Università degli Studi di Cagliari

DOTTORATO DI RICERCA

Tossicologia – Indirizzo Farmacologia e Farmacoterapia delle

Tossicodipendenze

Ciclo XXVIII

Influence of different neuroprotective drugs on dopamine

neurotoxicity induced by 3,4-methylenedioxymethamphetamine

and MPTP in mice

Settore scientifico disciplinare di afferenza

BIO/14

Presentata da: Dott.ssa Pier Francesca Porceddu

Coordinatore Dottorato Prof. Gaetano Di Chiara

Tutor Prof.ssa Micaela Morelli

Esame finale anno accademico 2014 – 2015

Abstract

Introduction: Parkinson‟s disease (PD) is characterized by a chronic progressive loss of

nigrostriatal dopaminergic neurons that is associated with chronic neuroinflammation. Current

treatments for PD can significantly improve symptoms but do not cure the disease or slow its

progression. An approach used in existing therapies is based on the inhibition of monoamine

oxidase (MAO), enzyme involved in the metabolic degradation of dopamine. Although, preclinical

studies showed that MAO-B inhibitors have neuroprotective activity in cellular and animal models

of PD, clinical trials did not completely confirm this result. Therefore a large number of new

molecules, with more potent MAO-B inhibitory activity and a possible neuroprotective effect, have

been proposed to replace the pre-existing MAO-B inhibitors. The profile of the recent MAO

inhibitor, SZV558, appears to be particularly interesting because of its pharmacodynamic, favorable

for disease-modifying properties and its irreversible MAO-B enzyme bind.

The enhancement of adult neurogenesis could be of great clinical interest in the management of

neurodegenerative disorders. In line with this, the metformin, a well-known antidiabetic drug, has

recently been proposed to promote neurogenesis and to have a neuroprotective effect on the

neurodegenerative processes induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP) in a mice PD model.

Although, PD has multiple origins, one hypothesis is that amphetamine-related drugs may be part of

the wide array of factors leading to the dopaminergic neuron degeneration that causes the disease.

These hypothesis are supported by different results that showed a persistent, long-term

dopaminergic toxicity induced by 3,4-methylenedioxymethamphetamine (MDMA) in mice.

Moreover, the MDMA, altering the dopaminergic transmission, may affect neurogenesis and

synaptogenesis. On these basis, considering that the young brain is particularly sensitive to drug-

induced neurotoxicity, the consumption of MDMA during the adolescence might increase the

vulnerability of dopaminergic neurons. However, the use of amphetamine-related drugs by

adolescent and young people is often combined with caffeinated energy drinks in order to amplify

their stimulant actions. Although caffeine use is safe, the combined treatment of caffeine and

MDMA increases not only the DA release but also the microglia and astroglia activation.

Aims: During my Ph.D. I studied the influence of neuroprotective drugs, such as MAO inhibitors

and metformin, or substances, such as caffeine, on the neurodegenerative effects of two

dopaminergic toxins, MDMA and MPTP, in mice.

1. In the first phase of my study, I evaluated the neuroprotective activity of the new MAO-B

inhibitor SZV558, compared with well-known rasagiline, in a chronic mouse model of MPTP

plus probenecid (MPTPp), which induces a progressive loss of nigrostriatal dopaminergic

neurons.

2. Previous results showed that when MDMA is associated with caffeine, a more pronounced

degeneration in adolescent compared with adult mice was observed. To better clarify the

molecular mechanism at the base of the different neurotoxic effect of this drug association at

different ages, I evaluated the neuronal nitric oxide synthase (nNOS) expression, which plays a

critical role in the integration of dopaminergic and glutamatergic transmissions, in the CPu of

adolescent or adult mice treated with MDMA, alone or in combination with caffeine.

3. Finally, I investigated the neuroprotective effect of metformin against dopaminergic

neurotoxicity induced by MDMA in the CPu and SNc of adult mice.

Conclusions: These results demonstrated that the dopaminergic neurodegenerative process may be

induced or conditioned by environment stressors or substances which influence, through different

ways, the development of neurodegenerative mechanisms. In the present study I evaluated the

effects of 3 substances, known as potentially neuroprotective, in combination with two different

neurotoxins that affect the nigrostriatal dopaminergic system. The SZV558 MAO-B inhibitor and

the metformin protected the nigrostriatal pathway, usually affected in PD, by MPTP- and MDMA-

induced neurotoxicity, respectively. On the other hand, caffeine, administrated with MDMA,

showed a neurotoxic potential depending on the age of consumers, confirming the vulnerability of

adolescent brain to consumption of drug and substances that affected the dopaminergic system. In

conclusion, the study of neurodegenerative processes may be relevant to understand the human

pharmacology, the origin and development of neurodegenerative disease and to predict the

neurotoxic effect of drug abuse.

List of abbreviations:

5-HIAA 5-hydroxyl indole acetic acid

5-HT 5-hydroxytryptamine

6-OHDA 6-hydroxidopamine

ADAGIO Attenuation of disease progression with azilect given once-daily

a-MeDA a-methyldopamine

AMPK AMP-activated protein kinase

ANOVA Analysis of variance

ATP Adenosine triphosphate

BBB Blood brain barrier

BDNF Brain-derived neurotrophic factor

cAMP cyclic adenosine monophosphate

CNS Central nervous system

COMT Catechol-o-methyltransferase

CPu Caudate-putamen

CREB cAMP cresponsive element binding protein

CSF Cerebrospinal fluid

DA Dopamine

DAPI 4′,6-diamidine-2′-phenylindole dihydrochloride

DAQ DA quinone

DAT DA transporter

DOPAC 3,4-dihydroxyphenylacetic acid

DOPAL 3,4-dihydroxyphenylacetaldehyde

FDA Food and drug administration

GABA Gamma-amino butyric acid

GDNF Glial cell-derived neurotrophic factor

GFAP Glial fibrillary acidic protein

GLU Glutamate

HHA Dihydroxyamphetamine

HHMA 3, 4 –dihydroxymethamphetamine

HVA Homovanilic acid

IL Interleukin

iNOS inducible nitric oxide synthase

JPND Join programme for neurodegenerative disease

L-DOPA Levo-DOPA

LPS Lipopolysaccharide

MAO Monoaminooxidase

MAO-B Monoaminooxidase-B

MDA 3,4-methylenedioxyamphetamine

MDMA 3,4-methylenedioxymethamphetamine

MPDP+ 1-methyl-4-phenyl-2,3-dihydropyridium

MPP+ 1-methyl-4-phenylpyridinium

MPPP 1-methyl-4-phenyl-4-propionoxypiperidine

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

MPTPp 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid

NA Noradrenaline

NET NA transporter

NMDA N-methyl-D-aspartic acid

N-Me-a-MeDA N-methyl-a-methyldopamine

nNOS neuronal nitric oxide synthase

NO Nitric oxide

PB Phosphate buffer

PBS Phosphate buffer solution

PD Parkinson‟s disease

PET Positron emission tomography

PKC–CREB Protein kinase C–cyclic-adenosine monophosphate (cAMP) response element-binding

protein

RNS Reactive nitrogen species

ROS Reactive oxygen species

SERT Serotonin transporter

SN Substantia nigra

SNc Substantia nigrapars compacta

SOD Superoxide dismutase

TH Tyrosine hydroxylase

TNF-α Tumour necrosis factor α

VMAT2 Vesicular monoamine transporter 2

Table of contents

INTRODUCTION ......................................................................................................................... 1

1. Neurodegeneration .................................................................................................................... 1

1.1 Definition of Neurodegeneration ....................................................................................... 1

1.2 Role of environmental factors exposure in Neurodegeneration .......................................... 1

1.3 Molecular mechanisms on the basis of Neurodegeneration................................................. 2

1.4 Dopamine vulnerability to neurodegenerative processes ........................................................ 3

2. Parkinson‟s disease .................................................................................................................... 6

2.1 PD pathophysiology ........................................................................................................... 6

2.2 PD animal models ............................................................................................................. 7

3. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ........................................................................... 8

3.1 MPTP: a doparminergic neurotoxin .................................................................................. 8

3.2 Mechanism involved in MPTP neurotoxicity ....................................................................... 9

3.2.1 Oxidative stress and neuroinflammation .................................................................... 10

3.3 Neurotoxicity in humans ........................................................................................................... 11

3.4 MPTP as PD mouse model ....................................................................................................... 12

4. 3,4-methylenedioxymethamphetamine ............................................................................................ 13

4.1 MDMA: a neurotoxic drug........................................................................................................ 13

4.2 Mechanism involved in MDMA neurotoxicity ......................................................................... 14

4.2.1 Oxidative stress ........................................................................................................ 15

4.2.2 Neuroinflammation and Hyperthermia ......................................................................... 16

4.3 Neurotoxicology in humans ..................................................................................................... 18

4.3 MDMA administration in mice: a model of dopaminergic neurotoxicity .............................. 19

4.5 MDMA neurotoxicity and adolescence .................................................................................... 20

5. PD therapy .......................................................................................................................................... 21

5.1 Current PD therapies ................................................................................................................ 21

5.2 Monoamine oxidase inhibitors therapy .................................................................................... 21

5.3 Novel (hetero)arylalkenyl propargylamine compounds.......................................................... 23

5.4 Metformin ................................................................................................................................... 24

5.5 Caffeine: Neuroprotective or Neurotoxic? ............................................................................. 25

AIMS ........................................................................................................................................... 28

MATERIALS AND METHODS ................................................................................................ 30

1. Drugs ....................................................................................................................................... 30

2. Treatments ............................................................................................................................... 30

2.1 Chronic protocol of MPTPplus probenecid MPTPp ......................................................... 30

2.2 Acute MDMA treatment in combination with caffeine ....................................................... 30

2.3 Acute MDMA treatment in combination with metformin ................................................... 31

3. Behavioral tests ......................................................................................................................... 31

3.1 Spontaneousmotor activity: Motility test ........................................................................... 31

3.2 Beam walking test............................................................................................................. 31

3.3 Inverted grid test .............................................................................................................. 32

3.4 Pellet retrieval olfactory test ............................................................................................ 32

4. Immunohistochemistry .............................................................................................................. 32

4.1 Immunohistochemistry and cresyl violet for Nissl staining ................................................ 32

4.2 Immunofluorescence for nNOS, IL-1β and TNF-α ............................................................ 33

4.3 Analisys of TH-positive cells and Nissl staining in the SNc ............................................... 33

4.4 Analisys of TH-positive fibers in CPu ............................................................................... 34

4.5 Analisys of nNOS-positive cells in CPu ............................................................................. 34

4.6 Analisys of IL-1β and TNF-α immunoreactivity in CPu ..................................................... 35

5. Statistical Analysis .................................................................................................................... 35

RESULTS .................................................................................................................................... 36

1. SZV558 administration reverts the motor impairments, olfactory dysfunction and dopaminergic

neuron degeneration induced by a chronic MPTPp treatment .................................................... 36

1.1 Changes in spontaneous motor activity: Motility test ........................................................ 36

1.2 Effect of SZV558 on motor impairment inducedby MPTPp: beam walking test ................. 36

1.3 Grasp-strength evaluation: inverted grid test.................................................................... 38

1.4 Effect of SZV558 on olfactory deficit induced by MPTPp:olfactory test ............................ 38

1.5 Effect of SZV558 on MPTPp-induced neurodegneration: TH immunohistochemistry and

Nissl staining in the SNc and CPu ................................................................................... 38

2. Effects of repeated MDMA+caffeine administration in adolescent and adult mice .................... 40

2.1 nNOS activation in the CPu of adolescent and adult mice ................................................. 40

2.2 IL-1β activation in the CPu of adolescent and adult mice ................................................. 41

2.3 TNF-α activation in the CPu of adolescent and adult mice................................................ 42

3. Neuroprotective effects of metformin administration on MDMA-induced neurodegeneration: TH

immunoreactivity and Nissl staining ......................................................................................... 44

DISCUSSION ............................................................................................................................. 46

1. The neuroprotective effects of SZV558 in a chronic MPTPp model of PD ................................ 46

2. The neurotoxic effect of caffeine on repeated MDMA administration in adult and adolescent

mice .............................................................................................................................................. 49

3. The neuroprotective effects of metformin on neurodegeneration induced by repeated MDMA

administration ........................................................................................................................... 52

CONCLUSIONS ......................................................................................................................... 54

REFERENCES............................................................................................................................ 55

1

INTRODUCTION

1. Neurodegeneration

1.1 Definition of Neurodegeneration

The term “Neurodegeneration” refers to a pathological condition characterized by dysfunction and /

or death of neurons in brain and spinal cord. Etymologically, the word is composed of the prefix

“neuro-,” which denotes relationship to the nervous system, and “degeneration,” which refers to, in

the case of tissues or organs, a process of losing structure or function (Jennekens 2014). Thus, in the

strict sense of the word, neurodegeneration corresponds to any pathological condition primarily

affecting neurons. However, this term is ever used to characterize a diverse group of neurological

disorders known as neurodegenerative disease (Przedborski et al. 2003). The European Joint

Programme for Neurodegenerative Disease (JPND) in the 2012 defined the neurodegenerative

disease as “an umbrella term for a range of conditions primarily involving neurodegeneration which

is the loss of structure or functions of neurons”. Definitions in the literature indicate that

neurodegenerative diseases are considered as to be age related, incurable, and largely untreatable

chronic progressive diseases of the central nervous system (Jennekens 2014). In general,

neurodegenerative diseases are defined as hereditary and sporadic conditions which are

characterized by progressive nervous system dysfunction. Although hundreds of neurological

disorders may fit the definition of a neurodegenerative disease, many are rare and have been found

to be caused by purely genetic factors. A small number of neurodegenerative diseases are relatively

common, such as Alzheimer‟s and Parkinson‟s disease (PD) and characterized by heterogeneous

clinical and pathological expressions affecting specific subsets of neurons in specific functional

anatomic systems (Cannon and Greenamyre 2011).

