pèzzola, A., Potenza, R.L.,

Morgese, M.G., Popoli, P., Alleva, E., 2008. Nonmotor symptoms in

Parkinson’s disease: investigating early-phase onset of behavioral

dysfunction in the 6-hydroxydopamine-lesioned rat model. J. Neurosci. Res.

86, 2050–2061. https://doi.org/10.1002/jnr.21642

178

Brooks, A.I., Chadwick, C.A., Gelbard, H.A., Cory-Slechta, D.A., Federoff, H.J.,

1999. Paraquat elicited neurobehavioral syndrome caused by dopaminergic

neuron loss. Brain Res. 823, 1–10.

Brotchie, J., Jenner, P., 2011. New approaches to therapy. Int Rev Neurobiol 98,

123–50. https://doi.org/10.1016/b978-0-12-381328-2.00005-5

Brotchie, J.M., Lee, J., Venderova, K., 2005. Levodopa-induced dyskinesia in

Parkinson’s disease. J Neural Transm Vienna 112, 359–91.

https://doi.org/10.1007/s00702-004-0251-7

Brouet, I., Ohshima, H., 1995. Curcumin, an anti-tumour promoter and anti-

inflammatory agent, inhibits induction of nitric oxide synthase in activated

macrophages. Biochem. Biophys. Res. Commun. 206, 533–540.

Brown, P., Eusebio, A., 2008. Paradoxes of functional neurosurgery: clues from

basal ganglia recordings. Mov Disord 23, 12–20; quiz 158.


Bruyn, G.W., 1983. Handbook of chemical neuroanatomy, vol. 1 (methods in

chemical neuroanatomy): By A. Björklund and T. Hökfelt (eds.), 560 pages,

Elsevier Biomedical Press, Amsterdam, 1983, US$ 140.50, Dfl 330.00. J.

Neurol. Sci. 62, 358. https://doi.org/10.1016/0022-510X(83)90212-5

Cadet, J.L., Krasnova, I.N., Jayanthi, S., Lyles, J., 2007. Neurotoxicity of substituted

amphetamines: molecular and cellular mechanisms. Neurotox. Res. 11, 183–

202.

Calabresi, P., Di Filippo, M., Ghiglieri, V., Picconi, B., 2008. Molecular mechanisms

underlying levodopa-induced dyskinesia. Mov Disord 23 Suppl 3, S570-9.


Callahan, P.M., Hutchings, E.J., Kille, N.J., Chapman, J.M., Terry, A.V., 2013.

Positive allosteric modulator of α7 nicotinic-acetylcholine receptors, PNU-

120596 augments the effects of donepezil on learning and memory in aged

rodents and non-human primates. Neuropharmacology 67, 201–212.

https://doi.org/10.1016/j.neuropharm.2012.10.019

Cannon, J.R., Greenamyre, J.T., 2010. Neurotoxic in vivo models of Parkinson’s

disease recent advances. Prog. Brain Res. 184, 17–33.

https://doi.org/10.1016/S0079-6123(10)84002-6

Cannon, J.R., Tapias, V., Na, H.M., Honick, A.S., Drolet, R.E., Greenamyre, J.T.,

2009. A highly reproducible rotenone model of Parkinson’s disease.

Neurobiol. Dis. 34, 279–290.

179

Cao, L., Liu, J., Zhang, L., Xiao, X., Li, W., 2016. Curcumin inhibits H2O2-induced

invasion and migration of human pancreatic cancer via suppression of the

ERK/NF-κB pathway. Oncol. Rep. 36, 2245–2251.

https://doi.org/10.3892/or.2016.5044

Carlsson, A., 1959. The occurrence, distribution and physiological role of

catecholamines in the nervous system. Pharmacol. Rev. 11, 490–493.

Carlsson, A., Lindqvist, M., Magnusson, T., 1957. 3,4-Dihydroxyphenylalanine and

5-hydroxytryptophan as reserpine antagonists. Nature 180, 1200.

Carroll, R.E., Benya, R.V., Turgeon, D.K., Vareed, S., Neuman, M., Rodriguez, L.,

Kakarala, M., Carpenter, P.M., McLaren, C., Meyskens, F.L., Brenner, D.E.,

2011. Phase IIa clinical trial of curcumin for the prevention of colorectal

neoplasia. Cancer Prev. Res. Phila. Pa 4, 354–364.

https://doi.org/10.1158/1940-6207.CAPR-10-0098

Carta, M., Carlsson, T., Munoz, A., Kirik, D., Bjorklund, A., 2008. Serotonin-

dopamine interaction in the induction and maintenance of L-DOPA-induced

dyskinesias. Prog Brain Res 172, 465–78. https://doi.org/10.1016/s0079-

6123(08)00922-9

Cenci, M.A., Lundblad, M., 2007. Ratings of L-DOPA-induced dyskinesia in the

unilateral 6-OHDA lesion model of Parkinson’s disease in rats and mice.

Curr Protoc Neurosci Chapter 9, Unit 9.25.

https://doi.org/10.1002/0471142301.ns0925s41

Cenci, M.A., Lundblad, M., 2006. Post- versus presynaptic plasticity in L-DOPA-

induced dyskinesia. J Neurochem 99, 381–92. https://doi.org/10.1111/j.1471-

4159.2006.04124.x

Changeux, J.-P., 2010. Allosteric receptors: from electric organ to cognition. Annu.

Rev. Pharmacol. Toxicol. 50, 1–38.

https://doi.org/10.1146/annurev.pharmtox.010909.105741

Changeux, J.P., Kasai, M., Lee, C.Y., 1970. Use of a snake venom toxin to

characterize the cholinergic receptor protein. Proc. Natl. Acad. Sci. U. S. A.

67, 1241–1247.

Chatzidaki, A., Millar, N.S., 2015. Allosteric modulation of nicotinic acetylcholine

receptors. Biochem. Pharmacol. 97, 408–417.

https://doi.org/10.1016/j.bcp.2015.07.028

Chen, A., Xu, J., Johnson, A.C., 2006. Curcumin inhibits human colon cancer cell

growth by suppressing gene expression of epidermal growth factor receptor

180

through reducing the activity of the transcription factor Egr-1. Oncogene 25,

278–287. https://doi.org/10.1038/sj.onc.1209019

Chen, X., Zong, C., Gao, Y., Cai, R., Fang, L., Lu, J., Liu, F., Qi, Y., 2014.

Curcumol exhibits anti-inflammatory properties by interfering with the JNK-

mediated AP-1 pathway in lipopolysaccharide-activated RAW264.7 cells.

Eur. J. Pharmacol. 723, 339–345.

https://doi.org/10.1016/j.ejphar.2013.11.007

Cheng, A.L., Hsu, C.H., Lin, J.K., Hsu, M.M., Ho, Y.F., Shen, T.S., Ko, J.Y., Lin,

J.T., Lin, B.R., Ming-Shiang, W., Yu, H.S., Jee, S.H., Chen, G.S., Chen,

T.M., Chen, C.A., Lai, M.K., Pu, Y.S., Pan, M.H., Wang, Y.J., Tsai, C.C.,

Hsieh, C.Y., 2001. Phase I clinical trial of curcumin, a chemopreventive

agent, in patients with high-risk or pre-malignant lesions. Anticancer Res. 21,

2895–2900.

Cheng, K.K., Yeung, C.F., Ho, S.W., Chow, S.F., Chow, A.H.L., Baum, L., 2013.

Highly stabilized curcumin nanoparticles tested in an in vitro blood-brain

barrier model and in Alzheimer’s disease Tg2576 mice. AAPS J. 15, 324–

336. https://doi.org/10.1208/s12248-012-9444-4

Cho, J.-W., Lee, K.-S., Kim, C.-W., 2007. Curcumin attenuates the expression of IL-

1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT

cells; NF-kappaB and MAPKs as potential upstream targets. Int. J. Mol. Med.

19, 469–474.

Choi, S.W., Kim, K.S., Shin, D.H., Yoo, H.Y., Choe, H., Ko, T.H., Youm, J.B., Kim,

W.K., Zhang, Y.H., Kim, S.J., 2013. Class 3 inhibition of hERG K+ channel

by caffeic acid phenethyl ester (CAPE) and curcumin. Pflugers Arch. 465,

1121–1134. https://doi.org/10.1007/s00424-013-1239-7

Chung, K.K., Zhang, Y., Lim, K.L., Tanaka, Y., Huang, H., Gao, J., Ross, C.A.,

Dawson, V.L., Dawson, T.M., 2001. Parkin ubiquitinates the alpha-

synuclein-interacting protein, synphilin-1: implications for Lewy-body

formation in Parkinson disease. Nat. Med. 7, 1144–1150.

https://doi.org/10.1038/nm1001-1144

Chung, Y.C., Ko, H.W., Bok, E., Park, E.S., Huh, S.H., Nam, J.H., Jin, B.K., 2010.

The role of neuroinflammation on the pathogenesis of Parkinson’s disease.

BMB Rep 43, 225–32.

Cicchetti, F., Drouin-Ouellet, J., Gross, R.E., 2009. Environmental toxins and

Parkinson’s disease: what have we learned from pesticide-induced animal

models? Trends Pharmacol. Sci. 30, 475–483.

https://doi.org/10.1016/j.tips.2009.06.005

181

Clementi, F., Fornasari, D., Gotti, C., 2000. Neuronal nicotinic receptors, important

new players in brain function. Eur J Pharmacol 393, 3–10.

Cohly, H.H., Taylor, A., Angel, M.F., Salahudeen, A.K., 1998. Effect of turmeric,

turmerin and curcumin on H2O2-induced renal epithelial (LLC-PK1) cell

injury. Free Radic. Biol. Med. 24, 49–54.

Cole, G.M., Teter, B., Frautschy, S.A., 2007. Neuroprotective effects of curcumin.

Adv. Exp. Med. Biol. 595, 197–212. https://doi.org/10.1007/978-0-387-

46401-5_8

Conney, A.H., Lysz, T., Ferraro, T., Abidi, T.F., Manchand, P.S., Laskin, J.D.,

Huang, M.T., 1991. Inhibitory effect of curcumin and some related dietary

compounds on tumor promotion and arachidonic acid metabolism in mouse

skin. Adv. Enzyme Regul. 31, 385–396.

Connolly, B.S., Lang, A.E., 2014. Pharmacological treatment of Parkinson disease: a

review. JAMA 311, 1670–1683. https://doi.org/10.1001/jama.2014.3654

Cormier, A., Morin, C., Zini, R., Tillement, J.P., Lagrue, G., 2003. Nicotine protects

rat brain mitochondria against experimental injuries. Neuropharmacology 44,

642–52.

Corradi, J., Andersen, N., Bouzat, C., 2011. A novel mechanism of modulation of 5-

HT₃A receptors by hydrocortisone. Biophys. J. 100, 42–51.

https://doi.org/10.1016/j.bpj.2010.10.046

Corradi, J., Bouzat, C., 2016. Understanding the Bases of Function and Modulation

of α7 Nicotinic Receptors: Implications for Drug Discovery. Mol. Pharmacol.

90, 288–299. https://doi.org/10.1124/mol.116.104240

Corrodi, H., Hanson, L.C., 1966. Central effects of an inhibitor of tyrosine

hydroxylation. Psychopharmacologia 10, 116–125.

Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N.,

Valera, S., Barkas, T., Ballivet, M., 1990. A neuronal nicotinic acetylcholine

receptor subunit (alpha 7) is developmentally regulated and forms a homo-

oligomeric channel blocked by alpha-BTX. Neuron 5, 847–856.

Creţu, E., Trifan, A., Vasincu, A., Miron, A., 2012. Plant-derived anticancer agents -

curcumin in cancer prevention and treatment. Rev. Med. Chir. Soc. Med. Nat.

Iasi 116, 1223–1229.

Dadhaniya, P., Patel, C., Muchhara, J., Bhadja, N., Mathuria, N., Vachhani, K., Soni,

M.G., 2011. Safety assessment of a solid lipid curcumin particle preparation:

acute and subchronic toxicity studies. Food Chem. Toxicol. Int. J. Publ. Br.

182

Ind. Biol. Res. Assoc. 49, 1834–1842.

https://doi.org/10.1016/j.fct.2011.05.001

Dajas-Bailador, F., Wonnacott, S., 2004. Nicotinic acetylcholine receptors and the

regulation of neuronal signalling. Trends Pharmacol Sci 25, 317–24.


Dani, J.A., 2001. Overview of nicotinic receptors and their roles in the central

nervous system. Biol Psychiatry 49, 166–74.

Dantuma, N.P., Bott, L.C., 2014. The ubiquitin-proteasome system in

neurodegenerative diseases: precipitating factor, yet part of the solution.

Front. Mol. Neurosci. 7, 70. https://doi.org/10.3389/fnmol.2014.00070

Darvesh, A.S., Carroll, R.T., Bishayee, A., Novotny, N.A., Geldenhuys, W.J., Van

der Schyf, C.J., 2012. Curcumin and neurodegenerative diseases: a

perspective. Expert Opin. Investig. Drugs 21, 1123–1140.

https://doi.org/10.1517/13543784.2012.693479

Dauer, W., Kholodilov, N., Vila, M., Trillat, A.-C., Goodchild, R., Larsen, K.E.,

Staal, R., Tieu, K., Schmitz, Y., Yuan, C.A., Rocha, M., Jackson-Lewis, V.,

Hersch, S., Sulzer, D., Przedborski, S., Burke, R., Hen, R., 2002. Resistance

of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc.

Natl. Acad. Sci. U. S. A. 99, 14524–14529.

https://doi.org/10.1073/pnas.172514599

Dauer, W., Przedborski, S., 2003. Parkinson’s disease: mechanisms and models.

Neuron 39, 889–909.

Davis, G.C., Williams, A.C., Markey, S.P., Ebert, M.H., Caine, E.D., Reichert, C.M.,

Kopin, I.J., 1979. Chronic Parkinsonism secondary to intravenous injection of

meperidine analogues. Psychiatry Res. 1, 249–254.

Dawson, T.M., Ko, H.S., Dawson, V.L., 2010. Genetic animal models of Parkinson’s

disease. Neuron 66, 646–661. https://doi.org/10.1016/j.neuron.2010.04.034

Day, B.J., Patel, M., Calavetta, L., Chang, L.-Y., Stamler, J.S., 1999. A mechanism

of paraquat toxicity involving nitric oxide synthase. Proc. Natl. Acad. Sci. U.

S. A. 96, 12760–12765.

de Lau, L.M.L., Breteler, M.M.B., 2006. Epidemiology of Parkinson’s disease.

Lancet Neurol. 5, 525–535. https://doi.org/10.1016/S1474-4422(06)70471-9

De Smet, F., Christopoulos, A., Carmeliet, P., 2014. Allosteric targeting of receptor

tyrosine kinases. Nat Biotechnol 32, 1113–20.

https://doi.org/10.1038/nbt.3028

183

Del Tredici, K., Braak, H., 2016. Review: Sporadic Parkinson’s disease:

development and distribution of α-synuclein pathology. Neuropathol. Appl.

Neurobiol. 42, 33–50. https://doi.org/10.1111/nan.12298

Deodhar, S.D., Sethi, R., Srimal, R.C., 1980. Preliminary study on antirheumatic

activity of curcumin (diferuloyl methane). Indian J. Med. Res. 71, 632–634.

Derecki, N.C., Cronk, J.C., Kipnis, J., 2013. The role of microglia in brain

maintenance: implications for Rett syndrome. Trends Immunol 34, 144–50.

https://doi.org/10.1016/j.it.2012.10.002

Descarries, L., Mechawar, N., 2000. Ultrastructural evidence for diffuse transmission

by monoamine and acetylcholine neurons of the central nervous system. Prog

Brain Res 125, 27–47. https://doi.org/10.1016/s0079-6123(00)25005-x

Dikshit, M., Rastogi, L., Shukla, R., Srimal, R.C., 1995. Prevention of ischaemia-

induced biochemical changes by curcumin & quinidine in the cat heart.

Indian J. Med. Res. 101, 31–35.

Doherty, K.M., van de Warrenburg, B.P., Peralta, M.C., Silveira-Moriyama, L.,

Azulay, J.-P., Gershanik, O.S., Bloem, B.R., 2011. Postural deformities in

Parkinson’s disease. Lancet Neurol. 10, 538–549.

https://doi.org/10.1016/S1474-4422(11)70067-9

Doty, R.L., Deems, D.A., Stellar, S., 1988. Olfactory dysfunction in parkinsonism: a

general deficit unrelated to neurologic signs, disease stage, or disease

duration. Neurology 38, 1237–1244.

Dunnett, S.B., Torres, E.M., 2011. Rotation in the 6-OHDA-Lesioned Rat, in:

Animal Models of Movement Disorders, Neuromethods. Humana Press, pp.