1.2 Role of environmental factors exposure in Neurodegeneration

The exact etiology at the basis of neurodegenerative processes is not well known. They involve

specific combinations of genetic predispositions and environmental stressors exposure, that trigger

oxidative and proteostasis dysfunction in vulnerable neurons, in critical ages for the development of

brain (Saxena and Caroni 2011). Instead, toxic environmental factors may be the prime suspects in

initiating neurodegenerative processes. The environmental hypothesis posits that

neurodegeneration, such as PD related degeneration, results from exposure to a neurotoxin.

Theoretically, the progressive neurodegeneration of PD could be produced by chronic neurotoxin

exposure or by limited exposure initiating a self-perpetuating cascade of deleterious events (Cannon

and Greenamyre 2011). The finding that people intoxicated with 1-methyl-4-phenyl-1,2,3,6-

2

tetrahydropyridine (MPTP) develop a syndrome nearly identical to PD (Langston et al. 1983) is a

prototypic example of how an exogenous toxin can mimic the clinical and pathological features of a

neurodegenerative disease. In fact, MPTP is able to enter and destroy dopaminergic neurons

producing a severe and irreversible parkinsonian syndrome, which is almost identical to PD

(Przedborski and Vila 2001). Numerous chemical agents may induce a behavioral phenotype known

as parkinsonism, which shares some of the behavioral features of PD, but often has different

mechanistic and pathological correlates. Consequently, the majority of the exposures may actually

bear limited relevance to the etiology of PD (Cannon and Greenamyre 2011). However, considering

the numerous environmental toxicants by which humans are exposed in the course of a life, it is

difficult to identify a single environmental factor accounting for a significant number of cases.

Netherless, recent several clinical reports founding that patients diagnosed with neurodegenerative

diseases, such as PD, had a higher rate of exposure to amphetamine-related drugs at a young age,

compared with the general population (Parrott et al. 2004; Callaghan et al. 2010; Christine et al.

2010; Curtin et al. 2015). These data suggest a possible correlation between amphetamine-related

drugs and the PD etiology.

1.3 Molecular mechanism on the basis of Neurodegeneration

Although the neurodegenerative diseases show different pathophysiology, they have in common

specific molecular mechanisms, such as mitochondrial dysfunctions and oxidative stress,

aggregated protein deposits, neuroinflammation.

The presence in tissue of proteinaceous deposits is a hallmark of all neurodegenerative disease.

Although the composition and localization (intra- or extracellular) of protein aggregates differ from

disease to disease, this common feature suggests that protein deposition per se, or some related

event, is toxic to neurons. Aggregated or soluble misfolded protein could be neurotoxic through a

variety of mechanisms. Protein aggregates could directly cause damage, perhaps by deforming the

cell or indirectly interfering with the proteosomal functions (Dauer and Prezedborki 2003).

Another important factor which has been associated with many chronic neurodegerative conditions

is the inflammatory response in the central nervous system (CNS) (Stoll and Jander 1999). In fact

recent studies have demonstrated that glial activation participates in the events that induce neuronal

damage (Barcia et al. 2011). Investigating the specific correlation between neuroinflammation and

neurodegeneration, several studies have showed the presence of a glial response both concurrently

and after the dopaminergic neurodegeneration (Araki et al. 2001; Wu et al. 2002; Barcia et al. 2004;

Novikova et al. 2006; Yazdani et al. 2006). Some researchers speculated that increased pro-

inflammatory response could result in a delayed and progressive loss in dopaminergic neurons in

3

the substantia nigra (SN), similar to that seen in PD (Qin et al. 2007). The chronic

neuroinflammation might be involved in neurodegenerative processes through the chronic release of

toxic mediators, such as proiflammatory cytokines, which would attack surrounding neurons

eventually contributing to their death through apoptotic mechanisms, potentiating

neurodegenerative processes (Wu et al. 2002; Hirsch et al. 2003; Hald and Lotharius 2005; Tansey

et al. 2007). On this basis, it is plausible to speculate that drugs which prevent or counteract the

detrimental consequences of stress on inflammatory pathways may offer novel treatments for a

variety of neurodegenerative pathologies (Hurley and Tizabi, 2013).

Finally, abnormalities in mitochondrial functions detected in a range of neurodegenerative disease,

and in evidences from disease models suggest that mitochondrial dysfunctions may play a role in

disease pathogenesis (Dauer and Przedborski 2003). In particular, these pathological dysfunctions

trigger the production of oxidative stress factors. The oxidative stress is the result of the imbalance

between reactive oxygen species (ROS), such as peroxidase and free radicals, and the ability of the

biological system to detoxify them. ROS causes lipid peroxidation, cytoskeleton disorganization

and DNA phenomena that convey in cell death (Luo et al. 1998).

1.4 Dopamine vulnerability to neurodegenerative processes

It has been speculated that dopaminergic neurons present an elevated vulnerability to

neurodegenerative processes. In vitro studies demonstrated that the application of dopamine (DA)

induces death of striatal cells (Cheng et al. 1996). At physiological concentrations DA do not

exhibits toxicity, but malfunctions on DA release and/or her metabolism could lead

neurodegeneration. Although, the mechanisms is still unclear, several evidences showed that the

DA neurotoxic effects are associated with the production of ROS caused by DA metabolites such

as, DA-quinone (DAQ) (Cadet et al. 1997; Blum et al. 2001; Wersinger et al. 2004). Dopaminergic

neurons may be a particularly fertile environment for the generation of ROS, due to the metabolism

of DA producing hydrogen peroxide and superoxide radicals, and the auto-oxidation of DA

producing DAQ (Graham 1978), a molecule that damages proteins by reacting with cysteine

residues and inhibits ROS scavenger enzymes (Hauser et al. 2013).

The oxidative stress seems initiating with the interaction of DA with mitochondrial oxidative

phosphorylation system causing inhibition of complex 1 and decreasing adenosine triphosphate

(ATP) (Ben-Shachar et al. 2004). In particular, inhibition of complex 1 increases the production of

the superoxide, which may form toxic hydroxyl radicals or react with nitric oxide (NO) to form

peroxynitrite. These molecules may cause cellular damage by reacting with nucleic acids, proteins,

and lipids. However, one target of these reactive species may be the electron transport chain itself

4

(Cohen 2000), leading to mitochondrial damage and further production of reactive oxygen species

(ROS). This mitochondria-related energy failure may disrupt vesicular storage of DA, causing the

free cytosolic concentration of DA to rise and allowing harmful DA-mediated reactions to damage

cellular macromolecules (Fig. 1).

Finally, DA activates apoptotic signaling through mechanisms of oxidation (Luo et al. 1998) and

necrotic cell death (Di Filippo et al. 2006).

Fig. 1. Factors that trigger oxidative stress in neurodegenerative processes.

Dopaminergic neurons of the substantia nigra pars compacta (SNc), the area most affected in PD,

appear to be particularly vulnerable to oxidative stress induced by mitochondrial dysfunction

(Biskup and Moore, 2006). Studies addressing the excitability properties of these neurons have

provided that the vulnerability is induced by particular channels (Cav1.3 low voltage-dependent L-

type Ca-channels) that open at relatively hyperpolarized membrane potentials, leading to high Ca

flux loads in DA SNc neurons (Chan et al. 2007), producing oxidative stress (Guzman et al. 2010).

However, this neuronal SNc property if associated with mitochondrial dysfunction that

compromises the ability of mitochondria to accumulate Ca++

, may induce a neurodegenerative

5

process (Autere et al. 2004). The susceptibility of dopaminergic neurons to mitochondrial

dysfunction, could even explain the ability of several toxins, such as MPTP and 3,4-

methylenedioxymethamphetamine (MDMA), to induce cell death in DA neuronal populations

(Biskup and Moore 2006).

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2. Parkinson‟s Disease

2.1 PD pathophysiology

PD is the second most common neurodegenerative disease, affecting 1% of the population over 55

years of age (Lees et al. 2009). The main features of PD are tremor, muscle rigidity, bradykinesia,

and postural instability, whose intensity increases as the neurodegenerative process progresses;

however, these motor manifestations can be accompanied by non-motor symptoms such as

olfactory deficits, sleep impairments, and neuropsychiatric disorders (Forno 1996; Chaudhuri et al.

2006). The evident disease is characterized by the loss of over 70% of the dopaminergic neurons in

the SNc, a profound decrease of DA in the striatum, and the presence of intracytoplasmic inclusions

called Lewy bodies, which are composed mainly of α-synuclein and ubiquitin (Lees et al. 2009).

Although a variety of possible pathogenetic mechanisms have been proposed over the years,

including excessive release of oxygen free radicals, dysfunction of protein degradation, activation

of glia and impairment of mitochondrial function, the pathogenesis of PD is still largely uncertain

(Pringsheim et al. 2014; Block and Hong 2005).

As mentioned before, the SNc is particularly vulnerable to neurodegeneration induced by

mitochondrial dysfunction (Biskup and Moore 2006).

The hypothesis that mitochondrial dysfunctions play a role in the pathogenesis of PD was fueled by

the discovery that MPTP block the mitochondrial electron transport chain by inhibiting complex 1

(Nicklas et al. 1987). Subsequently, several studies identified abnormalities in complex 1 activity in

PD (Greenamyre et al. 2001). In vitro studies indicate that complex 1 defect may subject cells to

oxidative stress and energy failure. However, several biological markers of oxidative damage,

consistent with increased ROS, are elevated in the SNc of PD brains (Sian et al. 1994).

Moreover, other neurodegenerative conditions, such as the neuroinflammation, play vital roles in

the degeneration of dopaminergic neurons (Dauer and Przedborski 2003). However, emerging

evidence indicates that sustained inflammatory responses, T cell infiltration and glial cell activation

are common features of both human PD patients and animal models of PD (Hirsch et al. 2012; Lv et

al. 2015). In particular the involvement of neuroinflammation in PD pathogenesis has been

suggested by positron emission tomography (PET) studies that showed in the SNc of PD patients, a

pronounced activation of microglia, one of the major cell types which are involved in the

inflammatory responses in the CNS (Bartels et al. 2010; Gerhard et al. 2006). Further biochemical

analysis reveals higher levels of proinflammatory mediators, released by reactive microglia,

including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), in the midbrain of PD patients

(Boca et al 1994; Mogi et al. 1994; Brodacki et al. 2008). These data confirm the involvement of

7

immune components in PD pathology. A great body of studies shows that even astrocytes play a

role in the neuroinflammatory processes in PD. Like microglia, astrocytes respond to the

inflammatory stimulations such as Lipopolysaccharide (LPS), producing pro-inflammatory

cytokines both in vitro and in vivo (Saijo et al. 2009; Tanaka et al. 2013). Finally, reactive

astrogliosis characterized by the increased expression levels of glial fibrillary acidic protein (GFAP)

and hypertrophy of cell body and cell extensions have been reported in various PD animal models.

2.2 PD animal models

The most direct method to understand etiology, pathology, and molecular mechanisms of PD is the

use of various animal models. For the past several decades, animal models of PD have come in a

variety of forms. Typically, they can be divided into those using environmental or synthetic

neurotoxins or those utilizing the in vivo expression of PD-related mutations. Although the

identification of different genetic mutations (α-synuclein, parkin, LRKK2, PINK1, DJ-1) has led to

the development of genetic models of PD (Dawson et al. 2010), it is important to remember that,

only ∼10% of PD cases are due to genetic mutations (Dauer and Przedborski 2003), while the vast

majority of PD cases are sporadic and from unknown origins.

In the neurotoxic models have been used compounds that produce both reversible (reserpine) and

irreversible (MPTP, 6-hydroxydopamine (6-OHDA), paraquat, rotenone) effects. Recent studies

have focused more on irreversible toxins to produce PD-related pathology and symptomatology.

Neurotoxin-based models produced by 6-OHDA and MPTP administration are the most widely

used toxic models, while paraquat and rotenone are more recent additions to the stable of toxic

agents used to PD model (Dauer and Przedborski 2003; Betarbet et al. 2002). This strategy quite

popular among PD researchers is based on the premise that dopaminergic neurons have a

stereotyped death cascade that can be activated by a range of insults, including neurotoxins. A

common feature of all toxin-induced models is their ability to cause an oxidative stress and cell

death in DA neuronal populations. As mentioned above, oxidative stress results from increased

production of extremely reactive free radicals, which may be formed during a number of cellular

processes, including mitochondrial oxidative respiration and DA metabolism.

In addition to oxidative stress and mitochondrial deficits, many pathogenic mechanisms such as

chronic neuroinflammation, autophagy, and proteasomal dysfunction are believed to sustain and

amplify the neurodegenerative process, in PD animal models (Barcia et al. 2003; Hirsch et al. 2003;

Olanow 2007; Zhou et al. 2008).

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

3.1 MPTP: a dopaminergic toxin

As mentioned before, over the years, a variety of toxins of uncertain relevance inducing PD have

been used as agents to destroy dopaminergic neurons. However, none of the validated toxic models

of PD is a homolog of the disease, even though these models replicate many, but never all, of the

features of PD. Having stated this limitation, it is fair to say that among the various toxic models of

PD, the MPTP model has become one of the most commonly used. In fact, administration of MPTP

to humans and experimental animals causes the degeneration of dopaminergic neurons in SNc and

in striatum, causing a clinical picture in both humans and monkeys, indistinguishable from PD

(Langston et al. 1983).

Figure 2. Chemical structure of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

MPTP is a by-product of the chemical synthesis of a meperidine analog with potent heroin-like

effects (Fig. 2). It was made by Barry Kidstone, a 23 years old graduate student who set up a home

laboratory to synthesize 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP), but after four

injections of what he thought to be MPPP, he started to show severe bradykinesia (Langston and

Ballard 1983). However, similar to patients with idiopathic PD, he responded to treatment with

Levo-DOPA (L-DOPA) and developed the same complications associated with L-DOPA therapy

(Langston and Ballard 1983). Successive investigations confirmed that he had unconsciously

synthesized a new dopaminergic neurotoxin capable to produce a reliable and reproducible lesion of

the nigrostriatal dopaminergic pathway after its systemic administration. Successive

neuropathologic studies of MPTP-exposed addicts have revealed a loss of dopaminergic neurons

restricted to SNc, similar to PD (Langston et al. 1999). The responses, as well as the complications,

to traditional antiparkinsonian therapies are virtually identical to those seen in PD. However,

previous data suggest that, following the main phase of neuronal death in PD, MPTP-induced

neurodegeneration may continue to progress “silently” over several decades, at least in humans

intoxicated with MPTP (Vingerhoets et al. 1994; Langston et al. 1999). The severity of MPTP-

9

induced lesion depends on the regimen and route of administration, and on the species considered.

Since its discovery, MPTP has been widely used to create animal models of PD in a variety of

species (Jakowec and Petzinger 2004; Kopin 1987; Kurosaki et al. 2004), though the most used

species are currently the non human primates and the mice. Non human primates are the species

most sensitive and rats the lowest sensitive to MPTP neurotoxicity, whereas mouse strains widely

vary in their sensitivity to the toxin, with the C57BL/6J being the most susceptible (Hamre et al.

1999; Sedelis et al. 2000).

3.2 Mechanism of action and neurotoxicity

The mechanism of MPTP toxicity is quite similar among humans, non-human primates and mice.

MPTP is a highly lipophilic protoxin which rapidly crosses the blood-brain barrier (BBB) after

systemic administration. Once in the brain, MPTP enters astrocytes and is bioactived to the unstable

intermediate 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP+) by monoamine oxidase-B (MAO-

B) (Ekblom et al. 1993). Subsequently, MPDP+ spontaneously oxidizes to 1-methyl-4-

phenylpyridinium (MPP+) at least in vitro (Chiba et al. 1985; Fritz et al. 1985), whereas it is not

clear if this reaction may occur in vivo. Another mechanism for MPDP+ oxidation to MPP+

involves HO• radicals (Castagnoli et al. 1985), which appears in line with the evidence showing that

transgenic mice expressing high levels of superoxide dismutase are resistant to MPTP (Przedborski

et al. 1992). Recent findings show that once released from the astrocytes into the extracellular space

via the Organic Cation Transporter 3 (Cui et al. 2009), MPP+ is taken up into the neuron by the DA

transporter (DAT) (Chiba et al. 1985; Heikkila et al.1985). Consequently, mice lacking the DAT are

protected from MPTP toxicity (Bezard et al. 1999; Gainetdinov et al. 1997). Once inside the

neuron, MPP+ can follow three routes. It can remain in the cytosol to interact with cytosolic

enzymes, especially those carrying negative charges (Klaidman et al. 1993). In the second way, the

MPP+ can be stored in vesicles via uptake by the vesicular monoamine transporter (VMAT2) (Del

Zompo et al. 1993; Wimalasena et al. 2008), inducing the efflux of DA out into the intercellular

space. Here DA can be metabolized into a number of compounds, including metabolites and

products which lead the formation of ROS (Fig. 3) (Burke et al. 2008; Panneton et al. 2010).

Finally, the MPP+ can be concentrated within the mitochondria by a mechanism that relies on the

mitochondrial transmembrane potential (Ramsay and Singer 1986). In the mitochondria, MPP+ is

able to inhibit complex 1, resulting in the release of ROS as well as reduced ATP production

(Mizuno et al. 1987; Richardson et al. 2007). The ability to interfere with mitochondrial respiration

at the level of complex 1 is a key mechanism in the toxic effects of MPP+ (Nicklas et al. 1987;

Suzuki et al. 1990). Importantly, the cytotoxic effects of MPP+ are marked in cells that are

10

particularly sensitive to a deficiency in aerobic energy metabolism, a condition that applies to

dopaminergic neurons (Marey-Semper et al. 1993 and 1995) (Fig. 3)

Figure 3. Neurotoxic mechanism of the MPTP metabolite, MPP+, on dopaminergic terminals.

3.2.1 Oxidative stress and neuroinflammation

Through his metabolites, MPDP+ and MPP

+, the MPTP induces the formation of ROS and reactive

nitrogen species (RNS) (Drechsel and Patel 2008; Smith and Bennett 1997), which results in

oxidative stress (Adams et al. 1993; Meredith and Kang 2006). In this regards, it is worth

mentioning that mice transgenic for superoxide dismutase-1 (SOD-1), a key ROS scavenging

enzyme, are resistant to MPTP-induced dopaminergic neurodegeneration (Przedborski et al. 1992).

An excitotoxic mechanism for MPTP neurotoxicity has also been proposed, based on the

observation that intrastriatal administration of MPP+ to rats can induce a marked increase in

extracellular glutamate (GLU) (Carboni et al. 1990). Overstimulation of N-methyl-D-aspartic acid

(NMDA) receptors can lead to increase the intracellular Ca++

levels, an effect that causes the

activation of variety of proteases and kinases and results in the breakdown of cytoskeletal proteins

and the formation of ROS (Sattler and Tymiansky 2000 and 2001). However, the NMDA induced

the activation of nitric oxide synthase (nNOS) and, consequently, the production of NO.

Concurrently, depending on the level of NO production and the oxidative conditions, NO may

interact with H2O2 to produce peroxynitrite, or with Fe++

and Cu++

to generate ROS and to increase

the oxidative stress (Fig. 1) (Itzhak and Ali 2006).

Another mechanism by which MPTP is able to increase the oxidative stress in neurons is the

chronic neuroinflammation. In chronic neuroinflammatory response, elevated levels of pro-

inflammatory cytokines trigger oxidative and nitrosative stress that can aggravate the

11

neurodegenerative process (Hemmerle et al. 2012). Several evidences indicate that an intense

inflammatory reaction has been detected in the SNc of post mortem brains from MPTP-lesioned

addicts and monkeys after MPTP exposure (Barcia et al. 2003; McGeer et al. 2003). In this regard,

Liberatore and colleagues (1999) have shown that microglial cells not only increase in number after

MPTP injection, but also can flood dopaminergic neurons with large amounts of RNS, supporting a

role of activated microglia in MPTP-induced neurotoxicity in mice (Fig. 1) (Liberatore et al. 1999).

However, Hirsch and Hunot (2000) suggested that MPTP acts directly on the induction of cytokines

that activate inducible nitric oxide synthase (iNOS) (Hirsch and Hunot 2000), triggering the toxic

NO effects. Finally, blockade of neuroinflammation has been associated to a neuroprotective effect

in several models of dopaminergic degeneration, such as the chronic MPTP model, confirming that

glia may participate in MPTP-induced neurotoxicity (Wu et al. 2002; Schintu et al. 2009).

3.3 Neurotoxicity in humans

In the first cases of MPTP intoxication, patients showed immobility, marked generalized increase in

tone, inability to speak intelligibly, a fixed stare, marked diminution of blinking and other motor

impairment such as short steppes, slow shuffling gait and generalized bradykinesia, briefly the

classical PD symptoms (Langston and Ballard 1984). As in experimental animals, MPTP

administration in humans causes the degeneration of dopaminergic neurons in SNc and the

depletion of DA in striatum (Javitch et al. 1984). All cases of MPTP intoxication in humans were

caused by one, or few repeated administrations of the toxin that could lead to active

neurodegeneration, years after the initial exposure (Langston 1987). The loss of dopaminergic

neurons restricted to the SNc is the most important similarity with PD and has been revealed for the

first time in human through a neuropathologic study of brains of three MPTP-exposed addicts

(Langston et al. 1999). Moreover, PET studies using [18F]-DOPA have revealed that MPTP-

intoxicated individuals display a severely reduced DA uptake similar to that of late-stage idiopathic

PD (Calne et al. 1985; Snow et al. 2000; Vingerhoets et al. 1994). Finally, the depletion of nigral

dopaminergic neurons was found to be consistently present together with gliosis and clustering of

microglia around nerve cells (Langston et al. 1999).

One typical neuropathologic feature of PD has, until now, been lacking in the MPTP model: the

eosinophilic intraneuronal inclusions, called Lewy bodies have not been convincingly observed in

MPTP-induced parkinsonism (Forno et al. 1993). Netherless, the absence of Lewy bodies may be

due to the young age at the onset of MPTP-induced parkinsonism, since age may be an important

factor for development of these aggregates (Gibb and Lees 1988).

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3.4 MPTP as PD mouse model

In general MPTP is administered to mice either in an acute or in subchronic regimen (Heikkila et al.

1984; Sonsalla and Heikkila 1986). In these models, MPTP can produce death of dopaminergic

neurons in SNc by at least the 40% in C57BL/6J mice and significant depletions in striatal level of

DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)

(Ricaurte et al. 1986). MPTP administration in mice induces a neuroinflammatory effect in the SNc,

striatum (Członkowska et al. 1996; Kohutnicka et al. 1998; Kurkowska-Jastrzebska et al. 1999), and

hippocampus (Luellen et al. 2003; Costa et al. 2014). Bradykinesia, akinesia, altered balance and

other motor features can be observed in MPTP-treated mice through various behavioral analyses

(Fleming et al. 2013; Sedelis et al. 2001; Tillerson et al. 2002). Whole-body tremor and postural

abnormalities also have been reported, but chiefly in the first day after lesioning (Sedelis et al.

2001). Despite the evidence of DA reductions, mice that receive MPTP acutely do not always

exhibit motor dysfunctions or motor abnormalities (Heikkila et al. 1989; Meredith and Rademacher

2011). Acute MPTP treatment induces a rapid and transient neurodegeneration, which does not

allow the development of chronic pathogenic mechanisms and/or motor disabilities. In contrast, the

chronic administration induces a gradual and persistent degeneration of nigrostriatal neurons

associated with motor deficit (Bezard et al. 2000, Fornai et al. 2005). Accordingly, chronic

exposure to low doses of MPTP over several weeks, in combination with the clearance inhibitor

probenecid, has been shown to reproduce several aspects of the human disease and to be a most

suitable model for studying drugs with neuroprotective potential (Carta et al. 2013, Petroske et al.

2001, Schintu et al. 2009). In addition, typical PD pathological features, such as chronic

inflammatory response in the SNc, alpha-synuclein positive inclusions, Lewy-bodies like deposits,

altered glutamate function, apoptotic neuronal demise, have been described in the chronic MPTP

model, suggesting that this model might extensively reproduced the neuropathology of PD

(Meredith at al. 2002; Dervan et al. 2004; Novikova et al. 2006).

Moreover, when MPTP is co-administered with probenecid (MPTPp), which retards the renal and

CNS clearance of the toxic metabolites of MPTP, the degeneration of dopaminergic neurons takes

place over a period of 5-8 weeks. This chronic regimen induces apoptosis, no mortality and mice

survive in a healthy state to the treatment. Moreover, the chronic treatment induces olfactory deficit,

one of the symptoms that characterizes the early as well as late stages of the disease. Progression in

a model with MPTP is a very important requirement since it allows studying the efficacy of

neuroprotective drugs during the progressive DA neurodegeneration, reproducing more closely the

human situation (Schintu et al. 2009).