299–315. https://doi.org/10.1007/978-1-61779-298-4_15

Dyer, J.L., Khan, S.Z., Bilmen, J.G., Hawtin, S.R., Wheatley, M., Javed, M. -u.-H.,

Michelangeli, F., 2002. Curcumin: a new cell-permeant inhibitor of the

inositol 1,4,5-trisphosphate receptor. Cell Calcium 31, 45–52.

Elbaz, A., Moisan, F., 2008. Update in the epidemiology of Parkinson’s disease. Curr

Opin Neurol 21, 454–60. https://doi.org/10.1097/WCO.0b013e3283050461

Emborg, M.E., 2004. Evaluation of animal models of Parkinson’s disease for

neuroprotective strategies. J. Neurosci. Methods 139, 121–143.

https://doi.org/10.1016/j.jneumeth.2004.08.004

Enyeart, J.A., Liu, H., Enyeart, J.J., 2009. Curcumin inhibits ACTH- and angiotensin

II-stimulated cortisol secretion and Ca(v)3.2 current. J. Nat. Prod. 72, 1533–

1537. https://doi.org/10.1021/np900227x

184

Enyeart, J.A., Liu, H., Enyeart, J.J., 2008. Curcumin inhibits bTREK-1 K+ channels

and stimulates cortisol secretion from adrenocortical cells. Biochem.

Biophys. Res. Commun. 370, 623–628.

https://doi.org/10.1016/j.bbrc.2008.04.001

Epelbaum, R., Schaffer, M., Vizel, B., Badmaev, V., Bar-Sela, G., 2010. Curcumin

and gemcitabine in patients with advanced pancreatic cancer. Nutr. Cancer

62, 1137–1141. https://doi.org/10.1080/01635581.2010.513802

Exner, N., Lutz, A.K., Haass, C., Winklhofer, K.F., 2012. Mitochondrial dysfunction

in Parkinson’s disease: molecular mechanisms and pathophysiological

consequences. EMBO J. 31, 3038–3062.

https://doi.org/10.1038/emboj.2012.170

Fagerström, K.O., Pomerleau, O., Giordani, B., Stelson, F., 1994. Nicotine may

relieve symptoms of Parkinson’s disease. Psychopharmacology (Berl.) 116,

117–119.

Fahn, S., 2010. Parkinson’s disease: 10 years of progress, 1997-2007. Mov Disord 25

Suppl 1, S2-14. https://doi.org/10.1002/mds.22796

Fahn, S., 2008. How do you treat motor complications in Parkinson’s disease:

Medicine, surgery, or both? Ann Neurol 64 Suppl 2, S56-64.

https://doi.org/10.1002/ana.21453

Faull, R.L., Laverty, R., 1969. Changes in dopamine levels in the corpus striatum

following lesions in the substantia nigra. Exp. Neurol. 23, 332–340.

Feany, M.B., Bender, W.W., 2000. A Drosophila model of Parkinson’s disease.

Nature 404, 394–398. https://doi.org/10.1038/35006074

Ferger, B., Spratt, C., Earl, C.D., Teismann, P., Oertel, W.H., Kuschinsky, K., 1998.

Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-

induced neurotoxicity in vivo. Naunyn Schmiedebergs Arch Pharmacol 358,

351–9.

Fernagut, P.O., Hutson, C.B., Fleming, S.M., Tetreaut, N.A., Salcedo, J., Masliah, E.,

Chesselet, M.F., 2007. Behavioral and histopathological consequences of

paraquat intoxication in mice: effects of alpha-synuclein over-expression.

Synap. N. Y. N 61, 991–1001. https://doi.org/10.1002/syn.20456

Ferreira, J.J., Katzenschlager, R., Bloem, B.R., Bonuccelli, U., Burn, D., Deuschl,

G., Dietrichs, E., Fabbrini, G., Friedman, A., Kanovsky, P., Kostic, V.,

Nieuwboer, A., Odin, P., Poewe, W., Rascol, O., Sampaio, C., Schüpbach,

M., Tolosa, E., Trenkwalder, C., Schapira, A., Berardelli, A., Oertel, W.H.,

185

2013. Summary of the recommendations of the EFNS/MDS-ES review on

therapeutic management of Parkinson’s disease. Eur. J. Neurol. 20, 5–15.

https://doi.org/10.1111/j.1468-1331.2012.03866.x

Fleming, S.M., Zhu, C., Fernagut, P.-O., Mehta, A., DiCarlo, C.D., Seaman, R.L.,

Chesselet, M.-F., 2004. Behavioral and immunohistochemical effects of

chronic intravenous and subcutaneous infusions of varying doses of rotenone.

Exp. Neurol. 187, 418–429. https://doi.org/10.1016/j.expneurol.2004.01.023

Flor, P.J., Acher, F.C., 2012. Orthosteric versus allosteric GPCR activation: the great

challenge of group-III mGluRs. Biochem Pharmacol 84, 414–24.


Formaggio, E., Fazzini, F., Dalfini, A.C., Di Chio, M., Cantu, C., Decimo, I., Fiorini,

Z., Fumagalli, G., Chiamulera, C., 2010. Nicotine increases the expression of

neurotrophin receptor tyrosine kinase receptor A in basal forebrain

cholinergic neurons. Neuroscience 166, 580–9.

https://doi.org/10.1016/j.neuroscience.2009.12.073

Forster, G.L., Blaha, C.D., 2003. Pedunculopontine tegmental stimulation evokes

striatal dopamine efflux by activation of acetylcholine and glutamate

receptors in the midbrain and pons of the rat. Eur J Neurosci 17, 751–62.

Fox, S.H., Chuang, R., Brotchie, J.M., 2009. Serotonin and Parkinson’s disease: On

movement, mood, and madness. Mov. Disord. Off. J. Mov. Disord. Soc. 24,

1255–1266. https://doi.org/10.1002/mds.22473

Fox, S.H., Chuang, R., Brotchie, J.M., 2008. Parkinson’s disease–opportunities for

novel therapeutics to reduce the problems of levodopa therapy. Prog Brain

Res 172, 479–94. https://doi.org/10.1016/s0079-6123(08)00923-0

Franco-Iborra, S., Vila, M., Perier, C., 2016. The Parkinson Disease Mitochondrial

Hypothesis: Where Are We at? Neurosci. Rev. J. Bringing Neurobiol. Neurol.

Psychiatry 22, 266–277. https://doi.org/10.1177/1073858415574600

Freitas, K., Carroll, F.I., Damaj, M.I., 2013a. The antinociceptive effects of nicotinic

receptors α7-positive allosteric modulators in murine acute and tonic pain

models. J. Pharmacol. Exp. Ther. 344, 264–275.

https://doi.org/10.1124/jpet.112.197871

Freitas, K., Ghosh, S., Ivy Carroll, F., Lichtman, A.H., Imad Damaj, M., 2013b.

Effects of α7 positive allosteric modulators in murine inflammatory and

chronic neuropathic pain models. Neuropharmacology 65, 156–164.


186

Friesner, R.A., Murphy, R.B., Repasky, M.P., Frye, L.L., Greenwood, J.R., Halgren,

T.A., Sanschagrin, P.C., Mainz, D.T., 2006. Extra precision glide: docking

and scoring incorporating a model of hydrophobic enclosure for protein-

ligand complexes. J. Med. Chem. 49, 6177–6196.

https://doi.org/10.1021/jm051256o

Gandhi, S., Wood-Kaczmar, A., Yao, Z., Plun-Favreau, H., Deas, E., Klupsch, K.,

Downward, J., Latchman, D.S., Tabrizi, S.J., Wood, N.W., Duchen, M.R.,

Abramov, A.Y., 2009. PINK1-associated Parkinson’s disease is caused by

neuronal vulnerability to calcium-induced cell death. Mol. Cell 33, 627–638.

https://doi.org/10.1016/j.molcel.2009.02.013

Garcea, G., Berry, D.P., Jones, D.J.L., Singh, R., Dennison, A.R., Farmer, P.B.,

Sharma, R.A., Steward, W.P., Gescher, A.J., 2005. Consumption of the

putative chemopreventive agent curcumin by cancer patients: assessment of

curcumin levels in the colorectum and their pharmacodynamic consequences.

Cancer Epidemiol. Biomark. Prev. Publ. Am. Assoc. Cancer Res.

Cosponsored Am. Soc. Prev. Oncol. 14, 120–125.

Garcea, G., Jones, D.J.L., Singh, R., Dennison, A.R., Farmer, P.B., Sharma, R.A.,

Steward, W.P., Gescher, A.J., Berry, D.P., 2004. Detection of curcumin and

its metabolites in hepatic tissue and portal blood of patients following oral

administration. Br. J. Cancer 90, 1011–1015.

https://doi.org/10.1038/sj.bjc.6601623

Gatto, G.J., Bohme, G.A., Caldwell, W.S., Letchworth, S.R., Traina, V.M., Obinu,

M.C., Laville, M., Reibaud, M., Pradier, L., Dunbar, G., Bencherif, M., 2004.

TC-1734: an orally active neuronal nicotinic acetylcholine receptor

modulator with antidepressant, neuroprotective and long-lasting cognitive

effects. CNS Drug Rev. 10, 147–166.

Gault, L.M., Ritchie, C.W., Robieson, W.Z., Pritchett, Y., Othman, A.A., Lenz, R.A.,

2015. A phase 2 randomized, controlled trial of the α7 agonist ABT-126 in

mild-to-moderate Alzheimer’s dementia. Alzheimers Dement. N. Y. N 1, 81–

90. https://doi.org/10.1016/j.trci.2015.06.001

Gazewood, J.D., Richards, D.R., Clebak, K., 2013a. Parkinson disease: an update.

Am. Fam. Physician 87, 267–273.

Gazewood, J.D., Richards, D.R., Clebak, K., 2013b. Parkinson Disease: An Update.

Am. Fam. Physician 87, 267–273.

Gehrmann, J., Matsumoto, Y., Kreutzberg, G.W., 1995. Microglia: intrinsic

immuneffector cell of the brain. Brain Res Brain Res Rev 20, 269–87.

187

Gerfen, C.R., Herkenham, M., Thibault, J., 1987. The neostriatal mosaic: II. Patch-

and matrix-directed mesostriatal dopaminergic and non-dopaminergic

systems. J Neurosci 7, 3915–34.

Ghalaut, V.S., Sangwan, L., Dahiya, K., Ghalaut, P.S., Dhankhar, R., Saharan, R.,

2012. Effect of imatinib therapy with and without turmeric powder on nitric

oxide levels in chronic myeloid leukemia. J. Oncol. Pharm. Pract. Off. Publ.

Int. Soc. Oncol. Pharm. Pract. 18, 186–190.

https://doi.org/10.1177/1078155211416530

Gill, J.K., Savolainen, M., Young, G.T., Zwart, R., Sher, E., Millar, N.S., 2011.

Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric

transmembrane site. Proc Natl Acad Sci U A 108, 5867–72.


Giovanni, A., Sieber, B.A., Heikkila, R.E., Sonsalla, P.K., 1994. Studies on species

sensitivity to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine. Part 1: Systemic administration. J. Pharmacol. Exp. Ther.

270, 1000–1007.

Goedert, M., Spillantini, M.G., Del Tredici, K., Braak, H., 2013. 100 years of Lewy

pathology. Nat. Rev. Neurol. 9, 13–24.

https://doi.org/10.1038/nrneurol.2012.242

Goel, A., Aggarwal, B.B., 2010. Curcumin, the golden spice from Indian saffron, is a

chemosensitizer and radiosensitizer for tumors and chemoprotector and

radioprotector for normal organs. Nutr. Cancer 62, 919–930.

https://doi.org/10.1080/01635581.2010.509835

Goel, A., Kunnumakkara, A.B., Aggarwal, B.B., 2008. Curcumin as “Curecumin”:

from kitchen to clinic. Biochem. Pharmacol. 75, 787–809.


Goetz, C.G., Fahn, S., Martinez-Martin, P., Poewe, W., Sampaio, C., Stebbins, G.T.,

Stern, M.B., Tilley, B.C., Dodel, R., Dubois, B., Holloway, R., Jankovic, J.,

Kulisevsky, J., Lang, A.E., Lees, A., Leurgans, S., LeWitt, P.A., Nyenhuis,

D., Olanow, C.W., Rascol, O., Schrag, A., Teresi, J.A., Van Hilten, J.J.,

LaPelle, N., 2007. Movement Disorder Society-sponsored revision of the

Unified Parkinson’s Disease Rating Scale (MDS-UPDRS): Process, format,

and clinimetric testing plan. Mov. Disord. Off. J. Mov. Disord. Soc. 22, 41–

47. https://doi.org/10.1002/mds.21198

Goetz, C.G., Poewe, W., Rascol, O., Sampaio, C., 2005. Evidence-based medical

review update: pharmacological and surgical treatments of Parkinson’s

188

disease: 2001 to 2004. Mov. Disord. Off. J. Mov. Disord. Soc. 20, 523–539.


Goldberg, M.S., Fleming, S.M., Palacino, J.J., Cepeda, C., Lam, H.A., Bhatnagar, A.,

Meloni, E.G., Wu, N., Ackerson, L.C., Klapstein, G.J., Gajendiran, M., Roth,

B.L., Chesselet, M.-F., Maidment, N.T., Levine, M.S., Shen, J., 2003. Parkin-

deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic

neurons. J. Biol. Chem. 278, 43628–43635.

https://doi.org/10.1074/jbc.M308947200

Goldberg, M.S., Pisani, A., Haburcak, M., Vortherms, T.A., Kitada, T., Costa, C.,

Tong, Y., Martella, G., Tscherter, A., Martins, A., Bernardi, G., Roth, B.L.,

Pothos, E.N., Calabresi, P., Shen, J., 2005. Nigrostriatal dopaminergic

deficits and hypokinesia caused by inactivation of the familial Parkinsonism-

linked gene DJ-1. Neuron 45, 489–496.

https://doi.org/10.1016/j.neuron.2005.01.041

Gorell, J.M., Rybicki, B.A., Johnson, C.C., Peterson, E.L., 1999. Smoking and

Parkinson’s disease: a dose-response relationship. Neurology 52, 115–9.

Grady, S.R., Salminen, O., Laverty, D.C., Whiteaker, P., McIntosh, J.M., Collins,

A.C., Marks, M.J., 2007. The subtypes of nicotinic acetylcholine receptors on

dopaminergic terminals of mouse striatum. Biochem Pharmacol 74, 1235–46.


Graham, D.G., 1978. Oxidative pathways for catecholamines in the genesis of

neuromelanin and cytotoxic quinones. Mol. Pharmacol. 14, 633–643.

Grant, H., Lantos, P.L., Parkinson, C., 1980. Cerebral damage in paraquat poisoning.

Histopathology 4, 185–195.

Greenamyre, J.T., Cannon, J.R., Drolet, R., Mastroberardino, P.-G., 2010. Lessons

from the rotenone model of Parkinson’s disease. Trends Pharmacol. Sci. 31,

141–142; author reply 142-143. https://doi.org/10.1016/j.tips.2009.12.006

Grosset, D.G., Macphee, G.J.A., Nairn, M., Guideline Development Group, 2010.

Diagnosis and pharmacological management of Parkinson’s disease:

summary of SIGN guidelines. BMJ 340, b5614.

https://doi.org/10.1136/bmj.b5614

Guan, Z.Z., Nordberg, A., Mousavi, M., Rinne, J.O., Hellstrom-Lindahl, E., 2002.

Selective changes in the levels of nicotinic acetylcholine receptor protein and

of corresponding mRNA species in the brains of patients with Parkinson’s

disease. Brain Res 956, 358–66.

189

Guerra-Álvarez, M., Moreno-Ortega, A.J., Navarro, E., Fernández-Morales, J.C.,

Egea, J., López, M.G., Cano-Abad, M.F., 2015. Positive allosteric modulation

of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release

from the endoplasmic reticulum. J. Neurochem. 133, 309–319.

https://doi.org/10.1111/jnc.13049

Gupta, S.C., Patchva, S., Koh, W., Aggarwal, B.B., 2012. Discovery of curcumin, a

component of golden spice, and its miraculous biological activities. Clin.

Exp. Pharmacol. Physiol. 39, 283–299. https://doi.org/10.1111/j.1440-

1681.2011.05648.x

Halliday, G., Herrero, M.T., Murphy, K., McCann, H., Ros-Bernal, F., Barcia, C.,

Mori, H., Blesa, F.J., Obeso, J.A., 2009. No Lewy pathology in monkeys with

over 10 years of severe MPTP Parkinsonism. Mov. Disord. Off. J. Mov.

Disord. Soc. 24, 1519–1523. https://doi.org/10.1002/mds.22481

Hartzell, C., Putzier, I., Arreola, J., 2005. Calcium-activated chloride channels.

Annu. Rev. Physiol. 67, 719–758.

https://doi.org/10.1146/annurev.physiol.67.032003.154341

Hasima, N., Aggarwal, B.B., 2012. Cancer-linked targets modulated by curcumin.

Int. J. Biochem. Mol. Biol. 3, 328–351.