13

4. 3,4-methylenedioxymethamphetamine

4.1 MDMA: a neurotoxic drug

Although PD has multiple origins, one hypothesis is that amphetamine-related drugs may be part of

the wide array of factors leading to the dopaminergic neuron degeneration that causes the disease

(Obeso et al. 2010). However, recent studies have showed that MDMA (ecstasy) has a neurotoxic

effect that is selective for the nigrostriatal pathway (Granado et al. 2008a). On these basis, it is

conceivable that prolonged exposure to MDMA, similar to that proposed for other amphetamine-

related drugs (Garwood et al. 2006), may damage the dopaminergic neurons in the human SNc

(Moratalla et al. 2015). Therefore, these damaged neurons could die earlier, depleting the reserve of

neural cells necessary for normal neurological functions and ending up in the manifestation of PD

(Garwood et al. 2006; Todd et al. 2013). On these bases, the study of MDMA neurotoxicity appears

particularly useful to understand the molecular mechanisms that induce the neuronal degeneration

which cause the disease.

Figure 4. Chemical structure of 3,4-methylenedioxymethamphetamine (MDMA).

Although it has been synthesized in 1912 (Fig. 4), MDMA got popular as a recreational drug since

the mid 1980s, because of its effects on mood and social relations (Hall and Henry 2006). Similar to

other amphetamine-related drugs, MDMA induces a state of “high”, mainly characterized by

disinhibition in social relations, openness of spirit, increased empathy towards other people,

increased self-esteem and self-confidence, euphoria, increased vigilance, improvement of mood,

and decrease of fatigue (Downing 1986; Greer and Tolbert 1986; Kirkpatrick et al. 2014). For these

positive properties of inducing feeling of well being and increasing communication (Watson and

Beck 1991), in the beginning of 1976, it was introduced in clinical psychotherapeutic practice

(Shulgin 1990). Although MDMA generally elicits “positive” effects, the 25% of MDMA users

report having had at least one adverse reaction to the substance (Davison and Parrott 1997; Green et

al. 2003; Morton 2005). Acute toxicity elicited by MDMA in humans and experimental animals

includes effects on the neuroendocrine and thermoregulatory systems, in particular induction of

hyperthermia, and on the cardiovascular system (Gordon et al. 1991; Vollenweider et al. 1998; de la

14

Torre et al. 2000). Following several cases-reported toxicity and death induced by exposure to large

dose of MDMA, the UK classified the MDMA as a Schedule 1 drug, thus prohibiting to posses, sell

or give away the substance (Dowling et al. 1987). Finally, on 1 July 1985 the Food and Drug

Administration (FDA) placed the compound on schedule I control substance. Netherless, actually

the MDMA is the most common psychostimulant consumed by adolescent and young people in

dance-clubs.

4.2 Mechanisms involved in the MDMA neurotoxicity

MDMA is usually consumed in pills and numerous studies have indicated that MDMA is well

absorbed by the oral route, and reaches its maximum concentration after 1.5-3 hours from its intake,

with a plasma half-life around 6-7 hours (de la Torre et al. 2004). MDMA is able to easily cross the

BBB and enter the brain without significant delay, by means of his highly lipid solubility.

A number of reports have demonstrated that, in the CNS, MDMA binds to all three presynaptic

monoamine transporters, whit an interspecies different affinity (Moratalla et al. 2015). As substrate

for the monoamine transporters the MDMA is translocated to the cytoplasm. Here, MDMA causes

the dissipation of the proton gradient between the vesicles and the cytosol that is necessary for the

proper functioning of the VMAT2. The VMAT2 dysfunction inhibits the influx and proper storage

of serotonin (5-HT), DA, and noradrenalin (NA) (Rudnick and Wall 1992; Cozzi et al. 1999).

Because its ability to cause functional reversal of both VMAT2 and monoamine transporters,

MDMA is able to increase the extracellular levels of 5-HT, DA, and NA in multiple brain regions

(Gudelsky and Yamamoto 2008). However, the partial inhibition of the MAO-B, located in the

outer mitochondrial membrane, boots the stay of monoamine in the neuronal terminal (Leonardi and

Azmitia, 1994). In this regard, it has been confirmed in the mouse striatum a MDMA dose-

dependently increase of both DA and 5HT release (Górska and Gołembiowska 2015) (Fig. 5).

Several studies have reported that the microinjection of MDMA in different brain areas does not

induce neurotoxicity, unless it was administrated at doses much higher than those having neurotoxic

effects when administered peripherally (Paris and Cunningham 1992; Esteban et al. 2001; Escobedo

et al. 2005). These latter findings indicate that MDMA has to be systemically metabolized to

produce its neurotoxic effects, and suggest that MDMA metabolites are responsible for these

effects.

As stated earlier, MDMA is N-demethylated to 3,4-methylenedioxyamphetamine (MDA). MDMA

and MDA are O-demethylenated respectively to N-methyl-a-methyldopamine (N-Me-a-MeDA),

also called 3,4-dihydroxymethamphetamine (HHMA) and a-methyldopamine (a-MeDA), also

called 3,4-dihydroxyamphetamine (HHA) (Lim and Foltz 1988; Kumagai et al. 1994). These

15

catechols can undergo oxidation to o-quinones that are highly redox-active molecules and produce

free radicals, ROS or RNS (Green et al. 2003; de la Torre et al. 2004; Farré et al. 2004). N-Me-a-

MeDA, a-MeDA and theo-quinones may be conjugated with glutathione to form a glutathionyl

adduct (Hiramatsu et al. 1990; Bai et al. 2001). This conjugate remains redox-active, being readily

oxidized to the quinone thioether and many of the metabolites pharmacologically active (Easton et

al. 2003; Easton and Marsden 2006; de la Torre et al. 2004; Capela et al. 2006).

Figure 5. Neurotoxic mechanisms of MDMA on dopaminergic terminals.

4.2.1 Oxidative stress and excitotoxicity

In addition to its toxic metabolites, MDMA is able to lead in the production of ROS through three

different known pathways. Firstly, the elevated synaptical DA concentration induced by MDMA,

may undergo over auto-enzymatic oxidation, resulting in the production of ROS and toxic

metabolites (Marchitti et al. 2007). The second pathway that lead in ROS production involves

glutamatergic system that through increasing of the intracellular Ca++

levels, triggers the formation

of ROS (Sattler and Tymiansky 2000 and 2001) and the production of NO (Itzhak and Ali 2006).

The mechanism through with NO mediate the MDMA toxicity is not completely understood,

although neuronal nitric oxide synthase (nNOS) inhibitors prevent the MDMA induced

dopaminergic neurotoxicity in mice (Colado et al. 2001).

More recent evidence suggests an important role of the mitochondrial electron transport chain

defect in mediating the MDMA toxic effects. In particular, Puerta and coworkers (2010) recently

reported that inhibition of complex 1 of the mitochondrial electron transport chain is one of the

16

earlier events that take place in MDMA-induced neurotoxicity in mice (Puerta et al. 2010).

Similarly to PD, the inhibition of complex 1 induced by MDMA increases the production of ROS

and others dangerous free-radicals. However, as mentioned above, the mitochondria-related energy

failure in dopaminergic cells, may disrupt vescicular storage of DA, increasing the DA release and

the formation of DA toxic metabolites (Fig. 5). In this regard, the inhibition of MAO-B induced by

MDMA, increases the cytosolic DA levels (Leonardi and Azmitia 1994), promoting the

autoxidation of DA in DAQ.

Taken together, these mechanisms leading to the formation of reactive intermediates, ROS, and/or

toxic oxidation products may represent the triggering factors responsible for the toxicity exerted by

this amphetamine. Finally, these elevated levels of free radicals and the reduced levels or

inactivation of antioxidant enzymes, such as catalase and superoxide dismutase (SOD), induced by

MDMA results in oxidative stress (Cadet et al. 2001; Sanchez et al. 2003). In line with this, MDMA

produces a less marked oxidative stress and striatal depletion of both DA and 5-HT in transgenic

mice overexpressing SOD than in wild-type mice (Jayanthi et al. 1999). Furthermore, treatment

with antioxidant agents has been found to afford neuroprotection in MDMA-treated rats (Gudelsky

1996; Aguirre et al. 1999; Shankaran et al. 2001).

4.2.2 Neuroinflammation and Hyperthermia

A relevant issue related to MDMA-induced neurotoxicity is that MDMA can trigger inflammatory

processes in those brain areas that exhibit dopaminergic and/or serotonergic terminal degeneration,

but not in brain areas where no modifications in either DA or 5-HT levels occur (Yamamoto et al.

2010). Several preclinical have demonstrated that MDMA elicits astroglial and microglial activation

in the striatum (Granado et al. 2008a; Costa et al. 2013; Frau et al. 2016), as well as in the cortex

(Herndon et al. 2014; Costa et al. 2014), and hippocampus (Costa et al. 2014; Lopez-Rodriguez et

al. 2014). However, elevated levels of glia-generated reactive species, such as NO, superoxide and

cytokines, have been shown to correlate with neurodegeneration induced by amphetamine-related

drugs (Castelli et al. 2014; Thomas et al. 2004). In support of the hypothesis that the

neuroinflammation is one of the factors involved in MDMA toxicity, Zhang and coworkers showed

that minocycline, an anti-inflammatory drug, has a neuroprotective effect against MDMA induced

neurotoxicity (Zhang et al. 2006).

Another mechanism that may be implicated in MDMA neurotoxicity is the hyperthermic response

that is influenced by dose, ambient temperature and other housing conditions (Moratalla et al.

2015). In this regard, it is worth mentioning that the typical environmental conditions featuring

dance clubs, where music is deafening and room temperatures are high due to crowding, together

17

with the fact that club-goers usually consume little water and considerable amounts of ethanol, are

crucial to amplifying MDMA-induced hyperthermia (Green et al. 2003; Moratalla et al. 2015).

The mechanism of MDMA-induced hyperthermia is complex, and involves not only serotonergic

and dopaminergic systems, but also adrenergic transmission (Sprague et al. 1998). The increased

release of these monoamines induced by MDMA administration may stimulate receptors involved

in thermoregulation (Shankaran and Gudelsky 1999). Although previous studies associated the

hyperthermia with the increased 5-HT release (Shankaran and Gudelsky 1999), recent evidences

have indicated that the primary mechanism involved in the hyperthermic response is the dopamine

release. In fact, several studies reported that the activation of D1 receptors induces hyperthermia in

mice (Zarrindast and Tabatabai 1992), while the inactivation of D1 receptors attenuated MDMA-

induced hyperthermia and prevented the striatal loss of DA (Granado et al. 2014).

However, a role of GLU has been suggested by a significant decrease of MDMA-induced

hyperthermia in rats treated with NMDA receptor antagonists (Nisijima et al. 2012).

Studies in experimental animals have indicated that hyperthermia induced by MDMA, being

harmful per se by leading to dehydration and altered hydrosaline homeostasis (Green et al. 2004;

Baylen and Rosenberg 2006), may be one of the factors that promote glial activation and

neurotoxicity caused by this amphetamine-related drug (Miller and O‟Callaghan 1995; Colado et al.

1998; Mechan et al. 2001; Touriño et al. 2010). With regard to the possible relationship among

MDMA-induced hyperthermia and glial activation it is interesting to mention a recent study by our

group, which demonstrated the existence of a positive correlation between the elevation in body

temperature and the degree of glial activation in mice treated with MDMA (Frau et al. 2013).

However, previous studies in rats have demonstrated that hyperthermia induced by MDMA is

associated with enhanced production of free radicals and proinflammatory cytokines, such as IL-1β,

IL 6, and TNF-α, (Green et al. 2004; Orio et al. 2004). At the moment, is not clear if the increased

concentrations of proinflammatory cytokines could be a consequence, or the cause of MDMA-

induced hyperthermia. Further support to a possible relationship among hyperthermia and

neurotoxicity comes from evidence showing that in experimental animals high body temperature

enhances the formation of toxic metabolites of MDMA, which are known to increase oxidative

stress (Cadet et al. 2007). On this basis, it has been speculated that hyperthermia may have an effect

on the other neurotoxic mechanisms of MDMA, such as ROS and reactive RNS production,

eventually causing nerve terminal damage (Goni-Allo et al. 2008; Malberg et al. 1998), and glial

activation (O'Callaghan and Miller 1994; Thomas et al. 2004).

18

4.3 Neurotoxicity in humans

To elucidate the toxic mechanisms which lead to higher incidence of PD in MDMA abusers

(Callaghan et al. 2012; Curtin et al. 2015), several researchers investigated the MDMA-induced

neurotoxic effect in both humans and experimental animals. The features of neurotoxic damage

induced by MDMA seem to vary, depending on the gender and strain of animals, which may

influence the response to different dosing regimens and administration routes of MDMA (Ricaurte

et al. 1988; Colado et al. 1995; Itzhak et al. 2003).

In humans, MDMA displays higher affinity for noradrenalin transporter (NET), and lower, but

similar, affinities for serotonin transporter (SERT) and DAT (Verrico et al. 2007). However, the

ability to release intracellular monoamines is higher in SERT-expressing cells than in either DAT-

or NET-expressing cells, and this may justify the reduction of SERT density observed in ecstasy

users compared to controls (Haddad et al. 2002; Reneman et al. 2001a,b; Semple et al. 1999).

Moreover, the neurotoxic potential of MDMA in humans has been evaluated indirectly by

measuring the concentration of 5-HIAA (5-hydroxyl indole acetic acid) in cerebrospinal fluid (CSF)

in recreational ecstasy users. Several studies reported significantly lower levels of 5-HIAA in

recreational ecstasy users compared to polydrug users who had never used ecstasy (McCann et al.

1999 and 1994; Ricaurte et al. 1990). In this regard, Kish and colleagues (2000) demonstrated

severe depletion (50-80%) of striatal 5-HT and 5-HIAA in the brain of a 26-year-old male who had

taken MDMA regularly for 9 years (Kish et al. 2000). More direct evidence supporting that MDMA

produces long-term neurotoxic effects on brain 5-HT systems emerged from neuroimaging studies:

increase in 5-HT2A receptor density detected by SPECT, in heavy MDMA abusers compared to

controls, showed a long-term compensatory response to 5-HT depletion (Reneman et al. 2000).

While several studies have suggested that MDMA may harm the serotonergic system in the human

brain, it is less clear whether MDMA may be toxic for human dopaminergic neurons.

In this regard, it is noteworthy that MDMA has significant affinity for DAT (Verrico et al. 2007)

and promotes the release of DA in multiple brain regions. This consideration with the reported

higher incidence of PD in heavy MDMA-abusers, suggest that MDMA may be toxic for the human

dopaminergic system (Moratalla et al. 2015).

The evaluation of the long-term effects of MDMA, including neurotoxicity, in humans is complex,

since MDMA users frequently consume it in the context of poly-drug abuse, together with other

psychoactive substances, such as ethanol, cannabis, and cocaine (Schifano et al. 1998). However,

there are many variables that could not be controlled, e.g. dose and purity of MDMA, number of

times MDMA was used, set and setting, and the time between interview and the last use of MDMA.

19

Thus, animal models afford the unique opportunity to evaluate the effects of MDMA without many

complicating factors.

4.4 MDMA administration in mice: a model of dopaminergic neurotoxicity

To evaluate the MDMA toxicity on dopaminergic system, the murine model is the most validated.

In fact, although in rats MDMA has the highest affinity for the SERT, and lower affinities for NET

and DAT (Steele et al. 1987; Rudnick and Wall 1992), in mice MDMA seems to act as a

dopaminergic neurotoxin (Kindlundh-Högberg et al. 2007). Therefore, the study of MDMA actions

on DA in mouse appears particularly interesting, since DA mediates several behaviors that occur

after MDMA administration, such as hyperactivity, alterations in mental state, hyperthermia

(Fantegrossi et al. 2003; Green et al. 2003; Miller and O‟Callaghan 1995) and the reported higher

incidence of PD in MDMA abusers (Callaghan et al. 2012; Curtin et al. 2015). When administered

to mice, MDMA produced damage of DA terminals and decreases the concentrations of DA,

DOPAC, and HVA in several brain regions (Moratalla et al. 2015). Moreover, and most

importantly, MDMA produces long-term degeneration of dopaminergic nerve terminals (Brodkin et

al. 1993; Colado et al. 2001; Izco et al. 2010; Costa et al. 2013) and a decrease in tyrosine

hydroxylase (TH), the rate-limiting enzyme for DA synthesis, in the striatum (Green et al. 2003;

Costa et al. 2013). Moreover, a study by Granado and coworkers demonstrated that MDMA

administration induces a significant decrease in TH-positive neurons in the SNc but not reduce the

synthesis of TH, supporting the toxic effect of MDMA on the nigrostriatal system. Interestingly, the

same group observed that MDMA induces a loss of TH and DAT fibers in the striatum, but not in

the nucleus accumbens, indicating that the dopaminergic neurotoxicity of MDMA targets the

nigrostriatal system, while sparing the mesolimbic pathway (Granado et al. 2008a). These results

are in line with earlier evidence describing dopaminergic terminal loss in the mouse striatum

following MDMA administration (Fornai et al. 2004). Moreover, it damages striatal dopaminergic

terminals, with an effect that appears more pronounced in the striosomal compartment than in the

matrix (Granado et al. 2008b). Furthermore, and most notably, a similar striosomal damage has

been observed following the MPTP administration (Iravani et al. 2005); this feature is one of the

similarities between the effects of MDMA and those of a dopaminergic neurotoxin (Moratalla et al.

2015). In fact, MDMA, as MPTP, is able to induce mitochondrial dysfunction that, as explained

above, trigger oxidative stress in the vulnerable dopaminergic neurons of SNc (fig. 3 and 5).

20

4.5 MDMA Neurotoxicity and adolescence

It must be considered that an increased risk of developing drug abuse and drug-related problems is

often associated with the age of consumers (Hawkins et al. 1992). In fact, MDMA is largely

consumed by adolescents, during raves and club parties. In both humans and experimental animals,

adolescence is a critical period for the development of the brain, which undergoes major

developmental changes (Casey et al. 2000; Piper and Meyer 2004). In fact, the adolescent brain

appears to be particularly vulnerable to the long-term noxious effects of exogenous substances,

including drugs of abuse (Cadoni et al. 2015; Daza-Losada et al. 2009; Sisk and Zehr 2005; Spear

2000). In this contest it is important to highlight that although the adolescent brain is particularly

vulnerable to neurotoxicity; a lower sensitivity to MDMA effects in adolescent compared with adult

mice, was described by previous studies (Reveron et al. 2005; Teixeira-Gomes et al. 2015; Frau et

al. 2016). In particular, repeated MDMA administration induced a loss of dopaminergic neurons in

SNc of adult and adolescent mice, whereas TH-positive fibers in striatum were decreased in adult

mice only (Reveron et al. 2005; Frau et al. 2016). These results are confirmed by microdialysis

experiments that showed lower DA extracellular concentrations in adult compared whit adolescent

mice, 7 days after repeated MDMA administrations (Reveron et al. 2005). Although the lower

striatal neurotoxic effect, the abuse of MDMA during adolescence, such as other amphetamine-

related drugs, may lead to long-lasting effects, which could eventually render the brain more

sensitive to toxic insults later in life. Considering the MDMA property to affect neurogenesis and

synaptogenesis, leading to the disruption of DA homeostasis, the consumption of MDMA during

the adolescence could increase the vulnerability of dopaminergic neurons (Rice and Barone 2000).

In particular, MDMA may interfere with dopamine synapse development in a phase of life crucial

to the development of the dopaminergic system (Fasano et al. 2008; Goffin et al. 2010), rendering

this system more vulnerable at a later time (Costa et al. 2014; Frau et al. 2016). The alteration in

dopaminergic transmission and function could be a negative factor in the development of

neurodegenerative diseases, such as PD, which principally involves the dopaminergic system. In

line with this, adult mice treated with MDMA during adolescence showed more marked

neuroinflammatory and neurotoxic effects MPTP-induced in motor (SNc and striatum), and non-

motor (hippocampus and medial prefrontal cortex) brain areas, compared with mice non-pretreated

with MDMA (Costa et al. 2013 and 2014).

21

5. Parkinson disease therapy

5.1 Current PD therapies

Current treatments for Parkinson disease, based fundamentally on the replacement of DA, can

significantly improve symptoms but, unfortunately, do not cure the disease or slow down its

progression. Although the number of drugs has increased and our sophistication in using them has

improved, L-DOPA and dopaminergic agonists, remain the principal current treatment of PD. A

necessary prerequisite for these approaches, however, is the presence of functioning dopaminergic

nerve terminals in the striatum, meaning that with its progression the disease becomes increasingly

refractory to pharmacological treatment. Moreover, these approaches have many side effects, such

as dyskinesia or motor fluctuations, which can not easily be tolerated during life-long treatment and

could be a starting point for further complications (dopamine dysregulation syndrome or impulse

control disorder) (Vitale et al. 2013).

Several non dopaminergic therapies have been explored in the treatment of PD and the adenosine

A2A receptors antagonists seem promising. Furthermore, epidemiological studies have shown that

the incidence of PD is lower in consumers of high doses of caffeine, an antagonist of adenosine A1

and A2A receptors (Ross et al. 2000). A2A receptor antagonists have also been shown to have a

neuroprotective role in several experimental rodent models of PD (Carta et al. 2009; Ikeda et al.

2002; Pierri et al. 2005). Antagonism of adenosine A2A receptors facilitates GABA release in

striatum, reducing striatopallidal neuronal overactivity. This reduction helps to increase indirect

inhibitory output from the striatum to the globus pallidus, restoring balance between the basal

ganglia output pathways (Ferré et al. 1997; Mori and Shindou 2003). Moreover, several data have

shown that A2A receptor antagonism counteracts neuroinflammatory processes (Carta et al. 2009;

Huang et al. 2006) by inhibiting astroglial and microglial activation (Ikeda et al. 2002; Pierri et al.

2005). These data have been confirmed by Carta and coworkers that showed a reduced astrogliosis

and microgliosis after subchronic MPTP administration in A2A receptor knockout mice (Carta et al.

2009). Finally, preclinical studies and clinical trials suggest that these compounds may increase the

therapeutic efficacy of L-DOPA without exacerbating L-DOPA-associated dyskinetic effects (Bara-

Jimenez et al. 2003; Grondin et al. 1999; Hauser et al. 2008; Kanda et al. 2000; LeWitt et al. 2008;

Morelli 2003; Schwarzschild et al. 2006; Stacy et al. 2008).

5.2 Monoamine oxidase inhibitors therapy

A different approach currently used as an add-on therapy, is based on the inhibition of enzymes

involved in the metabolic degradation of dopamine (monoamine oxidase, (MAO), catechol-O-

methyltransferase, (COMT)) (Youdim and Riederer 2004). Selective MAO-B inhibitors block the

22

enzyme responsible for the intracellular degradation of DA, thereby increasing DA availability in

the nigrostriatal pathway and prolonging the effectiveness of L-DOPA replacement therapy.

a b

Figure 6. Chemical structures of (R)-N-methyl-N-(1-phenylpropan-2-yl) prop-2-yn-1-amine (selegiline) (a)

and N-propargyl-1(R)-aminoindan (rasagiline) (b).

During the last decade, the well-known MAO-B inhibitors, selegiline [deprenyl, (R)-N-methyl-N-

(1-phenylpropan-2-yl) prop-2-yn-1-amine] and rasagiline [Azilect, N-propargyl-1(R)-aminoindan]

(Fig. 6a and b), have been shown to be neuroprotective against dopaminergic cell death. In line with

these results, several reports demonstrated the neuroprotective effects of the MAO-B inhibitors in

parkinsonian animal models (Mandel et al. 2007). Their neuroprotection has been attributed to both

antioxidant and antiapoptotic properties of the parent compounds or their metabolites (Reznichenko

et al. 2010; Wu et al. 1996). Inhibition of MAO-B may afford neuroprotection through a reduced

production of ROS and aldehydes, protecting against oxidative stress (Youdim et al. 2006;

Hauptmann et al. 1996; Youdim and Lavie 1994; Mallajosyula et al. 2008). In particular, Youdim

and coworkers showed that rasagiline, as other propargylamine compounds, prevents the fall in the

mitochondrial potential induced by oxidative stress and increases the activity of antiapoptotic

factors and antioxidant enzymes (Youdim et al. 2001 and 2006). Although the primary effect of

rasagiline in PD is presumably done by MAO-B inhibition, resulting in a slower metabolism of

endogenous and exogenous DA and thus providing symptomatic benefits (Finberg et al. 1996 and

1998), it also possesses neuroprotective activity unrelated to its MAO-B inhibition. In particular

rasagiline has been shown to increase the levels of brain-derived- and glial cell line-derived

neurotrophic factors (BDNF, GDNF) (Maruyama et al. 2004; Weinreb et al. 2004) and it is able to

increase cell survival in cell culture and animal models of PD (Bar-Am et al. 2007; Zheng et al.

2005). The neurorestorative activity of this MAO-B inhibitor was demonstrated in a progressive

apoptotic model of neuronal death induced by long-term serum deprivation (Bar-Am et al. 2005 and

2007), and also in a post-MPTP-administration model in mice (Mandel et al. 2007; Sagi et al.

2007).