Healy, D.G., Falchi, M., O’Sullivan, S.S., Bonifati, V., Durr, A., Bressman, S., Brice,

A., Aasly, J., Zabetian, C.P., Goldwurm, S., Ferreira, J.J., Tolosa, E., Kay,

D.M., Klein, C., Williams, D.R., Marras, C., Lang, A.E., Wszolek, Z.K.,

Berciano, J., Schapira, A.H.V., Lynch, T., Bhatia, K.P., Gasser, T., Lees,

A.J., Wood, N.W., International LRRK2 Consortium, 2008. Phenotype,

genotype, and worldwide genetic penetrance of LRRK2-associated

Parkinson’s disease: a case-control study. Lancet Neurol. 7, 583–590.

https://doi.org/10.1016/S1474-4422(08)70117-0

Hefti, F., Melamed, E., Sahakian, B.J., Wurtman, R.J., 1980a. Circling behavior in

rats with partial, unilateral nigro-striatal lesions: effect of amphetamine,

apomorphine, and DOPA. Pharmacol. Biochem. Behav. 12, 185–188.

Hefti, F., Melamed, E., Wurtman, R.J., 1980b. Partial lesions of the dopaminergic

nigrostriatal system in rat brain: biochemical characterization. Brain Res.

195, 123–137.

Hendrickson, L.M., Guildford, M.J., Tapper, A.R., 2013. Neuronal nicotinic

acetylcholine receptors: common molecular substrates of nicotine and alcohol

dependence. Front Psychiatry 4, 29. https://doi.org/10.3389/fpsyt.2013.00029

190

Hess, A., Desiderio, C., McAuliffe, W.G., 1990. Acute neuropathological changes in

the caudate nucleus caused by MPTP and methamphetamine:

immunohistochemical studies. J. Neurocytol. 19, 338–342.

Hickey, P., Stacy, M., 2013. AAV2-neurturin (CERE-120) for Parkinson’s disease.

Expert Opin Biol Ther 13, 137–45.

https://doi.org/10.1517/14712598.2013.754420

Hinkle, K.M., Yue, M., Behrouz, B., Dächsel, J.C., Lincoln, S.J., Bowles, E.E.,

Beevers, J.E., Dugger, B., Winner, B., Prots, I., Kent, C.B., Nishioka, K., Lin,

W.-L., Dickson, D.W., Janus, C.J., Farrer, M.J., Melrose, H.L., 2012. LRRK2

knockout mice have an intact dopaminergic system but display alterations in

exploratory and motor co-ordination behaviors. Mol. Neurodegener. 7, 25.

https://doi.org/10.1186/1750-1326-7-25

Hirayama, M., 2006. Sweating dysfunctions in Parkinson’s disease. J. Neurol. 253

Suppl 7, VII42-47. https://doi.org/10.1007/s00415-006-7010-7

Hisahara, S., Shimohama, S., 2011. Dopamine Receptors and Parkinson’s Disease.

Int. J. Med. Chem. 2011. https://doi.org/10.1155/2011/403039

Hoehle, S.I., Pfeiffer, E., Sólyom, A.M., Metzler, M., 2006. Metabolism of

curcuminoids in tissue slices and subcellular fractions from rat liver. J. Agric.

Food Chem. 54, 756–764. https://doi.org/10.1021/jf058146a

Holder, G.M., Plummer, J.L., Ryan, A.J., 1978. The metabolism and excretion of

curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)

in the rat. Xenobiotica Fate Foreign Compd. Biol. Syst. 8, 761–768.

Home - ClinicalTrials.gov [WWW Document], n.d. URL https://clinicaltrials.gov/

(accessed 12.15.18).

Hong, D.H., Son, Y.K., Choi, I.-W., Park, W.S., 2013. The inhibitory effect of

curcumin on voltage-dependent K+ channels in rabbit coronary arterial

smooth muscle cells. Biochem. Biophys. Res. Commun. 430, 307–312.


Hosur, V., Loring, R.H., 2011. alpha4beta2 nicotinic receptors partially mediate anti-

inflammatory effects through Janus kinase 2-signal transducer and activator

of transcription 3 but not calcium or cAMP signaling. Mol Pharmacol 79,

167–74. https://doi.org/10.1124/mol.110.066381

Hu, C.-W., Sheng, Y., Zhang, Q., Liu, H.-B., Xie, X., Ma, W.-C., Huo, R., Dong, D.-

L., 2012. Curcumin inhibits hERG potassium channels in vitro. Toxicol. Lett.

208, 192–196. https://doi.org/10.1016/j.toxlet.2011.11.005

191

Huang, H.C., Jan, T.R., Yeh, S.F., 1992. Inhibitory effect of curcumin, an anti-

inflammatory agent, on vascular smooth muscle cell proliferation. Eur. J.

Pharmacol. 221, 381–384.

Huang, M., Felix, A.R., Flood, D.G., Bhuvaneswaran, C., Hilt, D., Koenig, G.,

Meltzer, H.Y., 2014. The novel α7 nicotinic acetylcholine receptor agonist

EVP-6124 enhances dopamine, acetylcholine, and glutamate efflux in rat

cortex and nucleus accumbens. Psychopharmacology (Berl.) 231, 4541–4551.

https://doi.org/10.1007/s00213-014-3596-0

Hudson, J.L., van Horne, C.G., Strömberg, I., Brock, S., Clayton, J., Masserano, J.,

Hoffer, B.J., Gerhardt, G.A., 1993. Correlation of apomorphine- and

amphetamine-induced turning with nigrostriatal dopamine content in

unilateral 6-hydroxydopamine lesioned rats. Brain Res. 626, 167–174.

Hughes, A.J., Daniel, S.E., Kilford, L., Lees, A.J., 1992. Accuracy of clinical

diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of

100 cases. J. Neurol. Neurosurg. Psychiatry 55, 181–184.

Hughes, J.T., 1988. Brain damage due to paraquat poisoning: a fatal case with

neuropathological examination of the brain. Neurotoxicology 9, 243–248.

Huot, P., Johnston, T.H., Koprich, J.B., Fox, S.H., Brotchie, J.M., 2013. The

pharmacology of L-DOPA-induced dyskinesia in Parkinson’s disease.

Pharmacol Rev 65, 171–222. https://doi.org/10.1124/pr.111.005678

Hurst, R., Rollema, H., Bertrand, D., 2013. Nicotinic acetylcholine receptors: from

basic science to therapeutics. Pharmacol Ther 137, 22–54.

https://doi.org/10.1016/j.pharmthera.2012.08.012

Ibrahim, A., El-Meligy, A., Lungu, G., Fetaih, H., Dessouki, A., Stoica, G.,

Barhoumi, R., 2011. Curcumin induces apoptosis in a murine mammary

gland adenocarcinoma cell line through the mitochondrial pathway. Eur. J.

Pharmacol. 668, 127–132. https://doi.org/10.1016/j.ejphar.2011.06.048

Iravani, M.M., McCreary, A.C., Jenner, P., 2012. Striatal plasticity in Parkinson’s

disease and L-dopa induced dyskinesia. Park. Relat Disord 18 Suppl 1, S123-

5. https://doi.org/10.1016/s1353-8020(11)70038-4

Itier, J.-M., Ibanez, P., Mena, M.A., Abbas, N., Cohen-Salmon, C., Bohme, G.A.,

Laville, M., Pratt, J., Corti, O., Pradier, L., Ret, G., Joubert, C., Periquet, M.,

Araujo, F., Negroni, J., Casarejos, M.J., Canals, S., Solano, R., Serrano, A.,

Gallego, E., Sanchez, M., Denefle, P., Benavides, J., Tremp, G., Rooney,

T.A., Brice, A., Garcia de Yebenes, J., 2003. Parkin gene inactivation alters

192

behaviour and dopamine neurotransmission in the mouse. Hum. Mol. Genet.

12, 2277–2291. https://doi.org/10.1093/hmg/ddg239

Jankovic, J., 2008. Parkinson’s disease: clinical features and diagnosis. J. Neurol.

Neurosurg. Psychiatry 79, 368–376.

https://doi.org/10.1136/jnnp.2007.131045

Jayakumary, M., Jayadevan, S., Ranade, A.V., Mathew, E., 2010. Prevalence and

pattern of dokha use among medical and allied health students in Ajman,

United Arab Emirates. Asian Pac J Cancer Prev 11, 1547–9.

Jenner, P., 2008. Molecular mechanisms of L-DOPA-induced dyskinesia. Nat Rev

Neurosci 9, 665–77. https://doi.org/10.1038/nrn2471

Jensen, A.A., Frolund, B., Liljefors, T., Krogsgaard-Larsen, P., 2005. Neuronal

nicotinic acetylcholine receptors: structural revelations, target identifications,

and therapeutic inspirations. J Med Chem 48, 4705–45.

https://doi.org/10.1021/jm040219e

Jeon, B.S., Jackson-Lewis, V., Burke, R.E., 1995. 6-Hydroxydopamine lesion of the

rat substantia nigra: time course and morphology of cell death. Neurodegener.

J. Neurodegener. Disord. Neuroprotection Neuroregeneration 4, 131–137.

Jeong, G.S., Oh, G.S., Pae, H.-O., Jeong, S.-O., Kim, Y.-C., Shin, M.-K., Seo, B.Y.,

Han, S.Y., Lee, H.S., Jeong, J.-G., Koh, J.-S., Chung, H.-T., 2006.

Comparative effects of curcuminoids on endothelial heme oxygenase-1

expression: ortho-methoxy groups are essential to enhance heme oxygenase

activity and protection. Exp. Mol. Med. 38, 393–400.

https://doi.org/10.1038/emm.2006.46

Joe, B., Vijaykumar, M., Lokesh, B.R., 2004. Biological properties of curcumin-

cellular and molecular mechanisms of action. Crit. Rev. Food Sci. Nutr. 44,

97–111. https://doi.org/10.1080/10408690490424702

Johnston, T.H., Huot, P., Fox, S.H., Koprich, J.B., Szeliga, K.T., James, J.W., Graef,

J.D., Letchworth, S.R., Jordan, K.G., Hill, M.P., Brotchie, J.M., 2013. TC-

8831, a nicotinic acetylcholine receptor agonist, reduces L-DOPA-induced

dyskinesia in the MPTP macaque. Neuropharmacology 73, 337–347.


Jones, A.K., Buckingham, S.D., Sattelle, D.B., 2010. Proteins interacting with

nicotinic acetylcholine receptors: expanding functional and therapeutic

horizons. Trends Pharmacol. Sci. 31, 455–462.


193

Joselin, A.P., Hewitt, S.J., Callaghan, S.M., Kim, R.H., Chung, Y.-H., Mak, T.W.,

Shen, J., Slack, R.S., Park, D.S., 2012. ROS-dependent regulation of Parkin

and DJ-1 localization during oxidative stress in neurons. Hum. Mol. Genet.

21, 4888–4903. https://doi.org/10.1093/hmg/dds325

Jost, W.H., 2003. Autonomic dysfunctions in idiopathic Parkinson’s disease. J.

Neurol. 250 Suppl 1, I28-30. https://doi.org/10.1007/s00415-003-1105-z

Jurado-Coronel, J.C., Avila-Rodriguez, M., Capani, F., Gonzalez, J., Moran, V.E.,

Barreto, G.E., 2016. Targeting the Nicotinic Acetylcholine Receptors

(nAChRs) in Astrocytes as a Potential Therapeutic Target in Parkinson’s

Disease. Curr. Pharm. Des. 22, 1305–1311.

Jurenka, J.S., 2009. Anti-inflammatory properties of curcumin, a major constituent of

Curcuma longa: a review of preclinical and clinical research. Altern. Med.

Rev. J. Clin. Ther. 14, 141–153.

Kalappa, B.I., Sun, F., Johnson, S.R., Jin, K., Uteshev, V.V., 2013. A positive

allosteric modulator of α7 nAChRs augments neuroprotective effects of

endogenous nicotinic agonists in cerebral ischaemia. Br. J. Pharmacol. 169,

1862–1878. https://doi.org/10.1111/bph.12247

Kalf, J.G., Bloem, B.R., Munneke, M., 2012. Diurnal and nocturnal drooling in

Parkinson’s disease. J. Neurol. 259, 119–123. https://doi.org/10.1007/s00415-

011-6138-2

Kalf, J.G., Borm, G.F., de Swart, B.J., Bloem, B.R., Zwarts, M.J., Munneke, M.,

2011. Reproducibility and validity of patient-rated assessment of speech,

swallowing, and saliva control in Parkinson’s disease. Arch. Phys. Med.

Rehabil. 92, 1152–1158. https://doi.org/10.1016/j.apmr.2011.02.011

Kawamata, J., Shimohama, S., 2011. Stimulating nicotinic receptors trigger multiple

pathways attenuating cytotoxicity in models of Alzheimer’s and Parkinson’s

diseases. J Alzheimers Dis 24 Suppl 2, 95–109. https://doi.org/10.3233/jad-

2011-110173

Kerr, D.S., 2010. Treatment of mitochondrial electron transport chain disorders: a

review of clinical trials over the past decade. Mol Genet Metab 99, 246–55.

https://doi.org/10.1016/j.ymgme.2009.11.005

Khatri, D.K., Juvekar, A.R., 2016. Neuroprotective effect of curcumin as evinced by

abrogation of rotenone-induced motor deficits, oxidative and mitochondrial

dysfunctions in mouse model of Parkinson’s disease. Pharmacol. Biochem.

Behav. 150–151, 39–47. https://doi.org/10.1016/j.pbb.2016.09.002

194

Khuwaja, G., Khan, M.M., Ishrat, T., Ahmad, A., Raza, S.S., Ashafaq, M., Javed, H.,

Khan, M.B., Khan, A., Vaibhav, K., Safhi, M.M., Islam, F., 2011.

Neuroprotective effects of curcumin on 6-hydroxydopamine-induced

Parkinsonism in rats: behavioral, neurochemical and immunohistochemical

studies. Brain Res. 1368, 254–263.

https://doi.org/10.1016/j.brainres.2010.10.023

Kim, R.H., Smith, P.D., Aleyasin, H., Hayley, S., Mount, M.P., Pownall, S.,

Wakeham, A., You-Ten, A.J., Kalia, S.K., Horne, P., Westaway, D., Lozano,

A.M., Anisman, H., Park, D.S., Mak, T.W., 2005. Hypersensitivity of DJ-1-

deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and

oxidative stress. Proc. Natl. Acad. Sci. U. S. A. 102, 5215–5220.


Kim, S.G., Veena, M.S., Basak, S.K., Han, E., Tajima, T., Gjertson, D.W., Starr, J.,

Eidelman, O., Pollard, H.B., Srivastava, M., Srivatsan, E.S., Wang, M.B.,

2011. Curcumin treatment suppresses IKKβ kinase activity of salivary cells

of patients with head and neck cancer: a pilot study. Clin. Cancer Res. Off. J.

Am. Assoc. Cancer Res. 17, 5953–5961. https://doi.org/10.1158/1078-

0432.CCR-11-1272

King, J.R., Nordman, J.C., Bridges, S.P., Lin, M.-K., Kabbani, N., 2015.

Identification and Characterization of a G Protein-binding Cluster in α7

Nicotinic Acetylcholine Receptors. J. Biol. Chem. 290, 20060–20070.

https://doi.org/10.1074/jbc.M115.647040

Kinon, B.J., Millen, B.A., Zhang, L., McKinzie, D.L., 2015. Exploratory analysis for

a targeted patient population responsive to the metabotropic glutamate 2/3

receptor agonist pomaglumetad methionil in schizophrenia. Biol. Psychiatry

78, 754–762. https://doi.org/10.1016/j.biopsych.2015.03.016

Kirik, D., Rosenblad, C., Björklund, A., 1998. Characterization of behavioral and

neurodegenerative changes following partial lesions of the nigrostriatal

dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp.

Neurol. 152, 259–277. https://doi.org/10.1006/exnr.1998.6848

Kiso, Y., Suzuki, Y., Watanabe, N., Oshima, Y., Hikino, H., 1983. Antihepatotoxic

principles of Curcuma longa rhizomes. Planta Med. 49, 185–187.

Kitada, T., Pisani, A., Karouani, M., Haburcak, M., Martella, G., Tscherter, A.,

Platania, P., Wu, B., Pothos, E.N., Shen, J., 2009. Impaired dopamine release

and synaptic plasticity in the striatum of parkin-/- mice. J. Neurochem. 110,

613–621. https://doi.org/10.1111/j.1471-4159.2009.06152.x

195

Kiuchi, F., Goto, Y., Sugimoto, N., Akao, N., Kondo, K., Tsuda, Y., 1993.

Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem.

Pharm. Bull. (Tokyo) 41, 1640–1643.

Kozlowski, D.A., Miljan, E.A., Bremer, E.G., Harrod, C.G., Gerin, C., Connor, B.,

George, D., Larson, B., Bohn, M.C., 2004. Quantitative analyses of

GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the

nigrostriatal system reveal bilateral compensatory changes following

unilateral 6-OHDA lesions in the rat. Brain Res. 1016, 170–181.

https://doi.org/10.1016/j.brainres.2004.05.003

Krasnova, I.N., Cadet, J.L., 2009. Methamphetamine toxicity and messengers of

death. Brain Res. Rev. 60, 379–407.

https://doi.org/10.1016/j.brainresrev.2009.03.002

Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS.

Trends Neurosci 19, 312–8.

Krüger, R., Kuhn, W., Müller, T., Woitalla, D., Graeber, M., Kösel, S., Przuntek, H.,

Epplen, J.T., Schöls, L., Riess, O., 1998. Ala30Pro mutation in the gene

encoding alpha-synuclein in Parkinson’s disease. Nat. Genet. 18, 106–108.

https://doi.org/10.1038/ng0298-106

Kunnumakkara, A.B., Bordoloi, D., Padmavathi, G., Monisha, J., Roy, N.K., Prasad,

S., Aggarwal, B.B., 2016. Curcumin, the golden nutraceutical: multitargeting

for multiple chronic diseases. Br. J. Pharmacol.

https://doi.org/10.1111/bph.13621

Kuttan, R., Sudheeran, P.C., Josph, C.D., 1987. Turmeric and curcumin as topical

agents in cancer therapy. Tumori 73, 29–31.

Lahrmann, H., Cortelli, P., Hilz, M., Mathias, C.J., Struhal, W., Tassinari, M., 2006.

EFNS guidelines on the diagnosis and management of orthostatic

hypotension. Eur. J. Neurol. 13, 930–936. https://doi.org/10.1111/j.1468-

1331.2006.01512.x

Langley, J.N., 1905. On the reaction of cells and of nerve-endings to certain poisons,

chiefly as regards the reaction of striated muscle to nicotine and to curari. J.

Physiol. 33, 374–413.

Langston, J.W., Ballard, P., Tetrud, J.W., Irwin, I., 1983. Chronic Parkinsonism in

humans due to a product of meperidine-analog synthesis. Science 219, 979–

980.

196

Langston, J.W., Forno, L.S., Tetrud, J., Reeves, A.G., Kaplan, J.A., Karluk, D., 1999.

Evidence of active nerve cell degeneration in the substantia nigra of humans

years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure. Ann.

Neurol. 46, 598–605.

Lao, C.D., Ruffin, M.T., Normolle, D., Heath, D.D., Murray, S.I., Bailey, J.M.,

Boggs, M.E., Crowell, J., Rock, C.L., Brenner, D.E., 2006. Dose escalation of

a curcuminoid formulation. BMC Complement. Altern. Med. 6, 10.

https://doi.org/10.1186/1472-6882-6-10

Le Novere, N., Zoli, M., Changeux, J.P., 1996. Neuronal nicotinic receptor alpha 6

subunit mRNA is selectively concentrated in catecholaminergic nuclei of the

rat brain. Eur J Neurosci 8, 2428–39.

Lewis, A.S., van Schalkwyk, G.I., Bloch, M.H., 2017. Alpha-7 nicotinic agonists for

cognitive deficits in neuropsychiatric disorders: A translational meta-analysis

of rodent and human studies. Prog. Neuropsychopharmacol. Biol. Psychiatry

75, 45–53. https://doi.org/10.1016/j.pnpbp.2017.01.001

Li, C., Zhang, Y., Su, T., Feng, L., Long, Y., Chen, Z., 2012. Silica-coated flexible

liposomes as a nanohybrid delivery system for enhanced oral bioavailability

of curcumin. Int. J. Nanomedicine 7, 5995–6002.

https://doi.org/10.2147/IJN.S38043

Li, Y., Revalde, J., Paxton, J.W., 2017. The effects of dietary and herbal

phytochemicals on drug transporters. Adv. Drug Deliv. Rev. 116, 45–62.

https://doi.org/10.1016/j.addr.2016.09.004

Lian, Y.-T., Yang, X.-F., Wang, Z.-H., Yang, Yong, Yang, Ying, Shu, Y.-W.,

Cheng, L.-X., Liu, K., 2013. Curcumin serves as a human kv1.3 blocker to

inhibit effector memory T lymphocyte activities. Phytother. Res. PTR 27,

1321–1327. https://doi.org/10.1002/ptr.4863

LigPrep | Schrödinger [WWW Document], n.d. URL

https://www.schrodinger.com/ligprep (accessed 1.15.17).

Limtrakul, P., Lipigorngoson, S., Namwong, O., Apisariyakul, A., Dunn, F.W., 1997.

Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer

Lett. 116, 197–203.

Linazasoro, G., Van Blercom, N., Ugedo, L., Ruiz Ortega, J.A., 2008.

Pharmacological treatment of Parkinson’s disease: life beyond dopamine

D2/D3 receptors? J Neural Transm Vienna 115, 431–41.

https://doi.org/10.1007/s00702-007-0852-z

197

Lindstrom, J.M., 2003. Nicotinic acetylcholine receptors of muscles and nerves:

comparison of their structures, functional roles, and vulnerability to

pathology. Ann N Acad Sci 998, 41–52.

Liu, B., Hong, J.S., 2003. Role of microglia in inflammation-mediated

neurodegenerative diseases: mechanisms and strategies for therapeutic

intervention. J Pharmacol Exp Ther 304, 1–7.

https://doi.org/10.1124/jpet.102.035048

Liu, H., Danthi, S.J., Enyeart, J.J., 2006. Curcumin potently blocks Kv1.4 potassium

channels. Biochem. Biophys. Res. Commun. 344, 1161–1165.


Liu, K., Gui, B., Sun, Y., Shi, N., Gu, Z., Zhang, T., Sun, X., 2013. Inhibition of L-

type Ca(2+) channels by curcumin requires a novel protein kinase-theta

isoform in rat hippocampal neurons. Cell Calcium 53, 195–203.

https://doi.org/10.1016/j.ceca.2012.11.014

Liu, Q., Huang, Y., Shen, J., Steffensen, S., Wu, J., 2012. Functional α7β2 nicotinic

acetylcholine receptors expressed in hippocampal interneurons exhibit high

sensitivity to pathological level of amyloid β peptides. BMC Neurosci. 13,

155. https://doi.org/10.1186/1471-2202-13-155

Liu, Q., Huang, Y., Xue, F., Simard, A., DeChon, J., Li, G., Zhang, J., Lucero, L.,

Wang, M., Sierks, M., Hu, G., Chang, Y., Lukas, R.J., Wu, J., 2009. A novel

nicotinic acetylcholine receptor subtype in basal forebrain cholinergic

neurons with high sensitivity to amyloid peptides. J. Neurosci. Off. J. Soc.

Neurosci. 29, 918–929. https://doi.org/10.1523/JNEUROSCI.3952-08.2009

Liu, Y., Zeng, X., Hui, Y., Zhu, C., Wu, J., Taylor, D.H., Ji, J., Fan, W., Huang, Z.,

Hu, J., 2015. Activation of α7 nicotinic acetylcholine receptors protects

astrocytes against oxidative stress-induced apoptosis: implications for

Parkinson’s disease. Neuropharmacology 91, 87–96.


Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P.,

1992. Glutamate-immunoreactive synapses on retrogradely-labelled

sympathetic preganglionic neurons in rat thoracic spinal cord. Brain Res. 581,

67–80.

Long-Smith, C.M., Sullivan, A.M., Nolan, Y.M., 2009. The influence of microglia

on the pathogenesis of Parkinson’s disease. Prog Neurobiol 89, 277–87.

https://doi.org/10.1016/j.pneurobio.2009.08.001

198

Low, V., Ben-Shlomo, Y., Coward, E., Fletcher, S., Walker, R., Clarke, C.E., 2015.

Measuring the burden and mortality of hospitalisation in Parkinson’s disease:

A cross-sectional analysis of the English Hospital Episodes Statistics

database 2009-2013. Parkinsonism Relat. Disord. 21, 449–454.

https://doi.org/10.1016/j.parkreldis.2015.01.017

Lu, X.-H., Fleming, S.M., Meurers, B., Ackerson, L.C., Mortazavi, F., Lo, V.,

Hernandez, D., Sulzer, D., Jackson, G.R., Maidment, N.T., Chesselet, M.-F.,

Yang, X.W., 2009. Bacterial artificial chromosome transgenic mice

expressing a truncated mutant parkin exhibit age-dependent hypokinetic

motor deficits, dopaminergic neuron degeneration, and accumulation of

proteinase K-resistant alpha-synuclein. J. Neurosci. Off. J. Soc. Neurosci. 29,

1962–1976. https://doi.org/10.1523/JNEUROSCI.5351-08.2009

Lücking, C.B., Dürr, A., Bonifati, V., Vaughan, J., De Michele, G., Gasser, T.,

Harhangi, B.S., Meco, G., Denèfle, P., Wood, N.W., Agid, Y., Brice, A.,

French Parkinson’s Disease Genetics Study Group, European Consortium on

Genetic Susceptibility in Parkinson’s Disease, 2000. Association between

early-onset Parkinson’s disease and mutations in the parkin gene. N. Engl. J.

Med. 342, 1560–1567. https://doi.org/10.1056/NEJM200005253422103

Luthman, J., Fredriksson, A., Sundström, E., Jonsson, G., Archer, T., 1989. Selective

lesion of central dopamine or noradrenaline neuron systems in the neonatal

rat: motor behavior and monoamine alterations at adult stage. Behav. Brain

Res. 33, 267–277.

Macleod, A.D., Taylor, K.S.M., Counsell, C.E., 2014. Mortality in Parkinson’s

disease: a systematic review and meta-analysis. Mov. Disord. Off. J. Mov.


Macor, J.E., Gurley, D., Lanthorn, T., Loch, J., Mack, R.A., Mullen, G., Tran, O.,

Wright, N., Gordon, J.C., 2001. The 5-HT3 antagonist tropisetron (ICS 205-

930) is a potent and selective alpha7 nicotinic receptor partial agonist.

Bioorg. Med. Chem. Lett. 11, 319–321.

Maestro 11 | Schrödinger [WWW Document], n.d. URL

https://www.schrodinger.com/maestro (accessed 1.15.17).

Mahmmoud, Y.A., 2007. Modulation of protein kinase C by curcumin; inhibition

and activation switched by calcium ions. Br. J. Pharmacol. 150, 200–208.

https://doi.org/10.1038/sj.bjp.0706970

Mak, S.K., McCormack, A.L., Manning-Bog, A.B., Cuervo, A.M., Di Monte, D.A.,

2010. Lysosomal degradation of alpha-synuclein in vivo. J. Biol. Chem. 285,

13621–13629. https://doi.org/10.1074/jbc.M109.074617

199

Malmfors, T., Sachs, C., 1968. Degeneration of adrenergic nerves produced by 6-

hydroxydopamine. Eur. J. Pharmacol. 3, 89–92.

Mameli-Engvall, M., Evrard, A., Pons, S., Maskos, U., Svensson, T.H., Changeux,

J.P., Faure, P., 2006. Hierarchical control of dopamine neuron-firing patterns

by nicotinic receptors. Neuron 50, 911–21.

https://doi.org/10.1016/j.neuron.2006.05.007

Manikandan, P., Sumitra, M., Aishwarya, S., Manohar, B.M., Lokanadam, B.,

Puvanakrishnan, R., 2004. Curcumin modulates free radical quenching in

myocardial ischaemia in rats. Int. J. Biochem. Cell Biol. 36, 1967–1980.

https://doi.org/10.1016/j.biocel.2004.01.030

Manning-Bog, A.B., McCormack, A.L., Li, J., Uversky, V.N., Fink, A.L., Di Monte,

D.A., 2002. The herbicide paraquat causes up-regulation and aggregation of

alpha-synuclein in mice: paraquat and alpha-synuclein. J. Biol. Chem. 277,

1641–1644. https://doi.org/10.1074/jbc.C100560200

Mansvelder, H.D., Keath, J.R., McGehee, D.S., 2002. Synaptic mechanisms underlie

nicotine-induced excitability of brain reward areas. Neuron 33, 905–19.

Marcus, M.M., Björkholm, C., Malmerfelt, A., Möller, A., Påhlsson, N.,

Konradsson-Geuken, Å., Feltmann, K., Jardemark, K., Schilström, B.,

Svensson, T.H., 2016. Alpha7 nicotinic acetylcholine receptor agonists and

PAMs as adjunctive treatment in schizophrenia. An experimental study. Eur.

Neuropsychopharmacol. J. Eur. Coll. Neuropsychopharmacol. 26, 1401–

1411. https://doi.org/10.1016/j.euroneuro.2016.07.004

Marubio, L.M., del Mar Arroyo-Jimenez, M., Cordero-Erausquin, M., Lena, C., Le

Novere, N., de Kerchove d’Exaerde, A., Huchet, M., Damaj, M.I., Changeux,

J.P., 1999. Reduced antinociception in mice lacking neuronal nicotinic

receptor subunits. Nature 398, 805–10. https://doi.org/10.1038/19756

Marubio, L.M., Gardier, A.M., Durier, S., David, D., Klink, R., Arroyo-Jimenez,

M.M., McIntosh, J.M., Rossi, F., Champtiaux, N., Zoli, M., Changeux, J.P.,

2003. Effects of nicotine in the dopaminergic system of mice lacking the

alpha4 subunit of neuronal nicotinic acetylcholine receptors. Eur J Neurosci

17, 1329–37.

Massey, K.A., Zago, W.M., Berg, D.K., 2006. BDNF up-regulates alpha7 nicotinic

acetylcholine receptor levels on subpopulations of hippocampal interneurons.

Mol Cell Neurosci 33, 381–8. https://doi.org/10.1016/j.mcn.2006.08.011

200

Matsui, H., Uemura, N., Yamakado, H., Takeda, S., Takahashi, R., 2014. Exploring

the pathogenetic mechanisms underlying Parkinson’s disease in medaka fish.

J. Park. Dis. 4, 301–310. https://doi.org/10.3233/JPD-130289

McCormack, A.L., Thiruchelvam, M., Manning-Bog, A.B., Thiffault, C., Langston,

J.W., Cory-Slechta, D.A., Di Monte, D.A., 2002. Environmental risk factors

and Parkinson’s disease: selective degeneration of nigral dopaminergic

neurons caused by the herbicide paraquat. Neurobiol. Dis. 10, 119–127.

McNally, S.J., Harrison, E.M., Ross, J.A., Garden, O.J., Wigmore, S.J., 2007.

Curcumin induces heme oxygenase 1 through generation of reactive oxygen

species, p38 activation and phosphatase inhibition. Int. J. Mol. Med. 19, 165–

172.

Meredith, G.E., Kang, U.J., 2006. Behavioral models of Parkinson’s disease in

rodents: A new look at an old problem. Mov. Disord. 21, 1595–1606.


Meredith, G.E., Sonsalla, P.K., Chesselet, M.-F., 2008. Animal models of

Parkinson’s disease progression. Acta Neuropathol. (Berl.) 115, 385–398.

https://doi.org/10.1007/s00401-008-0350-x

Metz, G.A., Whishaw, I.Q., 2002. Drug-induced rotation intensity in unilateral

dopamine-depleted rats is not correlated with end point or qualitative

measures of forelimb or hindlimb motor performance. Neuroscience 111,

325–336.

Mihalak, K.B., Carroll, F.I., Luetje, C.W., 2006. Varenicline is a partial agonist at

alpha4beta2 and a full agonist at alpha7 neuronal nicotinic receptors. Mol

Pharmacol 70, 801–5. https://doi.org/10.1124/mol.106.025130

Miledi, R., Potter, L.T., 1971. Acetylcholine receptors in muscle fibres. Nature 233,

599–603.

Millar, N.S., Gotti, C., 2009. Diversity of vertebrate nicotinic acetylcholine

receptors. Neuropharmacology 56, 237–246.


Mineur, Y.S., Picciotto, M.R., 2008. Genetics of nicotinic acetylcholine receptors:

Relevance to nicotine addiction. Biochem Pharmacol 75, 323–33.


Mizuno, Y., Sone, N., Saitoh, T., 1987. Effects of 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine and 1-methyl-4-phenylpyridinium ion on activities of the

201

enzymes in the electron transport system in mouse brain. J. Neurochem. 48,

1787–1793.

MOE: Molecular Operating Environment [WWW Document], n.d. URL

http://www.chemcomp.com/MOE-Molecular_Operating_Environment.htm

(accessed 1.12.17).

Monderer, R., Thorpy, M., 2009. Sleep disorders and daytime sleepiness in

Parkinson’s disease. Curr. Neurol. Neurosci. Rep. 9, 173–180.

Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M., 2005. Molecular

pathophysiology of Parkinson’s disease. Annu Rev Neurosci 28, 57–87.

https://doi.org/10.1146/annurev.neuro.28.061604.135718

Morais, V.A., Haddad, D., Craessaerts, K., De Bock, P.-J., Swerts, J., Vilain, S.,

Aerts, L., Overbergh, L., Grünewald, A., Seibler, P., Klein, C., Gevaert, K.,

Verstreken, P., De Strooper, B., 2014. PINK1 loss-of-function mutations

affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling.