23

Nevertheless, in the recent ADAGIO (Attenuation of Disease Progression with Azilect Given Once-

Daily) trials, a delayed start study design was applied to separate symptomatic benefit from disease-

modifying effect, and the results failed to define a clear, dose-dependent disease-modifying action

of rasagiline (Ahlskog et al. 2010).

5.3 Novel (hetero)arylalkenyl propargylamine compounds

On the basis of the controversial neuroprotective activity of the more useful irreversible MAO-B

inhibitors (Olanow and Rascol 2010; Weinreb et al. 2010), a large number of new molecules, that

contain a propargyl ring and showed more potent MAO-B inhibitory activity and neuroprotective

effects, have been proposed to replace the pre-existing MAO-B inhibitors. In particular, a novel

series of (hetero)arylalkenylpropargylamine compounds that demonstrate highly potent MAO-B

inhibitory activity with remarkable selectivity over other types of amine oxidase enzymes, were

synthesized (Huleatt et al. 2015). The design of these novel propargylamines is based on the

knowledge that the propargylamino group has been demonstrated to be a potent anti-apoptotic agent

(Maruyama et al. 2002), while MAO-B selectivity and potency as well as overall neuroprotective

profile were tuned through judicious choice of the skeleton and its substituents (Huleatt et al. 2015).

Figure 7. Chemical structure of N-Methyl-2-phenyl-N-(prop-2-yn-1-yl)prop-2-en-1-amine Hydrochloride

(SZV558).

A number of compounds among this series displayed cytoprotective properties against 6-OHDA-

and rotenone-induced cell death in PC12 cells (Huleatt et al. 2015). In particular, to elicit cell death

was used in the first series of experiments 6-OHDA, whereas in the second series, rotenone. The

compounds effects were compared to rasagiline treated-cells. In line with literature data (Zheng et

al. 2005; Milusheva et al. 2010; Bar-Am et al. 2007), rasagiline exerted significant 48% protective

effect. Of the compounds tested, the SZV558 [N-Methyl-2-phenyl-N-(prop-2-yn-1-yl)prop-2-en-1-

amine Hydrochloride] (Fig. 7) showed >40% protection against 6-OHDA-induced cell death

(Huleatt et al. 2015). Specifically, the profile of SZV558 appears to be particularly interesting

because of its pharmacodynamic, which is favorable for disease-modifying properties, since it

24

irreversibly binds the MAO-B enzyme, and has a long half-life. In particular the peak of the effect

of SZV558 was obtained 18 hours after the administration, and a tendency of protection was present

up to 42 hours (Huleatt et al. 2015). This compound appears to be superior to rasagiline with respect

to both selectivity (human MAOA/B selectivity ratio: 58) and potency, having an IC50 of 60 nM

(Huleatt et al. 2015).

However, culture-based cell survival assays represent oversimplified experimental systems, and

they might not reflect the complexity of in vivo systems. On these bases, in a recent study, the

protective activity of SZV558 was evaluated in various in vivo models of PD, using different

neurotoxins (rotenone and MPTP). The results displayed that SZV558 inhibited selectively the

oxidative stress induced by pathological DA release in the rotenone-treated rat striatum slices. In

this case, the new compound appeared to provide higher protective efficacy than rasagiline (Baranyi

et al. 2016). Moreover, the effect of SZV558 was dose-dependent and, at low doses, its efficacy was

higher than rasagiline against the depletion of DA, even after the acute administration of MPTP.

These data were confirmed by behavioral tests, such as the open-field test and rotarod test, used to

evaluate respectively locomotor activity and motor coordination (Hu et al. 1991; Shiotsuki et al.

2010; Meredith and Kang 2006). Therefore, SZV558 fulfills the criterion of a multi-target anti-PD

compound.

5.4 Metformin

Several therapies have been explored in the treatment of PD, such as the identifying well-tolerated,

medications that enhance adult neurogenesis that could be of great clinical interest in the

management of neurodegenerative disorders. In line with this, the metformin, a well-known

antidiabetic drug, has recently been proposed to promote neurogenesis and to have a

neuroprotective effect on the neurodegenerative processes induced by MPTP in a mice PD model

(Patil et al. 2014).

Figure 8. Chemical structure of metformin (1,1-dimetilbiguanida).

Metformin is an antidiabetic medication, member of biguanide class (Fig. 8). It is the first-line drug

of choice for the treatment of type 2 diabetes, that effectively lowers plasma glucose levels

25

primarily by decreasing hepatic glucose production and by improving lipid metabolism in both liver

and muscle tissues (Perriello et al. 1994; Stumvoll et al. 1995). At cellular level, metformin

activates AMP-activated protein kinase (AMPK), that is the key mechanism by which this drug acts

on liver and muscle but also on the intestine (Pieri et al. 2010; Sakar et al. 2010).

Moreover, study conducted by Labuzek et al. (2010 a, b) demonstrated that orally administered

metformin rapidly crosses the BBB and is carried in the neurons by organic cation transporter 1

(Łabuzek et al. 2010a, b), usually involved in the carriage of endogenous compounds, such as DA

(Becker et al. 2011). On this base, in addition to its antidiabetic potential, the therapeutical effects

of metformin on various CNS disorders, such as PD or Huntington disease, has been studied. (Ma et

al. 2007; Ng et al. 2012).

Clinical trials conducted in a Taiwanese population cohort showed that incident PD risk in type 2

diabetes increases 2.2-fold. While, the sulfonylureas treatment further increases risk by 57%, the

combination with metformin avoids completely the risk (Wahlqvist et al. 2012). In line with this,

Wang and colleagues demonstrated that the metformin activates in adult neural stem cells (Wang et

al. 2012), the protein kinase C–cyclic-adenosine monophosphate (cAMP) response element-binding

protein (PKC–CREB) pathway, which responds to a wide range of neurological disorders, such as

PD and Alzheimer‟s disease (Emsley et al. 2005; Potts and Lim 2012).

In the CNS, metformin is able to stimulate AMPK, increasing glucose uptake by neurons (Amato

and Man 2011) and affecting neural metabolism (Potts and Lim 2012). Recent studies suggest that

AMPK activation might prevent neuronal cell death and play a pivotal role as a survival factor in

PD. In fact, inhibition of AMPK increases MPP+-induced cell death while overexpression of

AMPK raises cell viability after exposure to MPP+ in SH-SY5Y cells (Choi et al. 2010). In line

with this, metformin was found to alleviate dopaminergic dysfunction and mitochondrial

abnormalities through the AMPK activation in drosophila models of PD (Ng et al. 2012). Moreover,

previous studies suggest that metformin prevents the oxidative stress-related cellular death in non-

neuronal cell lines (Łabuzek et al. 2010a, b), conserving the activities of antioxidant enzymes

(Pavlovic et al. 2000; Chakraborty et al. 2011; Esteghamati et al. 2013). Finally, metformin was

found to be neuroprotective by inhibiting apoptosis in neuronal cortical cells (El-Mir et al. 2008)

and more recently by promoting neurogenesis in the olfactory bulb and in hippocampus of adult

mice by PKC-CBP pathway (Wang et al. 2012).

5.5 Caffeine: neuroprotective or neurotoxic?

As mentioned above, several epidemiological evidences showed that caffeine as well as coffee

consumption is linked to a reduced risk of developing PD (Ross et al. 2000; Ascherio et al. 2001;

26

Schwarzschild et al. 2003). In this regard, considerable research data have suggested that the

caffeine may protect against the PD neurodegeneration by A2A receptor blockade (Morelli et al.

2010). The primary action of caffeine is the blockade of A1 and A2A receptors which leads to

secondary effects on many classes of neurotransmitters (Fredholm et al. 1999). The A2A receptor

are implicated as mediators or modulators of dopaminergic neurodegeneration (Morelli et al. 2010),

consequently the blockade of this receptor by caffeine might reduce the neurodegenerative

progression. Moreover, caffeine is able to attenuate the loss of striatal DA induced by acute MPTP

administration in mice, confirming the neuroprotective potential of A2A receptor blockade in PD

(Chen et al. 2001).

Although caffeine use is safe (Ascherio et al. 2001; Fredholm et al. 1999; Schwarzschild et al.

2003), the consumption of caffeinated energy drinks might influence the adverse effects of other

substances which are taken concomitantly (Mohamed et al. 2011; Morelli and Simola 2011; Simola

et al. 2006). The use of amphetamine-related drugs is often combined with beverages containing a

high quantity of caffeine in order to amplify their stimulant actions (Mohamed et al. 2011; Reissig

et al. 2009). Caffeine has been reported to influence the toxicity of amphetamine-related drugs, as

shown by potentiation of seizures, hyperthermia, tachycardia, and mortality (Camarasa et al. 2006;

Derlet et al. 1992; McNamara et al. 2006, 2007; Vanattou-Saïfoudine et al. 2012). When co-

administered with MDMA, caffeine potentiated the MDMA effect not only on the DA but also on

5-HT release, as measured by an in vivo microdialysis in mice (Gorska and Gołembiowska 2015).

Gorska and co-workers, using selective antagonists of A1 and A2A receptors, showed that both

adenosine receptors markedly enhanced the DA and 5-HT release induced by MDMA in the mouse

striatum (Gorska and Gołembiowska 2015). These data suggest that DA release induced by

combined treatment of MDMA and caffeine is mediated by blockade of both adenosine receptors.

Moreover, hyperthermia and neuroinflammation after acute but not chronic administration of

caffeine and MDMA have been reported to cause neurotoxic effects in rodents (Khairnar et al.

2010; McNamara et al. 2006; Ruiz-Medina et al. 2013; Vanattou-Saïfoudine et al. 2011). On the

other hand, exacerbation of MDMA-induced hyperthermia by caffeine is proposed to result from

the inhibition of A2A receptors (Vanattou-Saïfoudine et al. 2010).

However, caffeine has been reported to potentiate the glia-activation in the striatum of adult and

adolescent mice, when it has given together with MDMA (Khairnar et al. 2010; Frau et al. 2016).

Probably, by blocking both A1 and A2A receptors, caffeine may facilitate neuroinflammatory

processes induced by MDMA. However, the promotion of temperature elevation induced by

caffeine (McNamara et al. 2006) might contribute to the increase in astroglia reactivity observed

after MDMA plus caffeine administration, suggesting another possible mechanism potentiating

27

MDMA effects (Moratalla et al. 2015). Interestingly, adolescent mice showed a higher sensitivity to

dopaminergic neurodegeneration induced by the association caffeine - MDMA both in striatum and

SNc, compared with adults, suggesting that caffeine may worsen the toxicity elicited by MDMA in

adolescents (Frau et al. 2016).

Finally, the caffeine ability to affect the absorption of MDMA in intestinal epithelial cells and

increase the area under the plasma concentration curve of MDMA (Kuwayama et al. 2007), as well

as the increased DA release, excessive oxidative stress or formation of toxic metabolites may have a

role in caffeine‟s potentiation of MDMA effects (Gołembiowska et al. 2009; Gołembiowska and

Dziubina 2012).

Contrary to the acute treatment, the chronic administration of caffeine before an acute MDMA

treatment exerts a protective role against MDMA induced neuroinflammation (Ruiz-Medina et al.

2013). One explanation of this paradox is that mice pretreated with caffeine did not experience

temperature increase after MDMA administration; in fact after 21 days of chronic daily caffeine

administration, tolerance to elevated body temperature induced by caffeine, may be developed.

These data demonstrated that depending on the dose and/or administration pattern (acute versus

chronic), caffeine could contribute or attenuate brain damage (Ruiz-Medina et al. 2013).

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AIMS

The therapy of PD is based on the treatment of symptoms while neuroprotective treatments, the

only able to stop the disease progression, are not present yet. Starting from the assumption that both

MPTP and MDMA in mice are able to affect the nigrostriatal pathway inducing a specific

dopaminergic neurodegeneration, and considering that one of the possible multiple PD origins is the

chronic exposure to dopaminergic neurotoxins, the nigrostriatal neurodogeneration induced by

either MDMA or MPTP administration were used as models to test the effects of substances

potentially neuroprotective.

On this basis, the present study evaluated the influence of drugs, such as MAO inhibitors and

metformin, or substances, such as caffeine, on the neurodegenerative effects of MDMA and MPTP

in mice.

1. In the first phase of my study, I evaluated the neuroprotective activity of the new MAO-B

inhibitor compound, SZV558, compared with well-known rasagiline, in a chronic mouse model

of MPTP plus probenecid administration, characterized by the progressive development of

parkinsonian symptoms, neurodegeneration, and neuroinflammation (Schintu et al. 2009).

Consequently, the first aim of my study was to investigate whether the SZV558 administration

could revert the motor impairments, the olfactory dysfunction and the loss of nigrostriatal

neurons, induced by MPTPp chronic treatment in adult mice.

To pursue this first aim, the following experiments were carried out:

- Assessment of the olfactory deficit, usually exhibited after a chronic MPTP treatment or in

the early PD stages and evaluated by pellet retrieval olfactory test.