Science 344, 203–207. https://doi.org/10.1126/science.1249161

Moretti, M., Zoli, M., George, A.A., Lukas, R.J., Pistillo, F., Maskos, U., Whiteaker,

P., Gotti, C., 2014. The novel α7β2-nicotinic acetylcholine receptor subtype

is expressed in mouse and human basal forebrain: biochemical and

pharmacological characterization. Mol. Pharmacol. 86, 306–317.

https://doi.org/10.1124/mol.114.093377

Morgante, L., Morgante, F., Moro, E., Epifanio, A., Girlanda, P., Ragonese, P.,

Antonini, A., Barone, P., Bonuccelli, U., Contarino, M.F., Capus, L.,

Ceravolo, M.G., Marconi, R., Ceravolo, R., D’Amelio, M., Savettieri, G.,

2007. How many parkinsonian patients are suitable candidates for deep brain

stimulation of subthalamic nucleus? Results of a questionnaire. Parkinsonism

Relat. Disord. 13, 528–531. https://doi.org/10.1016/j.parkreldis.2006.12.013

Motterlini, R., Foresti, R., Bassi, R., Green, C.J., 2000. Curcumin, an antioxidant and

anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial

cells against oxidative stress. Free Radic. Biol. Med. 28, 1303–1312.

Moustapha, A., Pérétout, P.A., Rainey, N.E., Sureau, F., Geze, M., Petit, J.-M.,

Dewailly, E., Slomianny, C., Petit, P.X., 2015. Curcumin induces crosstalk

between autophagy and apoptosis mediated by calcium release from the

endoplasmic reticulum, lysosomal destabilization and mitochondrial events.

Cell Death Discov. 1, 15017. https://doi.org/10.1038/cddiscovery.2015.17

Mowrey, D.D., Liu, Q., Bondarenko, V., Chen, Q., Seyoum, E., Xu, Y., Wu, J.,

Tang, P., 2013. Insights into distinct modulation of α7 and α7β2 nicotinic

202

acetylcholine receptors by the volatile anesthetic isoflurane. J. Biol. Chem.

288, 35793–35800. https://doi.org/10.1074/jbc.M113.508333

Munro, G., Hansen, R., Erichsen, H., Timmermann, D., Christensen, J., Hansen, H.,

2012. The α7 nicotinic ACh receptor agonist compound B and positive

allosteric modulator PNU-120596 both alleviate inflammatory hyperalgesia

and cytokine release in the rat. Br. J. Pharmacol. 167, 421–435.

https://doi.org/10.1111/j.1476-5381.2012.02003.x

Nagatsu, T., Sawada, M., 2005. Inflammatory process in Parkinson’s disease: role

for cytokines. Curr Pharm Des 11, 999–1016.

Nakagawa, T., Touhara, K., 2013. Functional Assays for Insect Olfactory Receptors

in Xenopus Oocytes, in: Pheromone Signaling, Methods in Molecular

Biology. Humana Press, Totowa, NJ, pp. 107–119.

https://doi.org/10.1007/978-1-62703-619-1_8

Napoli, I., Neumann, H., 2010. Protective effects of microglia in multiple sclerosis.

Exp Neurol 225, 24–8. https://doi.org/10.1016/j.expneurol.2009.04.024

Newman, M.B., Arendash, G.W., Shytle, R.D., Bickford, P.C., Tighe, T., Sanberg,

P.R., 2002. Nicotine’s oxidative and antioxidant properties in CNS. Life Sci

71, 2807–20.

Ng, H.J., Whittemore, E.R., Tran, M.B., Hogenkamp, D.J., Broide, R.S., Johnstone,

T.B., Zheng, L., Stevens, K.E., Gee, K.W., 2007. Nootropic alpha7 nicotinic

receptor allosteric modulator derived from GABAA receptor modulators.

Proc. Natl. Acad. Sci. U. S. A. 104, 8059–8064.


Nicklas, W.J., Vyas, I., Heikkila, R.E., 1985. Inhibition of NADH-linked oxidation

in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the

neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. Life Sci. 36,

2503–2508.

Nicoletti, A., Pugliese, P., Nicoletti, G., Arabia, G., Annesi, G., Mari, M.D.,

Lamberti, P., Grasso, L., Marconi, R., Epifanio, A., Morgante, L., Cozzolino,

A., Barone, P., Torchia, G., Quattrone, A., Zappia, M., 2010. Voluptuary

habits and clinical subtypes of Parkinson’s disease: the FRAGAMP case-

control study. Mov Disord 25, 2387–94. https://doi.org/10.1002/mds.23297

Nirmala, C., Puvanakrishnan, R., 1996a. Protective role of curcumin against

isoproterenol induced myocardial infarction in rats. Mol. Cell. Biochem. 159,

85–93.

203

Nirmala, C., Puvanakrishnan, R., 1996b. Effect of curcumin on certain lysosomal

hydrolases in isoproterenol-induced myocardial infarction in rats. Biochem.

Pharmacol. 51, 47–51.

Nishikawa, T., Takashima, M., Toru, M., 1983. Increased [3H]kainic acid binding in

the prefrontal cortex in schizophrenia. Neurosci. Lett. 40, 245–250.

Nordberg, A., 1992. Neuroreceptor changes in Alzheimer disease. Cerebrovasc Brain

Metab Rev 4, 303–28.

Nosalski, R., Guzik, T.J., 2017. Perivascular adipose tissue inflammation in vascular

disease. Br. J. Pharmacol. https://doi.org/10.1111/bph.13705

Noyce, A.J., Bestwick, J.P., Silveira-Moriyama, L., Hawkes, C.H., Giovannoni, G.,

Lees, A.J., Schrag, A., 2012. Meta-analysis of early nonmotor features and

risk factors for Parkinson disease. Ann Neurol 72, 893–901.

https://doi.org/10.1002/ana.23687

Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V.,

Changeux, J.-P., Sonner, J.M., Delarue, M., Corringer, P.-J., 2011. X-ray

structures of general anaesthetics bound to a pentameric ligand-gated ion

channel. Nature 469, 428–431. https://doi.org/10.1038/nature09647

Nys, M., Kesters, D., Ulens, C., 2013. Structural insights into Cys-loop receptor

function and ligand recognition. Biochem. Pharmacol. 86, 1042–1053.


Obeso, J.A., Lanciego, J.L., 2011. Past, present, and future of the pathophysiological

model of the Basal Ganglia. Front Neuroanat 5, 39.


Okun, M.S., 2012. Deep-brain stimulation for Parkinson’s disease. N. Engl. J. Med.

367, 1529–1538. https://doi.org/10.1056/NEJMct1208070

Olanow, C.W., McNaught, K., 2011. Parkinson’s disease, proteins, and prions:

milestones. Mov. Disord. Off. J. Mov. Disord. Soc. 26, 1056–1071.


Olanow, C.W., Obeso, J.A., Stocchi, F., 2006. Continuous dopamine-receptor

treatment of Parkinson’s disease: scientific rationale and clinical implications.

Lancet Neurol 5, 677–87. https://doi.org/10.1016/s1474-4422(06)70521-x

Olincy, A., Blakeley-Smith, A., Johnson, L., Kem, W.R., Freedman, R., 2016. Brief

Report: Initial Trial of Alpha7-Nicotinic Receptor Stimulation in Two Adult

Patients with Autism Spectrum Disorder. J. Autism Dev. Disord. 46, 3812–

3817. https://doi.org/10.1007/s10803-016-2890-6

204

Oliveras-Salvá, M., Macchi, F., Coessens, V., Deleersnijder, A., Gérard, M., Van der

Perren, A., Van den Haute, C., Baekelandt, V., 2014. Alpha-synuclein-

induced neurodegeneration is exacerbated in PINK1 knockout mice.

Neurobiol. Aging 35, 2625–2636.

https://doi.org/10.1016/j.neurobiolaging.2014.04.032

Onaran, H.O., Costa, T., 2009. Allosteric coupling and conformational fluctuations

in proteins. Curr. Protein Pept. Sci. 10, 110–115.

O’Neill, M.J., Murray, T.K., Lakics, V., Visanji, N.P., Duty, S., 2002. The role of

neuronal nicotinic acetylcholine receptors in acute and chronic

neurodegeneration. Curr. Drug Targets CNS Neurol. Disord. 1, 399–411.

Ono, K., Hasegawa, K., Naiki, H., Yamada, M., 2004. Curcumin has potent anti-

amyloidogenic effects for Alzheimer’s beta-amyloid fibrils in vitro. J.

Neurosci. Res. 75, 742–750. https://doi.org/10.1002/jnr.20025

Onofrj, M., Thomas, A., Bonanni, L., 2007. New approaches to understanding

hallucinations in Parkinson’s disease: phenomenology and possible origins.

Expert Rev. Neurother. 7, 1731–1750.

https://doi.org/10.1586/14737175.7.12.1731

Orr, C.F., Rowe, D.B., Halliday, G.M., 2002. An inflammatory review of

Parkinson’s disease. Prog Neurobiol 68, 325–40.

Ottolini, D., Calì, T., Negro, A., Brini, M., 2013. The Parkinson disease-related

protein DJ-1 counteracts mitochondrial impairment induced by the tumour

suppressor protein p53 by enhancing endoplasmic reticulum-mitochondria

tethering. Hum. Mol. Genet. 22, 2152–2168.

https://doi.org/10.1093/hmg/ddt068

Oz, M., Melia, M.T., Soldatov, N.M., Abernethy, D.R., Morad, M., 1998. Functional

coupling of human L-type Ca2+ channels and angiotensin AT1A receptors

coexpressed in xenopus laevis oocytes: involvement of the carboxyl-terminal

Ca2+ sensors. Mol. Pharmacol. 54, 1106–1112.

Oz, M., Renaud, L.P., 2002. Angiotensin AT(1)-receptors depolarize neonatal spinal

motoneurons and other ventral horn neurons via two different conductances.

J. Neurophysiol. 88, 2857–2863. https://doi.org/10.1152/jn.00978.2001

Oz, M., Ravindran, A., Diaz-Ruiz, O., Zhang, L., Morales, M., 2003. The

endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine

receptor-mediated responses in Xenopus oocytes. J. Pharmacol. Exp. Ther.

306, 1003–1010. https://doi.org/10.1124/jpet.103.049981

205

Oz, M., Zakharova, I., Dinc, M., Shippenberg, T., 2004. Cocaine inhibits

cromakalim-activated K+ currents in follicle-enclosed Xenopus oocytes.

Naunyn. Schmiedebergs Arch. Pharmacol. 369, 252–259.

https://doi.org/10.1007/s00210-003-0838-9

Pan, M.H., Huang, T.M., Lin, J.K., 1999. Biotransformation of curcumin through

reduction and glucuronidation in mice. Drug Metab. Dispos. Biol. Fate Chem.

27, 486–494.

Pan-Montojo, F., Anichtchik, O., Dening, Y., Knels, L., Pursche, S., Jung, R.,

Jackson, S., Gille, G., Spillantini, M.G., Reichmann, H., Funk, R.H.W., 2010.

Progression of Parkinson’s disease pathology is reproduced by intragastric

administration of rotenone in mice. PloS One 5, e8762.

https://doi.org/10.1371/journal.pone.0008762

Panneton, W.M., Kumar, V.B., Gan, Q., Burke, W.J., Galvin, J.E., 2010. The

neurotoxicity of DOPAL: behavioral and stereological evidence for its role in

Parkinson disease pathogenesis. PloS One 5, e15251.


Parashos, S.A., Maraganore, D.M., O’Brien, P.C., Rocca, W.A., 2002. Medical

services utilization and prognosis in Parkinson disease: a population-based

study. Mayo Clin. Proc. 77, 918–925. https://doi.org/10.4065/77.9.918

Park, H.J., Lee, P.H., Ahn, Y.W., Choi, Y.J., Lee, G., Lee, D.Y., Chung, E.S., Jin,

B.K., 2007a. Neuroprotective effect of nicotine on dopaminergic neurons by

anti-inflammatory action. Eur J Neurosci 26, 79–89.

https://doi.org/10.1111/j.1460-9568.2007.05636.x

Parker, W.D., Boyson, S.J., Parks, J.K., 1989. Abnormalities of the electron transport

chain in idiopathic Parkinson’s disease. Ann. Neurol. 26, 719–723.

https://doi.org/10.1002/ana.410260606

Parkinson, J., 2002. An essay on the shaking palsy. 1817. J. Neuropsychiatry Clin.

Neurosci. 14, 223–236; discussion 222. https://doi.org/10.1176/jnp.14.2.223

Patrick, J., Ballivet, M., Boas, L., Claudio, T., Forrest, J., Ingraham, H., Mason, P.,

Stengelin, S., Ueno, S., Heinemann, S., 1983. Molecular cloning of the

acetylcholine receptor. Cold Spring Harb. Symp. Quant. Biol. 48 Pt 1, 71–78.

Paulo, J.A., Brucker, W.J., Hawrot, E., 2009. Proteomic analysis of an alpha7

nicotinic acetylcholine receptor interactome. J. Proteome Res. 8, 1849–1858.

https://doi.org/10.1021/pr800731z

206

Pérez-Lara, A., Corbalán-García, S., Gómez-Fernández, J.C., 2011. Curcumin

modulates PKCα activity by a membrane-dependent effect. Arch. Biochem.

Biophys. 513, 36–41. https://doi.org/10.1016/j.abb.2011.06.010

Perez-Lloret, S., Nègre-Pagès, L., Ojero-Senard, A., Damier, P., Destée, A., Tison,

F., Merello, M., Rascol, O., COPARK Study Group, 2012. Oro-buccal

symptoms (dysphagia, dysarthria, and sialorrhea) in patients with Parkinson’s

disease: preliminary analysis from the French COPARK cohort. Eur. J.

Neurol. 19, 28–37. https://doi.org/10.1111/j.1468-1331.2011.03402.x

Periquet, M., Latouche, M., Lohmann, E., Rawal, N., De Michele, G., Ricard, S.,

Teive, H., Fraix, V., Vidailhet, M., Nicholl, D., Barone, P., Wood, N.W.,

Raskin, S., Deleuze, J.-F., Agid, Y., Dürr, A., Brice, A., French Parkinson’s

Disease Genetics Study Group, European Consortium on Genetic

Susceptibility in Parkinson’s Disease, 2003. Parkin mutations are frequent in

patients with isolated early-onset parkinsonism. Brain J. Neurol. 126, 1271–

1278.

Perkins, S., Verschoyle, R.D., Hill, K., Parveen, I., Threadgill, M.D., Sharma, R.A.,

Williams, M.L., Steward, W.P., Gescher, A.J., 2002. Chemopreventive

efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of

familial adenomatous polyposis. Cancer Epidemiol. Biomark. Prev. Publ.

Am. Assoc. Cancer Res. Cosponsored Am. Soc. Prev. Oncol. 11, 535–540.

Pham-Huy, L.A., He, H., Pham-Huy, C., 2008. Free radicals, antioxidants in disease

and health. Int. J. Biomed. Sci. IJBS 4, 89–96.

Picciotto, M.R., Zoli, M., 2008. Neuroprotection via nAChRs: the role of nAChRs in

neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease.

Front Biosci 13, 492–504.

Pickel, V.M., Beckley, S.C., Joh, T.H., Reis, D.J., 1981. Ultrastructural

immunocytochemical localization of tyrosine hydroxylase in the neostriatum.

Brain Res 225, 373–85.

Poewe, W., 2006. The natural history of Parkinson’s disease. J. Neurol. 253 Suppl 7,

VII2-6. https://doi.org/10.1007/s00415-006-7002-7

Pont-Sunyer, C., Hotter, A., Gaig, C., Seppi, K., Compta, Y., Katzenschlager, R.,

Mas, N., Hofeneder, D., Brücke, T., Bayés, A., Wenzel, K., Infante, J., Zach,

H., Pirker, W., Posada, I.J., Álvarez, R., Ispierto, L., De Fàbregues, O.,

Callén, A., Palasí, A., Aguilar, M., Martí, M.J., Valldeoriola, F., Salamero,

M., Poewe, W., Tolosa, E., 2015. The onset of nonmotor symptoms in

Parkinson’s disease (the ONSET PD study). Mov. Disord. Off. J. Mov.


207

Posadas, I., Lopez-Hernandez, B., Cena, V., 2013. Nicotinic receptors in

neurodegeneration. Curr Neuropharmacol 11, 298–314.

https://doi.org/10.2174/1570159x11311030005

Postuma, R.B., Berg, D., Stern, M., Poewe, W., Olanow, C.W., Oertel, W., Obeso, J.,

Marek, K., Litvan, I., Lang, A.E., Halliday, G., Goetz, C.G., Gasser, T.,

Dubois, B., Chan, P., Bloem, B.R., Adler, C.H., Deuschl, G., 2015. MDS

clinical diagnostic criteria for Parkinson’s disease. Mov. Disord. Off. J. Mov.


Prasad, S., Tyagi, A.K., Aggarwal, B.B., 2014. Recent developments in delivery,

bioavailability, absorption and metabolism of curcumin: the golden pigment

from golden spice. Cancer Res. Treat. Off. J. Korean Cancer Assoc. 46, 2–18.

https://doi.org/10.4143/crt.2014.46.1.2

Priyadarsini, K.I., 2014. The chemistry of curcumin: from extraction to therapeutic

agent. Mol. Basel Switz. 19, 20091–20112.

https://doi.org/10.3390/molecules191220091

Priyadarsini, K.I., 2009. ChemInform Abstract: Photophysics, Photochemistry and

Photobiology of Curcumin: Studies from Organic Solutions, Biomimetics and

Living Cells. ResearchGate 40. https://doi.org/10.1002/chin.200951234

Prokop, S., Miller, K.R., Heppner, F.L., 2013. Microglia actions in Alzheimer’s

disease. Acta Neuropathol 126, 461–77.

Przedborski, S., Jackson-Lewis, V., Naini, A.B., Jakowec, M., Petzinger, G., Miller,

R., Akram, M., 2001. The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP): a technical review of its utility and safety. J.

Neurochem. 76, 1265–1274.

Przedborski, S., Levivier, M., Jiang, H., Ferreira, M., Jackson-Lewis, V., Donaldson,

D., Togasaki, D.M., 1995. Dose-dependent lesions of the dopaminergic

nigrostriatal pathway induced by intrastriatal injection of 6-

hydroxydopamine. Neuroscience 67, 631–647.

Purisai, M.G., McCormack, A.L., Langston, W.J., Johnston, L.C., Di Monte, D.A.,

2005. Alpha-synuclein expression in the substantia nigra of MPTP-lesioned

non-human primates. Neurobiol. Dis. 20, 898–906.

https://doi.org/10.1016/j.nbd.2005.05.028

Puschmann, A., 2013. Monogenic Parkinson’s disease and parkinsonism: clinical

phenotypes and frequencies of known mutations. Parkinsonism Relat. Disord.

19, 407–415. https://doi.org/10.1016/j.parkreldis.2013.01.020

208

Pyakurel, P., Shin, M., Venton, B.J., 2018. Nicotinic acetylcholine receptor (nAChR)

mediated dopamine release in larval Drosophila melanogaster. Neurochem.

Int. 114, 33–41. https://doi.org/10.1016/j.neuint.2017.12.012

Quarta, D., Naylor, C.G., Barik, J., Fernandes, C., Wonnacott, S., Stolerman, I.P.,

2009. Drug discrimination and neurochemical studies in alpha7 null mutant

mice: tests for the role of nicotinic alpha7 receptors in dopamine release.

Psychopharmacology (Berl.) 203, 399–410. https://doi.org/10.1007/s00213-

008-1281-x

Quik, M., Campos, C., Bordia, T., Strachan, J.P., Zhang, J., McIntosh, J.M.,

Letchworth, S., Jordan, K., 2013. alpha4beta2 Nicotinic receptors play a role

in the nAChR-mediated decline in L-dopa-induced dyskinesias in

parkinsonian rats. Neuropharmacology 71, 191–203.


Quik, M., Cox, H., Parameswaran, N., O’Leary, K., Langston, J.W., Di Monte, D.,

2007. Nicotine reduces levodopa-induced dyskinesias in lesioned monkeys.

Ann Neurol 62, 588–96. https://doi.org/10.1002/ana.21203

Quik, M., Kulak, J.M., 2002. Nicotine and nicotinic receptors; relevance to

Parkinson’s disease. Neurotoxicology 23, 581–94.

Quik, M., O’Leary, K., Tanner, C.M., 2008. Nicotine and Parkinson’s disease:

implications for therapy. Mov Disord 23, 1641–52.


Quik, M., Parameswaran, N., McCallum, S.E., Bordia, T., Bao, S., McCormack, A.,

Kim, A., Tyndale, R.F., Langston, J.W., Di Monte, D.A., 2006. Chronic oral

nicotine treatment protects against striatal degeneration in MPTP-treated

primates. J Neurochem 98, 1866–75. https://doi.org/10.1111/j.1471-

4159.2006.04078.x

Quik, M., Perez, X.A., Bordia, T., 2012. Nicotine as a potential neuroprotective

agent for Parkinson’s disease. Mov Disord 27, 947–57.


Quik, M., Wonnacott, S., 2011. α6β2* and α4β2* nicotinic acetylcholine receptors as

drug targets for Parkinson’s disease. Pharmacol. Rev. 63, 938–966.

https://doi.org/10.1124/pr.110.003269

Quik, M., Zhang, D., Perez, X.A., Bordia, T., 2014. Role for the nicotinic cholinergic

system in movement disorders; therapeutic implications. Pharmacol Ther

144, 50–9. https://doi.org/10.1016/j.pharmthera.2014.05.004

209

Rachmawati, H., Edityaningrum, C.A., Mauludin, R., 2013. Molecular inclusion

complex of curcumin-β-cyclodextrin nanoparticle to enhance curcumin skin

permeability from hydrophilic matrix gel. AAPS PharmSciTech 14, 1303–

1312. https://doi.org/10.1208/s12249-013-0023-5

Rainey, N., Motte, L., Aggarwal, B.B., Petit, P.X., 2015. Curcumin hormesis

mediates a cross-talk between autophagy and cell death. Cell Death Dis. 6,

e2003. https://doi.org/10.1038/cddis.2015.343

Rajan, S., 2012.

https://commons.wikimedia.org/wiki/File:Lewy_bodies_(alpha_synuclein_in

clusions).svg

Rangel-Barajas, C., Coronel, I., Florán, B., 2015. Dopamine Receptors and

Neurodegeneration. Aging Dis. 6, 349–368.

https://doi.org/10.14336/AD.2015.0330

Rao, C.V., Rivenson, A., Simi, B., Reddy, B.S., 1995. Chemoprevention of colon

carcinogenesis by dietary curcumin, a naturally occurring plant phenolic

compound. Cancer Res. 55, 259–266.

Ravindran, J., Prasad, S., Aggarwal, B.B., 2009. Curcumin and cancer cells: how

many ways can curry kill tumor cells selectively? AAPS J. 11, 495–510.

https://doi.org/10.1208/s12248-009-9128-x

Ravindranath, V., Chandrasekhara, N., 1981. Metabolism of curcumin--studies with

[3H]curcumin. Toxicology 22, 337–344.

Reddy, A.C., Lokesh, B.R., 1994. Studies on the inhibitory effects of curcumin and

eugenol on the formation of reactive oxygen species and the oxidation of

ferrous iron. Mol. Cell. Biochem. 137, 1–8.

Reddy, A.C., Lokesh, B.R., 1992. Studies on spice principles as antioxidants in the

inhibition of lipid peroxidation of rat liver microsomes. Mol. Cell. Biochem.

111, 117–124.

Reijnders, J.S.A.M., Ehrt, U., Weber, W.E.J., Aarsland, D., Leentjens, A.F.G., 2008.

A systematic review of prevalence studies of depression in Parkinson’s

disease. Mov. Disord. Off. J. Mov. Disord. Soc. 23, 183–189; quiz 313.


Rizzone, M.G., Fasano, A., Daniele, A., Zibetti, M., Merola, A., Rizzi, L., Piano, C.,

Piccininni, C., Romito, L.M., Lopiano, L., Albanese, A., 2014. Long-term

outcome of subthalamic nucleus DBS in Parkinson’s disease: from the

210

advanced phase towards the late stage of the disease? Parkinsonism Relat.

Disord. 20, 376–381. https://doi.org/10.1016/j.parkreldis.2014.01.012

Robinson, T.P., Ehlers, T., Hubbard IV, R.B., Bai, X., Arbiser, J.L., Goldsmith, D.J.,

Bowen, J.P., 2003. Design, synthesis, and biological evaluation of

angiogenesis inhibitors: aromatic enone and dienone analogues of curcumin.

Bioorg. Med. Chem. Lett. 13, 115–117.

Rodríguez Díaz, M., Abdala, P., Barroso-Chinea, P., Obeso, J., González-Hernández,

T., 2001. Motor behavioural changes after intracerebroventricular injection of

6-hydroxydopamine in the rat: an animal model of Parkinson’s disease.

Behav. Brain Res. 122, 79–92.

Roncarati, R., Scali, C., Comery, T.A., Grauer, S.M., Aschmi, S., Bothmann, H.,

Jow, B., Kowal, D., Gianfriddo, M., Kelley, C., Zanelli, U., Ghiron, C.,

Haydar, S., Dunlop, J., Terstappen, G.C., 2009. Procognitive and

neuroprotective activity of a novel alpha7 nicotinic acetylcholine receptor

agonist for treatment of neurodegenerative and cognitive disorders. J.

Pharmacol. Exp. Ther. 329, 459–468. https://doi.org/10.1124/jpet.108.150094

Rosenblad, C., Kirik, D., Devaux, B., Moffat, B., Phillips, H.S., Björklund, A., 1999.

Protection and regeneration of nigral dopaminergic neurons by neurturin or

GDNF in a partial lesion model of Parkinson’s disease after administration

into the striatum or the lateral ventricle. Eur. J. Neurosci. 11, 1554–1566.

Rudenko, I.N., Cookson, M.R., 2014. Heterogeneity of leucine-rich repeat kinase 2

mutations: genetics, mechanisms and therapeutic implications. Neurother. J.

Am. Soc. Exp. Neurother. 11, 738–750. https://doi.org/10.1007/s13311-014-

0284-z

Samadi, P., Bedard, P.J., Rouillard, C., 2006. Opioids and motor complications in

Parkinson’s disease. Trends Pharmacol Sci 27, 512–7.


Sanchez, G., Varaschin, R.K., Büeler, H., Marcogliese, P.C., Park, D.S., Trudeau, L.-

E., 2014. Unaltered striatal dopamine release levels in young Parkin

knockout, Pink1 knockout, DJ-1 knockout and LRRK2 R1441G transgenic

mice. PloS One 9, e94826. https://doi.org/10.1371/journal.pone.0094826

Sands, S.B., Costa, A.C., Patrick, J.W., 1993. Barium permeability of neuronal

nicotinic receptor alpha 7 expressed in Xenopus oocytes. Biophys. J. 65,

2614–2621. https://doi.org/10.1016/S0006-3495(93)81296-7

Sandur, S.K., Pandey, M.K., Sung, B., Ahn, K.S., Murakami, A., Sethi, G.,

Limtrakul, P., Badmaev, V., Aggarwal, B.B., 2007. Curcumin,

211

demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and

turmerones differentially regulate anti-inflammatory and anti-proliferative

responses through a ROS-independent mechanism. Carcinogenesis 28, 1765–

1773. https://doi.org/10.1093/carcin/bgm123

Saner, A., Thoenen, H., 1971. Model experiments on the molecular mechanism of

action of 6-hydroxydopamine. Mol. Pharmacol. 7, 147–154.

Sastry, G.M., Adzhigirey, M., Day, T., Annabhimoju, R., Sherman, W., 2013.

Protein and ligand preparation: parameters, protocols, and influence on

virtual screening enrichments. J. Comput. Aided Mol. Des. 27, 221–234.

https://doi.org/10.1007/s10822-013-9644-8

Sauer, H., Oertel, W.H., 1994. Progressive degeneration of nigrostriatal dopamine

neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a

combined retrograde tracing and immunocytochemical study in the rat.

Neuroscience 59, 401–415.

Sauguet, L., Shahsavar, A., Poitevin, F., Huon, C., Menny, A., Nemecz, À., Haouz,

A., Changeux, J.-P., Corringer, P.-J., Delarue, M., 2014. Crystal structures of

a pentameric ligand-gated ion channel provide a mechanism for activation.



Sawada, H., Oeda, T., Kuno, S., Nomoto, M., Yamamoto, K., Yamamoto, M.,

Hisanaga, K., Kawamura, T., 2010. Amantadine for dyskinesias in

Parkinson’s disease: a randomized controlled trial. PLoS One 5, e15298.


Sax’s Dangerous Properties of Industrial Materials, 5 Volume Set, 12th Edition

[WWW Document], n.d. . Wiley.com. URL https://www.wiley.com/en-

ae/Sax%27s_Dangerous_Properties_of_Industrial_Materials%2C_5+Volume

_Set%2C_12th_Edition-p-9780470623251 (accessed 12.3.18).

Scarffe, L.A., Stevens, D.A., Dawson, V.L., Dawson, T.M., 2014. Parkin and

PINK1: much more than mitophagy. Trends Neurosci. 37, 315–324.

https://doi.org/10.1016/j.tins.2014.03.004

Schapira, A.H., Cooper, J.M., Dexter, D., Jenner, P., Clark, J.B., Marsden, C.D.,

1989. Mitochondrial complex I deficiency in Parkinson’s disease. Lancet

Lond. Engl. 1, 1269.

Schapira, A.H., Jenner, P., 2011. Etiology and pathogenesis of Parkinson’s disease.



212

Schwarting, R.K., Bonatz, A.E., Carey, R.J., Huston, J.P., 1991. Relationships

between indices of behavioral asymmetries and neurochemical changes

following mesencephalic 6-hydroxydopamine injections. Brain Res. 554, 46–

55.

Schwarting, R.K., Huston, J.P., 1996. The unilateral 6-hydroxydopamine lesion

model in behavioral brain research. Analysis of functional deficits, recovery

and treatments. Prog. Neurobiol. 50, 275–331.

Séguéla, P., Wadiche, J., Dineley-Miller, K., Dani, J.A., Patrick, J.W., 1993.

Molecular cloning, functional properties, and distribution of rat brain alpha 7:

a nicotinic cation channel highly permeable to calcium. J. Neurosci. Off. J.

Soc. Neurosci. 13, 596–604.

Sgambato-Faure, V., Cenci, M.A., 2012. Glutamatergic mechanisms in the

dyskinesias induced by pharmacological dopamine replacement and deep

brain stimulation for the treatment of Parkinson’s disease. Prog. Neurobiol.

96, 69–86. https://doi.org/10.1016/j.pneurobio.2011.10.005

Shehab, S. a. S., Spike, R.C., Todd, A.J., 2003. Evidence against cholera toxin B

subunit as a reliable tracer for sprouting of primary afferents following

peripheral nerve injury. Brain Res. 964, 218–227.

Shehab, S., Anwer, M., Galani, D., Abdulkarim, A., Al-Nuaimi, K., Al-Baloushi, A.,

Tariq, S., Nagelkerke, N., Ljubisavljevic, M., 2015. Anatomical evidence that

the uninjured adjacent L4 nerve plays a significant role in the development of

peripheral neuropathic pain after L5 spinal nerve ligation in rats. J. Comp.

Neurol. 523, 1731–1747. https://doi.org/10.1002/cne.23750

Sherer, T.B., Kim, J.H., Betarbet, R., Greenamyre, J.T., 2003. Subcutaneous

rotenone exposure causes highly selective dopaminergic degeneration and

alpha-synuclein aggregation. Exp. Neurol. 179, 9–16.

Shimizu, M., Miyazaki, I., Higashi, Y., Eslava-Alva, M.J., Diaz-Corrales, F.J.,

Asanuma, M., Ogawa, N., 2008. Specific induction of PAG608 in cranial and

spinal motor neurons of L-DOPA-treated parkinsonian rats. Neurosci. Res.

60, 355–363. https://doi.org/10.1016/j.neures.2007.12.006

Shimohama, S., 2009. Nicotinic receptor-mediated neuroprotection in

neurodegenerative disease models. Biol Pharm Bull 32, 332–6.

Shimoji, M., Zhang, L., Mandir, A.S., Dawson, V.L., Dawson, T.M., 2005. Absence

of inclusion body formation in the MPTP mouse model of Parkinson’s

disease. Brain Res. Mol. Brain Res. 134, 103–108.

https://doi.org/10.1016/j.molbrainres.2005.01.012

213

Shishodia, S., Singh, T., Chaturvedi, M.M., 2007. Modulation of transcription factors

by curcumin. Adv. Exp. Med. Biol. 595, 127–148.

https://doi.org/10.1007/978-0-387-46401-5_4

Shoba, G., Joy, D., Joseph, T., Majeed, M., Rajendran, R., Srinivas, P.S., 1998.

Influence of piperine on the pharmacokinetics of curcumin in animals and

human volunteers. Planta Med. 64, 353–356. https://doi.org/10.1055/s-2006-

957450

Shytle, R.D., Mori, T., Townsend, K., Vendrame, M., Sun, N., Zeng, J., Ehrhart, J.,

Silver, A.A., Sanberg, P.R., Tan, J., 2004. Cholinergic modulation of

microglial activation by alpha 7 nicotinic receptors. J Neurochem 89, 337–43.

https://doi.org/10.1046/j.1471-4159.2004.02347.x

Singh, A., Naidu, P.S., Kulkarni, S.K., 2003. Quercetin potentiates L-Dopa reversal

of drug-induced catalepsy in rats: possible COMT/MAO inhibition.