- Assessment of the spontaneous motor activity, the motor performance and coordination,

and finally the skilled forepaw use, evaluated by, respectively, motility test, beam walking

test and inverted grid test after the last MPTP administration.

- Assessment of the dopaminergic neuronal damage in SNc and caudate-putamen (CPu)

studied by the TH immunohistochemistry and the neuronal death in SNc by Nissl staining.

2. In a second phase of my studies, on the basis of previous results that showed a more

pronounced degeneration in adolescent compared with adult mice when MDMA is associated

with caffeine, I studied the neurotoxic proprieties of MDMA and caffeine administrated in

association in adolescent or adult mice.

Consequently, the second aim was to clarify the molecular mechanism at the base of the

different neurotoxic effect of this drug association at different ages. To pursue this second aim,

I measured by immunofluorescent evaluation, the nNOS expression, which plays a critical role

29

in the integration of dopaminergic and glutamatergic transmissions, and the levels of

proinflammatory cytokines IL-1β and TNF-α in the CPu of adolescent or adult mice treated

with MDMA, alone or in combination with caffeine.

3. In the third part of my PhD, starting from the assumptions that the antidiabetic drug metformin

is able to decrease the loss of dopaminergic neurons induced by MPTP, I investigated whether

the neuroprotective effect of metformin is able to counteract the dopaminergic neurotoxicity

induced by repeated MDMA administration in adult mice. The third aim of my thesis was to

evaluate the effects of metformin administration on nigrostriatal dopaminergic loss of neurons

induced by acute MDMA treatment.

To pursue this third aim, I assess the dopaminergic neuronal damage in SNc and CPu by TH

immunohistochemistry and the neuronal death in SNc by Nissl staining.

30

MATERIALS AND METHODS

1. Drugs

The following drugs were used:

- (hetero)arylalkenylpropargylamine derivative (SZV558) and (R)-N-(prop-2-ynyl)-2,3-

dihydro-1H-inden-1-amine (rasagiline),which were synthesized according to the method

used by Huleatt and coworkers (Huleatt et al. 2015).

- Probenecid, MPTP-HCl, metformin and caffeine were obtained from Sigma (Sigma-

Aldrich, Milan, Italy).

- MDMA–HCl was synthesized at the Department of Life and Environmental Sciences,

University of Cagliari (Frau et al. 2013).

SZV558, rasagiline, MPTP and metformin were dissolved in distillated water. While probenecid

was dissolved in 5% NaHCO3 and MDMA was dissolved in saline. All solutions were freshly

prepared on the day of use.

2. Treatments

Experimental procedures were approved by the Ethics Committee of the University of Cagliari in

compliance with the European Communities Council Directives (2010/63/EU; L.276; 22/09/2010).

Efforts were made to minimize the number of animals used and to maximize humane treatment.

2.1 Chronic protocol of MPTP plus probenecid (MPTPp)

Experiments were performed on adult (12-week-old) male C57Bl/6J mice (Charles River, Italy)

treated with vehicle (saline solution) plus probenecid, or MPTP (25 mg/kg i.p.) plus probenecid

(100 mg/kg i.p.), administered 30 min before each MPTP administration (MPTPp), twice a week for

5 weeks, alone, or in the presence of the SZV558 (1 mg/kg i.p.) or rasagiline (1 mg/kg i.p.)

administered 18 hours before each MPTPp administration. At the end of treatment, mice were tested

behaviorally (motility test, beam-walking test, inverted grid test and olfactory test) to evaluate the

motor and olfactory performance impairments. Animals were sacrificed 3 days after the last

administration of MPTPp.

2.2 Acute MDMA treatment in combination with caffeine

Adult and adolescent (28-day-old) male C57BL/6J mice, were treated with vehicle, caffeine (2×10

mg/kg, 4-hour interval, i.p.), MDMA (4×20 mg/kg, 2-hour intervals, i.p.) or MDMA (4×20 mg/kg,

2-hour intervals) plus caffeine (2×10 mg/kg, before the first and third MDMA administration). On

31

the second day, mice received vehicle or caffeine (2×10 mg/kg, 12-hour interval), and on the third

day, mice received vehicle or caffeine (1×10 mg/kg). Animals were sacrificed 48 hours after the last

MDMA administration.

2.3 Acute MDMA treatment in combination with metformin

Following the previous MDMA treatment, adult male mice, were treated with metformin (2×200

mg/kg, 10-hour interval, o.s.) or MDMA plus metformin (2×200 mg/kg, 1h before the first MDMA

administration and 4h after the last). On the next two days, mice received one daily administration

of vehicle or metformin (200 mg/kg). 48 hours after the last administration of MDMA, mice were

sacrificed and transcardially perfused.

3. Behavioral tests

3.1 Spontaneous motor activity: motility test

Spontaneous motility was assessed 2 days before the MPTPp treatment and 1 day after the last

MPTPp administration, in a quiet isolated room. Mice were placed individually in plexiglass cages

(length 47 cm, height 19 cm, width 27 cm), with a metal grid over the floor, and equipped with

infrared photocell emitters-detectors situated along the long axis of each cage (Opto-Varimex Mini;

Columbus Instruments). The interruption of a photocell beam was detected by a counter that

recorded the total number of photocell beam interruptions. The counter recorded two different types

of motor activity: locomotor activity due to the locomotion of the mouse along the axes of the cage

and total motor activity due to locomotion plus non-finalized movements (stereotyped behaviors,

such as grooming, rearing, and sniffing). The counter recognized the stereotyped movements

because of the continuous interruption of the same photocell beam, whereas locomotion along the

cage produced interruptions of different photocell beams. Motility was detected as soon as the

mouse entered into the cage and was evaluated for 60 min (Baiguera et al. 2012).

3.2 Beam-walking test

The motor performance and coordination of mice were evaluated with the beam-walking test

(Fleming et al. 2004). In this test, mice were trained to traverse the length of a plexiglass beam that

was divided into four sections of 25 cm each (1 m total length). Each section of the beam had a

different width: 4, 3, 2, and 1 cm; the beam was placed on a table and ended in the animal‟s home

cage. Mice received 2 days of training before testing. On the first day, mice received two assisted

trials, involving the placement of the mouse on one extremity of the beam with the home cage in

close proximity to the animal. This encourages forward movement along the beam. After two

32

assisted trials, mice were able to traverse the entire length of the beam unassisted. Days 1 and 2 of

training ended when all animals had completed five unassisted runs across the entire length of the

beam. To increase the difficulty further, on the day of the test, a mesh grid (1 cm squares) of

corresponding width was placed over the beam surface. The test was performed 2 days after the last

MPTPp administration and mice were videotaped for a total of five trials. An error was counted

when, during a forward movement, a limb slipped through the grid. By scoring each limb slip

individually, the severity of the error could be measured. The number of steps and the number of

errors were calculated across all five trials and averaged for each group (Fleming et al. 2004).

3.3 Inverted grid test

The inverted grid test was used to assess skilled forepaw use, especially related to the distal

musculature and digit manipulations. Mice were placed in the center of a horizontal square grid (15

cm2) consisting of a wire mesh (mesh 0.5 cm

2) surrounded by wooden walls. The grid was placed

20 cm above a tabletop and was rotated upside down allowing mice to move freely. The time the

mice took before falling down was recorded. If a mouse fell from the mesh grid within 10 s,

additional trials were allowed (maximum: three trials) within an interval of 5 min; in this case,

latencies before falling were measured. The mean ± SEM of three trials was calculated. Moreover,

for each trial, the number of steps and forelimb fault per step was rated and compared with controls.

No training was performed, but a pre-test was carried out (Meredith and Kang 2006). The time the

mice took before falling down was measured 2 days after the last MPTPp administration.

3.4 Pellet retrieval olfactory test

Mice were food-deprived for 20 hours before the olfactory test. The test was conducted in a clean

plastic cage (length 42 cm, height 15 cm, width 24 cm). A smelling pellet was buried under the

bedding (1 cm) in a cage corner. The mouse was positioned in the center of the cage and the time to

retrieve the pellet and bite it was measured (Schintu et al. 2009). The retrieval time of the buried

pellet was measured 3 days after the last MPTPp administration.

4. Immunohistochemistry

4.1 Immunohistochemistry and cresyl violet for Nissl staining

Three days after the last dose of MPTPp or 48 hours after the last MDMA administration, the mice

were anesthetized with chloral hydrate (400 mg/kg i.p.), transcardially perfused with 4%

paraformaldehyde in phosphate buffered (PB 0.1 M, pH 7.4), and their brains removed and used for

33

immunohistochemistry. Coronal sections (40 μm thick) were cut on a vibratome. Free-floating

sections were incubated overnight with TH antibody (polyclonal rabbit anti-TH, 1:1000, Millipore,

USA). The primary antibody was prepared in Phosphate buffer solution (PBS) plus Triton solution

containing normal goat serum. After careful washing, the sections were incubated in proper

biotinylated secondary antibody (Vector, UK). For visualization, avidin-peroxidase protocol (ABC,

Vector, UK) was applied, using 3,3′-diaminobenzidine (Sigma, Italy) as chromogen. After washing,

the sections were mounted on gelatin-coated slides, air-dried, dehydrated in ascending

concentrations of ethanol, and cleared with xylene (Frau et al 2011).

Adjacent SNc sections were stained with cresyl violet for the Nissl staining to evaluate cell death in

this area. For TH and cresyl-violet-stained cells immunohistochemistry in the SNc, three sections

were sampled (anterior–posterior: -2.92 to -3.28 mm from bregma) according to the atlas of Paxinos

and Franklin 2001. For each mouse, three sections from the CPu (anterior-posterior: 1.10 mm to

0.62 mm from bregma) were analyzed for TH.

4.2 Immunofluorescence for nNOS, IL-1β and TNF-α

Sections from the CPu (50 μm thick) were cut coronally on a vibratome and incubated with the

primary antibodies for IL-1β, TNF-α and nNOS, (polyclonal goat anti-IL-1β and polyclonal goat

anti-TNF-α, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA; polyclonal rabbit anti-nNOS,

1:3000, Millipore, Temecula, CA, USA) for 48 h at 4°C in PBS containing Triton X-100, BSA, and

NGS. For fluorescent immunostaining of nNOS, AlexaFluor® 488-labeled goat anti-rabbit IgG

(1:400, Jackson ImmunoResearch Europe, Suffolk, UK) was used as a secondary antibody, while a

three-step detection was used to increase the signal of IL-1β and TNF-α antibodies by combining

biotin-conjugated IgG (1:200, rabbit anti-goat, Vector, Peterborough, UK) and AlexaFluor® 488-

labeled streptavidin–fluorescein (1:200, Jackson ImmunoResearch Europe, Suffolk, UK). To allow

visualization of cell nuclei, sections were finally incubated for 10 min with 4′,6-diamidine-2′-

phenylindole dihydrochloride (DAPI, 1:10,000, Sigma-Aldrich, Milan, Italy). Finally, all sections

were rinsed and mounted on slides using VectaShield anti-fade mounting media (Vector Inc.).

Standard control experiments were performed by omission of either the primary or secondary

antibody, and yielded no cellular labeling.

4.3 Analysis of TH-positive cells and Nissl staining in the SNc

Three sections of SNc were captured at 10x magnification; in each section, the whole left and right

SNc area were analyzed. Images were digitized (PL-A686 video camera, Pixelink, Canada) under

constant-light conditions. The stained cells were counted manually by a blind experimenter. The

34

number of TH-positive cells and Nissl stained neurons was obtained separately for each SNc level.

Thereafter, in order to obtain an average value from all levels analyzed, the number of cells/level

from each mouse was normalized with respect to the vehicle. Values from the three levels were than

averaged to generate a mean.

4.4 Analysis of TH-positive fibers in CPu

Images were digitized in gray scale with a video camera (Pixelink PL-A686) and TH

immunoreactivity analysis was performed using the Scion Image analysis program (Scion Corp.

USA). The average gray values from white matter were subtracted from each section to correct for

background immunoreactivity. For each level of CPu, the obtained value was first normalized with

respect to vehicle, and values from different levels were averaged thereafter.

4.5 Analysis of nNOS-positive cells in CPu

Analysis of nNOS immunoreactivity for each animal was performed on one tissue section out of

every 3 successive sections for a total of 6 sections containing the CPu. We have chosen the total

size of the examined area in which the nNOS-positive neurons were counted in order to include

almost all of the CPu, according to the extension of the region under analysis. The selected coronal

levels of these sections corresponded to the levels of plates 11–29 for the CPu (AP: +1.1 to +0.02).

The number of nNOS-positive neurons was counted bilaterally in 6 sections per animal. In these

sections, 12 non-overlapping randomly selected ROIs of 0.15 mm2, were examined with a 20×

objective of Olympus IX 61 microscope by two trained observers blind to drug treatment. In order

to ensure that the ROIs did not overlap, their limits were defined based on structural details within

the tissue sections. We did not count the nNOS-positive neurons touching the inferior or the right

sides of the ROIs. The number of nNOS-positive neurons was expressed as mean/mm2 ± SEM.