Pharmacology 68, 81–88. https://doi.org/10.1159/000069533

Singh, S., Kumar, P., 2017. Neuroprotective potential of curcumin in combination

with piperine against 6-hydroxy dopamine induced motor deficit and

neurochemical alterations in rats. Inflammopharmacology 25, 69–79.

https://doi.org/10.1007/s10787-016-0297-9

Sinkus, M.L., Graw, S., Freedman, R., Ross, R.G., Lester, H.A., Leonard, S., 2015.

The human CHRNA7 and CHRFAM7A genes: A review of the genetics,

regulation, and function. Neuropharmacology 96, 274–288.


Small-Molecule Drug Discovery Suite | Schrödinger [WWW Document], n.d. URL

https://www.schrodinger.com/suites/small-molecule-drug-discovery-suite

(accessed 1.15.17).

Snyder, S.H., D’Amato, R.J., 1985. Predicting Parkinson’s disease. Nature 317, 198–

199.

Song, S., Nie, Q., Li, Z., Du, G., 2016. Curcumin improves neurofunctions of 6-

OHDA-induced parkinsonian rats. Pathol. Res. Pract. 212, 247–251.

https://doi.org/10.1016/j.prp.2015.11.012

Sonsalla, P.K., Jochnowitz, N.D., Zeevalk, G.D., Oostveen, J.A., Hall, E.D., 1996.

Treatment of mice with methamphetamine produces cell loss in the substantia

nigra. Brain Res. 738, 172–175.

214

Soung, Y.H., Chung, J., 2011. Curcumin inhibition of the functional interaction

between integrin α6β4 and the epidermal growth factor receptor. Mol. Cancer

Ther. 10, 883–891. https://doi.org/10.1158/1535-7163.MCT-10-1053

Spector, S., Sjoerdsma, A., Udenfriend, S., 1965. BLOCKADE OF ENDOGENOUS

NOREPINEPHRINE SYNTHESIS BY ALPHA-METHYL-TYROSINE,

AN INHIBITOR OF TYROSINE HYDROXYLASE. J. Pharmacol. Exp.

Ther. 147, 86–95.

Spivak, C.E., Lupica, C.R., Oz, M., 2007. The endocannabinoid anandamide inhibits

the function of alpha4beta2 nicotinic acetylcholine receptors. Mol.

Pharmacol. 72, 1024–1032. https://doi.org/10.1124/mol.107.036939

Sreejayan, N., Rao, M.N., 1996. Free radical scavenging activity of curcuminoids.

Arzneimittelforschung. 46, 169–171.

Srinivasan, M., 1972. Effect of curcumin on blood sugar as seen in a diabetic subject.

Indian J. Med. Sci. 26, 269–270.

Srivastava, R., Dikshit, M., Srimal, R.C., Dhawan, B.N., 1985. Anti-thrombotic

effect of curcumin. Thromb. Res. 40, 413–417.

Steinlein, O.K., Mulley, J.C., Propping, P., Wallace, R.H., Phillips, H.A., Sutherland,

G.R., Scheffer, I.E., Berkovic, S.F., 1995. A missense mutation in the

neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with

autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 11, 201–3.

https://doi.org/10.1038/ng1095-201

Stence, N., Waite, M., Dailey, M.E., 2001. Dynamics of microglial activation: a

confocal time-lapse analysis in hippocampal slices. Glia 33, 256–66.

Sullivan, J.P., Decker, M.W., Brioni, J.D., Donnelly-Roberts, D., Anderson, D.J.,

Bannon, A.W., Kang, C.H., Adams, P., Piattoni-Kaplan, M., Buckley, M.J.,

al, et, 1994. (+/-)-Epibatidine elicits a diversity of in vitro and in vivo effects

mediated by nicotinic acetylcholine receptors. J Pharmacol Exp Ther 271,

624–31.

Sun, F., Jin, K., Uteshev, V.V., 2013. A Type-II Positive Allosteric Modulator of α7

nAChRs Reduces Brain Injury and Improves Neurological Function after

Focal Cerebral Ischemia in Rats. PLoS ONE 8.


Swinney, D.C., 2004. Biochemical mechanisms of drug action: what does it take for

success? Nat. Rev. Drug Discov. 3, 801–808. https://doi.org/10.1038/nrd1500

215

Tadaiesky, M.T., Dombrowski, P.A., Figueiredo, C.P., Cargnin-Ferreira, E., Da

Cunha, C., Takahashi, R.N., 2008. Emotional, cognitive and neurochemical

alterations in a premotor stage model of Parkinson’s disease. Neuroscience

156, 830–840. https://doi.org/10.1016/j.neuroscience.2008.08.035

Takikawa, M., Kurimoto, Y., Tsuda, T., 2013. Curcumin stimulates glucagon-like

peptide-1 secretion in GLUTag cells via Ca2+/calmodulin-dependent kinase

II activation. Biochem. Biophys. Res. Commun. 435, 165–170.


Talwar, S., Lynch, J.W., 2014. Phosphorylation mediated structural and functional

changes in pentameric ligand-gated ion channels: implications for drug

discovery. Int. J. Biochem. Cell Biol. 53, 218–223.

https://doi.org/10.1016/j.biocel.2014.05.028

Tambasco, N., Simoni, S., Marsili, E., Sacchini, E., Murasecco, D., Cardaioli, G.,

Rossi, A., Calabresi, P., 2012. Clinical aspects and management of levodopa-

induced dyskinesia. Park. Dis. 2012, 745947.

https://doi.org/10.1155/2012/745947

Tanner, C.M., 2010. Advances in environmental epidemiology. Mov Disord 25

Suppl 1, S58-62. https://doi.org/10.1002/mds.22721

Tanner, C.M., Goldman, S.M., Aston, D.A., Ottman, R., Ellenberg, J., Mayeux, R.,

Langston, J.W., 2002. Smoking and Parkinson’s disease in twins. Neurology

58, 581–8.

Tasneem, A., Iyer, L.M., Jakobsson, E., Aravind, L., 2005. Identification of the

prokaryotic ligand-gated ion channels and their implications for the

mechanisms and origins of animal Cys-loop ion channels. Genome Biol. 6,

R4. https://doi.org/10.1186/gb-2004-6-1-r4

Thiruchelvam, M., McCormack, A., Richfield, E.K., Baggs, R.B., Tank, A.W., Di

Monte, D.A., Cory-Slechta, D.A., 2003. Age-related irreversible progressive

nigrostriatal dopaminergic neurotoxicity in the paraquat and maneb model of

the Parkinson’s disease phenotype. Eur. J. Neurosci. 18, 589–600.

Thomas, A., Iacono, D., Luciano, A.L., Armellino, K., Di Iorio, A., Onofrj, M.,

2004. Duration of amantadine benefit on dyskinesia of severe Parkinson’s

disease. J Neurol Neurosurg Psychiatry 75, 141–3.

Thomas, B., Mandir, A.S., West, N., Liu, Y., Andrabi, S.A., Stirling, W., Dawson,

V.L., Dawson, T.M., Lee, M.K., 2011. Resistance to MPTP-neurotoxicity in

α-synuclein knockout mice is complemented by human α-synuclein and

216

associated with increased β-synuclein and Akt activation. PloS One 6,

e16706. https://doi.org/10.1371/journal.pone.0016706

Thomsen, M.S., Zwart, R., Ursu, D., Jensen, M.M., Pinborg, L.H., Gilmour, G., Wu,

J., Sher, E., Mikkelsen, J.D., 2015. α7 and β2 Nicotinic Acetylcholine

Receptor Subunits Form Heteromeric Receptor Complexes that Are

Expressed in the Human Cortex and Display Distinct Pharmacological

Properties. PloS One 10, e0130572.


Thrash, B., Thiruchelvan, K., Ahuja, M., Suppiramaniam, V., Dhanasekaran, M.,

2009. Methamphetamine-induced neurotoxicity: the road to Parkinson’s

disease. Pharmacol. Rep. PR 61, 966–977.

Tieu, K., 2011. A guide to neurotoxic animal models of Parkinson’s disease. Cold

Spring Harb. Perspect. Med. 1, a009316.

https://doi.org/10.1101/cshperspect.a009316

Timofeeva, O.A., Levin, E.D., 2011. Glutamate and nicotinic receptor interactions in

working memory: importance for the cognitive impairment of schizophrenia.

Neuroscience 195, 21–36. https://doi.org/10.1016/j.neuroscience.2011.08.038

Toda, S., Miyase, T., Arichi, H., Tanizawa, H., Takino, Y., 1985. Natural

antioxidants. III. Antioxidative components isolated from rhizome of

Curcuma longa L. Chem. Pharm. Bull. (Tokyo) 33, 1725–1728.

Tolosa, E., Compta, Y., 2006. Dystonia in Parkinson’s disease. J. Neurol. 253 Suppl

7, VII7-13. https://doi.org/10.1007/s00415-006-7003-6

Toulorge, D., Guerreiro, S., Hild, A., Maskos, U., Hirsch, E.C., Michel, P.P., 2011.

Neuroprotection of midbrain dopamine neurons by nicotine is gated by

cytoplasmic Ca2+. Faseb J 25, 2563–73. https://doi.org/10.1096/fj.11-182824

Tripanichkul, W., Jaroensuppaperch, E., 2012. Curcumin protects nigrostriatal

dopaminergic neurons and reduces glial activation in 6-hydroxydopamine

hemiparkinsonian mice model. Int. J. Neurosci. 122, 263–270.

https://doi.org/10.3109/00207454.2011.648760

Tripanichkul, W., Jaroensuppaperch, E.-O., 2013. Ameliorating effects of curcumin

on 6-OHDA-induced dopaminergic denervation, glial response, and SOD1

reduction in the striatum of hemiparkinsonian mice. Eur. Rev. Med.

Pharmacol. Sci. 17, 1360–1368.

Trulson, M.E., Cannon, M.S., Faegg, T.S., Raese, J.D., 1985. Effects of chronic

methamphetamine on the nigral-striatal dopamine system in rat brain:

217

tyrosine hydroxylase immunochemistry and quantitative light microscopic

studies. Brain Res. Bull. 15, 569–577.

Truong, L., Allbutt, H., Kassiou, M., Henderson, J.M., 2006. Developing a

preclinical model of Parkinson’s disease: a study of behaviour in rats with

graded 6-OHDA lesions. Behav. Brain Res. 169, 1–9.

https://doi.org/10.1016/j.bbr.2005.11.026

Turek, J.W., Kang, C.H., Campbell, J.E., Arneric, S.P., Sullivan, J.P., 1995. A

sensitive technique for the detection of the alpha 7 neuronal nicotinic

acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and

brain. J. Neurosci. Methods 61, 113–118.

Umana, I.C., Daniele, C.A., McGehee, D.S., 2013. Neuronal nicotinic receptors as

analgesic targets: it’s a winding road. Biochem Pharmacol 86, 1208–14.


Ungerstedt, U., 1971. Striatal dopamine release after amphetamine or nerve

degeneration revealed by rotational behaviour. Acta Physiol. Scand. Suppl.

367, 49–68.

Ungerstedt, U., Arbuthnott, G.W., 1970. Quantitative recording of rotational

behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal

dopamine system. Brain Res. 24, 485–493.

Uteshev, V.V., 2014. The therapeutic promise of positive allosteric modulation of

nicotinic receptors. Eur. J. Pharmacol. 727, 181–185.

https://doi.org/10.1016/j.ejphar.2014.01.072

Uteshev, V.V., 2012. α7 nicotinic ACh receptors as a ligand-gated source of Ca(2+)

ions: the search for a Ca(2+) optimum. Adv. Exp. Med. Biol. 740, 603–638.

https://doi.org/10.1007/978-94-007-2888-2_27

Uversky, V.N., 2004. Neurotoxicant-induced animal models of Parkinson’s disease:

understanding the role of rotenone, maneb and paraquat in

neurodegeneration. Cell Tissue Res. 318, 225–241.

https://doi.org/10.1007/s00441-004-0937-z

Verhagen Metman, L., Del Dotto, P., van den Munckhof, P., Fang, J., Mouradian,

M.M., Chase, T.N., 1998. Amantadine as treatment for dyskinesias and motor

fluctuations in Parkinson’s disease. Neurology 50, 1323–6.

Vila, M., Vukosavic, S., Jackson-Lewis, V., Neystat, M., Jakowec, M., Przedborski,

S., 2000. Alpha-synuclein up-regulation in substantia nigra dopaminergic

218

neurons following administration of the parkinsonian toxin MPTP. J.

Neurochem. 74, 721–729.

Virmani, T., Moskowitz, C.B., Vonsattel, J.-P., Fahn, S., 2015. Clinicopathological

characteristics of freezing of gait in autopsy-confirmed Parkinson’s disease.



Voorn, P., Vanderschuren, L.J., Groenewegen, H.J., Robbins, T.W., Pennartz, C.M.,

2004. Putting a spin on the dorsal-ventral divide of the striatum. Trends

Neurosci 27, 468–74. https://doi.org/10.1016/j.tins.2004.06.006

Wahlström, B., Blennow, G., 1978. A study on the fate of curcumin in the rat. Acta

Pharmacol. Toxicol. (Copenh.) 43, 86–92.

Wang, D., Tang, B., Zhao, G., Pan, Q., Xia, K., Bodmer, R., Zhang, Z., 2008.

Dispensable role of Drosophila ortholog of LRRK2 kinase activity in survival

of dopaminergic neurons. Mol. Neurodegener. 3, 3.

https://doi.org/10.1186/1750-1326-3-3

Wang, Hong, Yu, M., Ochani, M., Amella, C.A., Tanovic, M., Susarla, S., Li, J.H.,

Wang, Haichao, Yang, H., Ulloa, L., Al-Abed, Y., Czura, C.J., Tracey, K.J.,

2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator

of inflammation. Nature 421, 384–388. https://doi.org/10.1038/nature01339

Wang, W.-H., Chiang, I.-T., Ding, K., Chung, J.-G., Lin, W.-J., Lin, S.-S., Hwang,

J.-J., 2012. Curcumin-induced apoptosis in human hepatocellular carcinoma

j5 cells: critical role of ca(+2)-dependent pathway. Evid.-Based Complement.

Altern. Med. ECAM 2012, 512907. https://doi.org/10.1155/2012/512907

Wang, X.-S., Zhang, Z.-R., Zhang, M.-M., Sun, M.-X., Wang, W.-W., Xie, C.-L.,

2017. Neuroprotective properties of curcumin in toxin-base animal models of

Parkinson’s disease: a systematic experiment literatures review. BMC

Complement. Altern. Med. 17, 412. https://doi.org/10.1186/s12906-017-

1922-x

Ward, R.J., Lallemand, F., de Witte, P., Dexter, D.T., 2008. Neurochemical

pathways involved in the protective effects of nicotine and ethanol in

preventing the development of Parkinson’s disease: potential targets for the

development of new therapeutic agents. Prog Neurobiol 85, 135–47.

https://doi.org/10.1016/j.pneurobio.2008.03.003

Warpman, U., Nordberg, A., 1995. Epibatidine and ABT 418 reveal selective losses

of alpha 4 beta 2 nicotinic receptors in Alzheimer brains. Neuroreport 6,

2419–23.

219

Ween, H., Thorin-Hagene, K., Andersen, E., Gronlien, J.H., Lee, C.H.,

Gopalakrishnan, M., Malysz, J., 2010. Alpha3* and alpha 7 nAChR-mediated

Ca2+ transient generation in IMR-32 neuroblastoma cells. Neurochem Int 57,

269–77. https://doi.org/10.1016/j.neuint.2010.06.005

Weiland, S., Witzemann, V., Villarroel, A., Propping, P., Steinlein, O., 1996. An

amino acid exchange in the second transmembrane segment of a neuronal

nicotinic receptor causes partial epilepsy by altering its desensitization

kinetics. FEBS Lett 398, 91–6.

West, M.J., 1999. Stereological methods for estimating the total number of neurons

and synapses: issues of precision and bias. Trends Neurosci. 22, 51–61

West, M.J., Ostergaard, K., Andreassen, O.A., Finsen, B., 1996. Estimation of the

number of somatostatin neurons in the striatum: an in situ hybridization study

using the optical fractionator method. J. Comp. Neurol. 370, 11–22.

https://doi.org/10.1002/(SICI)1096-9861(19960617)370:1<11::AID-

CNE2>3.0.CO;2-O

West, M.J., 1993. New stereological methods for counting neurons. Neurobiol.

Aging 14, 275–285.

West, M.J., Slomianka, L., Gundersen, H.J., 1991. Unbiased stereological estimation

of the total number of neurons in thesubdivisions of the rat hippocampus

using the optical fractionator. Anat. Rec. 231, 482–497.

https://doi.org/10.1002/ar.1092310411

Widmann, R., Sperk, G., 1986. Topographical distribution of amines and major

amine metabolites in the rat striatum. Brain Res. 367, 244–249.

Williams, D.R., Lees, A.J., 2009. How do patients with parkinsonism present? A

clinicopathological study. Intern. Med. J. 39, 7–12.

https://doi.org/10.1111/j.1445-5994.2008.01635.x

Williams, D.R., Watt, H.C., Lees, A.J., 2006. Predictors of falls and fractures in

bradykinetic rigid syndromes: a retrospective study. J. Neurol. Neurosurg.

Psychiatry 77, 468–473. https://doi.org/10.1136/jnnp.2005.074070

Williams, M., Raddatz, R., 2006. Receptors as drug targets. Curr. Protoc. Pharmacol.

Chapter 1, Unit 1.1. https://doi.org/10.1002/0471141755.ph0101s32

Williams-Gray, C.H., Foltynie, T., Lewis, S.J.G., Barker, R.A., 2006. Cognitive

deficits and psychosis in Parkinson’s disease: a review of pathophysiology

and therapeutic options. CNS Drugs 20, 477–505.

220

Wirdefeldt, K., Adami, H.O., Cole, P., Trichopoulos, D., Mandel, J., 2011.

Epidemiology and etiology of Parkinson’s disease: a review of the evidence.

Eur J Epidemiol 26 Suppl 1, S1-58. https://doi.org/10.1007/s10654-011-

9581-6

Wolf, E., Seppi, K., Katzenschlager, R., Hochschorner, G., Ransmayr, G.,

Schwingenschuh, P., Ott, E., Kloiber, I., Haubenberger, D., Auff, E., Poewe,

W., 2010. Long-term antidyskinetic efficacy of amantadine in Parkinson’s

disease. Mov. Disord. Off. J. Mov. Disord. Soc. 25, 1357–1363.


Wozniak, K.L., Phelps, W.A., Tembo, M., Lee, M.T., Carlson, A.E., 2018. The

TMEM16A channel mediates the fast polyspermy block in Xenopus laevis. J.

Gen. Physiol. https://doi.org/10.1085/jgp.201812071

wwPDB: Worldwide Protein Data Bank [WWW Document], n.d. URL

http://www.wwpdb.org/ (accessed 1.15.17).

Xie, X., Tao, Q., Zou, Y., Zhang, F., Guo, M., Wang, Y., Wang, H., Zhou, Q., Yu,

S., 2011. PLGA nanoparticles improve the oral bioavailability of curcumin in

rats: characterizations and mechanisms. J. Agric. Food Chem. 59, 9280–9289.

https://doi.org/10.1021/jf202135j

Xie, Y.X., Bezard, E., Zhao, B.L., 2005. Investigating the receptor-independent

neuroprotective mechanisms of nicotine in mitochondria. J Biol Chem 280,

32405–12. https://doi.org/10.1074/jbc.M504664200

Yang, J., Song, S., Li, J., Liang, T., 2014. Neuroprotective effect of curcumin on

hippocampal injury in 6-OHDA-induced Parkinson’s disease rat. Pathol. Res.

Pract. 210, 357–362. https://doi.org/10.1016/j.prp.2014.02.005

Yang, J.-S., Seo, S.W., Jang, S., Jung, G.Y., Kim, S., 2012. Rational engineering of

enzyme allosteric regulation through sequence evolution analysis. PLoS

Comput. Biol. 8, e1002612. https://doi.org/10.1371/journal.pcbi.1002612

Yang, T., Xiao, T., Sun, Q., Wang, K., 2017. The current agonists and positive

allosteric modulators of α7 nAChR for CNS indications in clinical trials. Acta

Pharm. Sin. B 7, 611–622. https://doi.org/10.1016/j.apsb.2017.09.001

Yang, Y., Wu, X., Wei, Z., Dou, Y., Zhao, D., Wang, T., Bian, D., Tong, B., Xia,

Ying, Xia, Yufeng, Dai, Y., 2015. Oral curcumin has anti-arthritic efficacy

through somatostatin generation via cAMP/PKA and Ca(2+)/CaMKII

signaling pathways in the small intestine. Pharmacol. Res. 95–96, 71–81.

https://doi.org/10.1016/j.phrs.2015.03.016

221

Yenari, M.A., Kauppinen, T.M., Swanson, R.A., 2010. Microglial activation in

stroke: therapeutic targets. Neurotherapeutics 7, 378–91.

https://doi.org/10.1016/j.nurt.2010.07.005

Yeon, K.Y., Kim, S.A., Kim, Y.H., Lee, M.K., Ahn, D.K., Kim, H.J., Kim, J.S.,

Jung, S.J., Oh, S.B., 2010. Curcumin produces an antihyperalgesic effect via

antagonism of TRPV1. J. Dent. Res. 89, 170–174.

https://doi.org/10.1177/0022034509356169

Young, G.T., Zwart, R., Walker, A.S., Sher, E., Millar, N.S., 2008. Potentiation of

alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.



Yu, S., Zheng, W., Xin, N., Chi, Z.-H., Wang, N.-Q., Nie, Y.-X., Feng, W.-Y.,

Wang, Z.-Y., 2010. Curcumin prevents dopaminergic neuronal death through

inhibition of the c-Jun N-terminal kinase pathway. Rejuvenation Res. 13, 55–

64. https://doi.org/10.1089/rej.2009.0908

Zaborszky, L., Vadasz, C., 2001. The midbrain dopaminergic system: anatomy and

genetic variation in dopamine neuron number of inbred mouse strains. Behav.

Genet. 31, 47–59.

Zbarsky, V., Datla, K.P., Parkar, S., Rai, D.K., Aruoma, O.I., Dexter, D.T., 2005.

Neuroprotective properties of the natural phenolic antioxidants curcumin and

naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson’s

disease. Free Radic. Res. 39, 1119–1125.

https://doi.org/10.1080/10715760500233113

Zhang, D., Bordia, T., McGregor, M., McIntosh, J.M., Decker, M.W., Quik, M.,

2014. ABT-089 and ABT-894 reduce levodopa-induced dyskinesias in a

monkey model of Parkinson’s disease. Mov. Disord. Off. J. Mov. Disord.

Soc. 29, 508–517. https://doi.org/10.1002/mds.25817

Zhang, D., McGregor, M., Bordia, T., Perez, X.A., McIntosh, J.M., Decker, M.W.,

Quik, M., 2015. α7 nicotinic receptor agonists reduce levodopa-induced

dyskinesias with severe nigrostriatal damage. Mov. Disord. Off. J. Mov.


Zhang, D.-W., Fu, M., Gao, S.-H., Liu, J.-L., 2013. Curcumin and diabetes: a

systematic review. Evid.-Based Complement. Altern. Med. ECAM 2013,

636053. https://doi.org/10.1155/2013/636053

Zhang, L., Oz, M., Weight, F.F., 1995. Potentiation of 5-HT3 receptor-mediated

responses by protein kinase C activation. Neuroreport 6, 1464–1468.

222

Zhang, X., Chen, Q., Wang, Y., Peng, W., Cai, H., 2014. Effects of curcumin on ion

channels and transporters. Front. Physiol. 5, 94.

https://doi.org/10.3389/fphys.2014.00094

Zhi, L., Dong, L., Kong, D., Sun, B., Sun, Q., Grundy, D., Zhang, G., Rong, W.,

2013. Curcumin acts via transient receptor potential vanilloid-1 receptors to

inhibit gut nociception and reverses visceral hyperalgesia.

Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc. 25, e429-440.

https://doi.org/10.1111/nmo.12145

Zhou, F.M., Liang, Y., Dani, J.A., 2001. Endogenous nicotinic cholinergic activity

regulates dopamine release in the striatum. Nat Neurosci 4, 1224–9.

https://doi.org/10.1038/nn769

Zhou, F.M., Wilson, C.J., Dani, J.A., 2002. Cholinergic interneuron characteristics

and nicotinic properties in the striatum. J Neurobiol 53, 590–605.

https://doi.org/10.1002/neu.10150

Zhou, H., Beevers, C.S., Huang, S., 2011. Targets of curcumin. Curr. Drug Targets

12, 332–347.

Zhou, X., Nai, Q., Chen, M., Dittus, J.D., Howard, M.J., Margiotta, J.F., 2004. Brain-

derived neurotrophic factor and trkB signaling in parasympathetic neurons:

relevance to regulating alpha7-containing nicotinic receptors and synaptic

function. J Neurosci 24, 4340–50. https://doi.org/10.1523/jneurosci.0055-

04.2004

Zoli, M., Pistillo, F., Gotti, C., 2015. Diversity of native nicotinic receptor subtypes

in mammalian brain. Neuropharmacology 96, 302–11.


223

List of Publications

El Nebrisi, E.G., Bagdas, D., Toma, W., Al Samri, H., Brodzik, A., Alkhlaif, Y.,

Yang, K.-H.S., Howarth, F.C., Damaj, I.M., Oz, M., 2018. Curcumin Acts as

a Positive Allosteric Modulator of α7-Nicotinic Acetylcholine Receptors and

Reverses Nociception in Mouse Models of Inflammatory Pain. J. Pharmacol.

Exp. Ther. 365, 190–200. https://doi.org/10.1124/jpet.117.245068

Nebrisi, E.E., Al Kury, L.T., Yang, K.-H.S., Jayaprakash, P., Howarth, F.C.,

Kabbani, N., Oz, M., 2018. Curcumin potentiates the function of human α7-

nicotinic acetylcholine receptors expressed in SH-EP1 cells. Neurochem. Int.

114, 80–84. https://doi.org/10.1016/j.neuint.2017.12.010

Oz, M., El Nebrisi, E.G., Yang, K.-H.S., Howarth, F.C., Al Kury, L.T., 2017.

Cellular and Molecular Targets of Menthol Actions. Front. Pharmacol. 8,

472. https://doi.org/10.3389/fphar.2017.00472

Sultan, A., Yang, K.-H.S., Isaev, D., Nebrisi, E.E., Syed, N., Khan, N., Howarth,

C.F., Sadek, B., Oz, M., 2017. Thujone inhibits the function of α7-nicotinic

acetylcholine receptors and impairs nicotine-induced memory enhancement

in one-trial passive avoidance paradigm. Toxicology 384, 23–32.

https://doi.org/10.1016/j.tox.2017.04.005

224

Appendix

Optimization

In-vitro

• Acetylcholine

- Stock solution of the Acetylcholine (100 µM) was prepared in ND96 solution

using the following formula:


= 181.7 x 0.2 L x 0.100

= 3.6 mg of ACh in 200 ml ND96

- Further dilutions were prepared using the Charles equation:

C1 x V1 = C2 x V2

- For preparation of 10 µM ACh;

100 x V1 = 10 x 100 ml (ND96)

V1 = 10 ml of 100 µM stock solution plus 90 ml ND96

= 100 ml ACh solution of 10 µM concentration, divided into two parts:

50 ml for first application line, and the remaining 50 ml was for the preparation of

the second application line solution.

Stock solutions and required dilutions were prepared freshly before

starting the experiments.

ACh was applied every five minutes. Shorter intervals would lead to stimulation

the receptor during the desensitized state (the channel is insensitive to agonist

stimulus).

225

• Curcumin

Curcumin is a highly hydrophobic compound and is soluble only in organic solvents

e.g., dimethyl sulfoxide (DMSO).

Stock solution of the curcumin (10 µM) was prepared in DMSO (0.001%) using

the following formula:


= 368.38 x 0.001 L x 10

= 3.68 mg of curcumin in 1 ml of DMSO

- Further dilutions were prepared using the Charles equation:

C1 x V1 = C2 x V2

- For preparation of 10 µM curcumin;

10 x V1 = 0.01 x 50 ml (ACh sol.)

V1 = 0.05 ml

= 50 µl of curcumin stock solution (10 µM) plus 50 ml ACh solution

prepared in the First step; second application line.

- First perfusion was ND96 physiological solution

- Second perfusion using curcumin stock solution (10 µM)

C1 x V1 = C2 x V2

10 x V1 = 0.01 x 600 ml ND96

V1 = 0.6 ml of curcumin stock solution (10 µM) plus 600 ml ND96.

Since the magnitude of the curcumin effect was time-dependent, 10-minute

curcumin application time was used routinely to ensure equilibrium conditions.

Same concept was applied for different concentrations of curcumin used in the study,

and also for other curcumin derivatives with the respect of the molecular weight of

each compound.

226

• Glycine

- Preparation of 30 µM from stock solution:

C1 x V1 = C2 x V2

100 x V1 = 0.03 x 100

V1 = 30 µl of Gly (100 mM) in 100 ml Ringer physiological solution.



To prepare 10 µM from 100 mM stock curcumin:

C1 x V1 = C2 x V2

100 x V1 = 0.01 x 50

V1 = 5 µl of curcumin stock solution (100 mM) in 50 ml of Gly (30 µM) Ringer

solution, as a second application line.

- First perfusion was Ringer physiological solution


C1 x V1 = C2 x V2

10 x V1 = 0.01 x 600 ml ND96

V1 = 0.6 ml of curcumin stock solution (10 µM) plus 600 ml Ringer solution.

• 5HT3

- Preparation of 1 µM from stock solution:

C1 x V1 = C2 x V2

100 x V1 = 0.001 x 100

V1 = 1 µl of 5HT (100 mM) in 100 ml Ringer physiological solution.



227

- To prepare 10 µM from 100 mM stock curcumin:

C1 x V1 = C2 x V2

100 x V1 = 0.01 x 50

V1 = 5 µl of curcumin stock solution (100 mM) in 50 ml of Gly (30 µM) Ringer

solution, as a second application line.

- First perfusion was Ringer physiological solution


C1 x V1 = C2 x V2

10 x V1 = 0.01 x 600 ml ND96

V1 = 0.6 ml of curcumin stock solution (10 µM) plus 600 ml Ringer solution.

• BAPTA

- Preparation of stock solution:


= 476.23 x 0.001 x 100

= 47.623 mg BAPTA in 1 ml D/W

pH was adjusted to 7.4 by CsOH, and stored at +4℃

• Barium

- Preparation of Barium-containing solution:

ND96 solution but with substitution of BaCl2 (1.8 mM) instead of CaCL2 (1.8 mM).

• Protein Kinases Inhibitors

Stock solution of (100 µM) for each protein kinase inhibitor, to prepare the working

concentration of 10 µM using the formula:

C1 x V1 = C2 x V2

228

100 x V1 = 0.1 x 10

V1 = 0.01 ml

V1 = 10µM of stock solution plus 1 ml ND96 physiological solution for pre-

incubation.

Oocytes were pre-incubated for 30 minutes before electrophysiological recording.

• Nicotine

- Preparation of 100 mM stock solution:


= 462.4 x 0.001x 100

= 46.24 mg in 1 ml D/W

- To prepare 100 µM from 100 mM stock solution of nicotine

C1 x V1 = C2 x V2

100 x V1= 0.1x100

V1= 0.1 ml of tock in 100 ml of D/W

In-vivo

• 6-hydroxydopamine (6-OHDA)

- Preparation of 0.9% NaCl:

0.9 NaCl in 100 ml H2O

- Preparation of 0.01% ascorbic acid solution:

10 mg of L-ascorbic acid in 0.9% NaCL

- Preparation of 6-OHDA injection solution:

229

Thirty-five milligram of 6-OHDA was dissolved in 1o ml of ice-cold 0.01%

ascorbate in 0.9% normal saline and aliquoted into 250 µl Eppendorf tubes and

kept in -40℃ until use.

• Curcumin

- Preparation of 0.5% CMC (vehicle):

0.5 gm CMC added to 100 ml distilled water, stirred, and heated up to 60℃ until it

dissolves completely.

- 200 mg of curcumin plus 50 µl of NaOH (10M) in 2 ml of 0.5% CMC.

• Methyllycaconitine (MLA)

Methyllycaconitine (MLA) is the most potent and selective competitive antagonist

of α7-nAChR (Turek et al., 1995). MLA was prepared in normal saline, no need for

pH adjustment, kept in +4℃ in fridge and its stable compound. MLA half-life is

around 19 min, and for in-vivo use its administered 10 min for maximal effect.

- Dose: 1 mg/kg/rat for 4 weeks, other research papers have used it 1-3 mg/kg,

but to avoid toxicity the daily dose should be 1/10th of the LD50 (LD50=5

mg/kg, 1/10th = 0.5 mg/kg), so we have used (1 mg/kg rat) = (1 µl/gram rat).

• Apomorphine

The dose of apomorphine that has been used in the study (0.25 mg/KG SC) (Kirik

et al., 1998) was significantly lower than the toxic dose (160 mg/kg) (“Sax’s

Dangerous Properties of Industrial Materials, 5 Volume Set, 12th Edition,” n.d.).

Apomorphine was dissolved in 0.1 ascorbic acid in saline and prepared on demand.

Animals were injected subcutaneously with apomorphine-HCL.

Documents

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