4.6 Analysis of IL-1β and TNF-α immunoreactivity in CPu

Images of single wavelengths were obtained with an epifluorescence microscope (Axio Scope. A1,

Zeiss, Oberkochen, Germany) connected to a digital camera (1.4 MPixels, Infinity 3-1, Lumenera,

Nepean, Canada). In each brain section, two portions from the CPu (dorsolateral and ventromedial),

left and right, were acquired using a 40× objective. The images of both IL-1β and TNF-α were

digitally merged with the images of DAPI, using the Image J software (U.S. National Institutes of

Health, USA). In the merged image, the levels of IL-1β and TNF-α were quantified. For each

animal, three sections from the CPu (A: 1.10 mm; 0.74 mm; 0.38 mm from bregma) were analyzed.

35

For each section of the CPu, the obtained value was first normalized with respect to vehicle, and

then values from different sections were averaged.

5. Statistical analysis

Behavioral results were statistically compared with a one-way analysis of variance (ANOVA),

followed by Newman-Keuls post hoc test, for comparison between experimental groups. For

immunohistochemistry experiments, results were statistically analyzed by one-way ANOVA

followed by Tukey‟s post hoc test (for unequal N). Results were considered significant at P< 0.05.

36

RESULTS

1. SZV558 administration reverts the motor impairments, olfactory dysfunction and dopaminergic

neuron degeneration induced by a chronic MPTPp treatment

1.1 Changes in spontaneous motor activity: motility test

To evaluate the modification of the spontaneous motor activity induced by the chronic MPTPp

treatment, the total activity and the ambulatory activity were evaluated through the motility test

before the treatment and 1 day after the last MPTPp administration. We observed no significant

changes for total activity and ambulatory activity (Table 1) after MPTPp administration. Moreover,

in animals treated with MPTPp compared with vehicle and MPTPp plus SZV558 or rasagiline

groups, the one-way ANOVA did not indicate a significant effect of treatment in the ambulatory

activity.

Table 1. Motility test pre- and post-chronic MPTPp treatment.

MOTILITY TEST

TOTAL ACTIVITY LOCOMOTOR ACTIVITY

Pre-treatment Post-treatment Pre-treatment Post-treatment

Mean SEM Mean SEM Mean SEM Mean SEM

Vehicle 11503.7 983.5 8737.5 1651.8 8727 983.5 3690.2 1651.8

MPTPp 5803.8 1162.2 6587 771.6 4004.6 916.6 2773.4 343.8

MPTPp+SZV558 9063.1 1055.9 7904.8 982.4 6929.8 791.6 3415.9 463.7

MPTPp+Rasagiline 9941.6 1055.6 8867.8 1214.2 7497 928.8 3938 639.8

Motor activity was measured 2 days before the first MPTPp administration and 1 day after the last MPTPp

administration. Values are expressed as mean ± SEM.

Data were statistically compared with one-way ANOVA, followed by the Newman–Keuls post hoc test.

Number of independent experiments: 4–7/group

1.2 Effect of SZV558 on motor impairment induced by MPTPp: beam-walking test

The number of steps and errors made by mice were recorded for each animal 2 days after the 10th

MPTPp administration, in order to evaluate, respectively, the motor performance and coordinat ion.

The analysis of the time to traverse the beam did not show any difference between the four groups

(data not shown). In contrast, the analysis of the number of steps showed an increase in the MPTPp

group, indicating that MPTPp-treated mice, as parkinsonian patient performed smaller steps.

37

Repeated treatment with SZV558 or rasagiline during MPTPp administration prevented the

impairment induced by MPTPp, as showed by the post hoc test (Newman-Keuls) (Fig. 9a).

Analysis of the total errors showed that MPTPp administration induced a significant increase in

total errors compared with vehicle, demonstrating motor function impairment (Fig. 9b). Motor

function improvement, demonstrated by the decrease in total errors, was observed in the groups

treated with SZV558 or rasagiline compared with the MPTPp group, as indicated in the post hoc

test.

Figure 9. The effect of SZV558 on the behavior in the chronic MPTPp induced PD in mice.

a-b Beam-walking test. To evaluate the number of steps (a) and the total errors (b), the mice were

videotaped for a total of five trials, 2 days after the 10th MPTPp administration. The number of steps and the

number of errors were calculated across all five trials and averaged for each group. *P< 0.05 compared with

vehicle, # P< 0.05 compared with MPTPp. (c) Inverted grid test. To assess skilled forepaw use, the latency

time of falling down was measured 2 days after the 10th MPTPp administration. (d) Olfactory test. To

evaluate the olfactory deficit, the retrieval time of a buried smelling pellet was measured 3 days after the 10th

MPTPp administration. *P< 0.05 compared with MPTPp. Values are expressed as mean ± SEM. Data were

statistically compared with one-way ANOVA, followed by the Newman-Keuls post hoc test.

38

1.3 Grasp-strength evaluation: inverted grid test

The inverted grid test was used to assess forepaw use, especially related to distal musculature and

digit manipulations. This test was done 2 days after the end of treatment. No significant changes

with either MPTPp or rasagiline were observed, indicating that the mice did not have any distal

musculature deficit (Fig. 9c).

1.4 Effect of SZV558 on olfactory deficit induced by MPTPp: Pellet retrieval olfactory test

To reveal any effect of the compounds on olfactory deficit, elicited by MPTPp, mice were evaluated

for olfactory function 3 days after the end of treatment, through the retrieval time of a buried

smelling pellet.

The results of olfactory test showed an increase of retrieval time of a buried smelling pellet,

demonstrating an olfactory deficit, in the MPTPp group. In contrast in mice treated with MPTPp

plus SZV558 or rasagiline, a decrease in retrieval time was observed compared with the MPTPp

group (Fig. 9d).

1.5 Effect of SZV558 on MPTPp-induced neurodegeneration: TH immunohistochemistry and Nissl

Staining in the SNc and CPu

To investigate the effect of SZV558 on dopaminergic cell death, TH immunohistochemistry and

Nissl Staining were performed in the SNc and CPu. MPTPp induced a significant loss of TH-

positive cells in the SNc, as measured by TH immunoreactivity (Fig. 10a). Analysis of Nissl-

positive nigral neurons showed a decrease in number of neurons demonstrating a neuronal death of

MPTPp treated mice compared to vehicle, confirming the dopaminergic neuron degeneration

induced by MPTPp. The combined treatment with MPTPp plus SZV558 or rasagiline prevented the

loss of dopaminergic neurons in the SNc, observed in the mice treated with MPTPp alone, as

demonstrated by the post hoc analysis (Tukey‟s test) of TH immunoreactivity (Fig. 10b). The cresyl

violet staining confirmed the 30% of reduction in number of neurons in the SNc in MPTPp treated

mice. However, this loss of dopaminergic neurons were not observed in Nissl stained SNc of mice

pretreated with SZV558 and rasagiline (Fig. 10c). The loss of TH-positive fibers in the CPu of mice

treated with MPTPp confirmed dopaminergic neuron degeneration (Fig. 10d and e). Both SZV558

and rasagiline prevented neurodegeneration in the CPu compared with the MPTPp group, as shown

by the post hoc analysis (Tukey‟s test) of TH-positive fibers in the CPu.

39

Figure 10. Immunoreactivity for TH in the SNc and CPu in chronic MPTPp treatment.

Representative coronal sections of SNc immunostained for TH show the TH positive neuronal reduction

induced by MPTP treatment (a). The graphs show the number of TH immunoreactive cells (b) and of Nissl-

stained cells (c) in the SNc, expressed as a percentage with respect to vehicle-treated mice. SZV558 and

rasagiline prevented the loss of TH-positive neurons in the SNc, as compared with the animals treated with

MPTPp. Representative coronal sections of striatum immunostained for TH at 5× magnification (d). The

graph shows the mean density of gray value of TH expressed as a percentage with respect to vehicle-treated

mice (e). The TH density reduction confirms the neurodegeneration induced by MPTP. The two compounds

prevent the loss of TH positive fiber induced by MPTPp. Values are expressed as mean ± SEM. *P< 0.05

compared with vehicle. # P< 0.05 compared with MPTPp. Data were statistically compared with one-way

ANOVA, followed by Tukey‟s post hoc test.

D

40

2. Effects of repeated MDMA+caffeine administration in the CPu of adolescent and adult mice

As MPTP, MDMA was shown to produce DA neuron degeneration offer acute-repeated or chronic

treatments. Moreover, caffeine was shown to potentiate DA neurons degeneration. To obtain a

better understanding of the possible molecular mechanism at the basis of the neurodegeneration

observed in CPu of adult and adolescent mice treated with MDMA+caffeine, I evaluated the nNOS,

IL-1β and TNF-α expression (Fig.11a, 12a and 13a).

2.1 nNOS activation in the CPu of adolescent and adult mice

Our results showed a significant increase in nNOS-positive neurons in the CPu of adult mice treated

with MDMA alone (Granado et al. 2008a), caffeine alone, and MDMA plus caffeine, compared

with vehicle (Fig. 11c). In contrast, in adolescent mice, MDMA administration did not increase the

number of nNOS-positive neurons, while caffeine alone or in combination with MDMA

significantly increased the number of nNOS-positive neurons (Fig. 11b).

A

B C

41

Figure 11. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine neuronal nitric

oxide synthase (nNOS) levels in the CPu.

Representative coronal sections of striatum immunostained for Nnos. Scale bar: 100μm (a). Histograms of

nNOS levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean

SEM.*P<0.05;**P<0.002 compared with vehicle-treated mice; by Tukey‟s post hoc test.

2.2 IL-1β activation in the CPu of adolescent and adult mice

Very low levels of IL-1β were observed in the CPu of adult and adolescent mice treated with

vehicle or caffeine alone. Conversely, an increase in the levels of IL-1β was found in adult mice

treated with MDMA alone, compared with mice treated with vehicle or caffeine alone (Fig. 12c),

whereas the same treatment induced no modification in the levels of IL-1β in adolescent mice (Fig.

12b). Combined treatment with MDMA plus caffeine did not further raise the levels of IL-1β

induced by MDMA alone in adults (Fig. 12c), whereas in adolescent mice, an increase in the levels

of IL-1β was observed, compared with vehicle-treated mice (Fig. 12b).

A

B C

42

Figure 12. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine IL-1β levels in

the CPu.

Representative coronal sections of striatum immunostained for IL-1β. Scale bar: 100μm (a). Histograms of

IL-1β levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean

SEM.*P<0.05;**P<0.002 compared with vehicle-treated mice; ^^P<0.002 compared with caffeine-treated

mice by Tukey‟s post hoc test.

2.3 TNF-α activation in the CPu of adolescent and adult mice

Accordingly with IL-1β results, vehicle or caffeine treated groups showed very low levels of TNF-α

in the CPu of adult and adolescent mice. Increased levels of TNF-α was observed in adult mice

treated with MDMA alone, compared with vehicle-treated mice (Fig. 13c), but adolescent MDMA-

treated mice did not show modification in the levels of TNF-α (Fig. 13b). Combined treatment with

MDMA plus caffeine elevated the levels of TNF-α induced by MDMA alone in both adult and

adolescent mice (Figs. 13b and c).

A

B C

43

Figure 13. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine neuronal TNF-α

levels in the CPu.

Representative coronal sections of striatum immunostained for TNF-α. Scale bar: 100μm (a). Histograms of

TNF-α levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean SEM.

**p<0.002 compared with vehicle-treated mice; ^^p<0.002 compared with caffeine-treated mice; ##

p<0.001

compared with MDMA-treated mice by Tukey‟s post hoc test.

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3. Neuroprotective effects of metformin administration on MDMA-induced neurodegeneration:

TH immunoreactivity and Nissl staining

Since the antidiabetic drug metformin has been shown to have neuroprotective activity, in order to

confirm the neuroprotective property of metformin on neurodegenerative processes, induced by an

acute MDMA treatment, I studied its effect on TH immunoreactivity.

In line with previous results, acute repeated MDMA administration induced a significant loss of

TH-positive neurons in the SNc (Fig 14a and b), as measured by TH immunoreactivity. Cresyl

violet staining confirmed a reduction in the number of neurons in the SNc of MDMA-treated mice

by about 30% compared with the vehicle group (data not shown). Moreover, a decrease in TH-

positive fibers in the CPu was observed in MDMA-treated mice compared with the vehicle group

(Fig 14c and d), confirming the MDMA-induced nigrostriatal neurodegeneration.

Metformin treatment during MDMA administration prevented the MDMA-induced decrease in TH-

positive neurons and fibers in the SNc (Fig 14b) and CPu (Fig 14d), respectively. Finally, analysis

of Nissl-positive neurons demonstrated a recovery in the number of neurons in the SNc of mice

treated with MDMA plus metformin compared with the MDMA group.

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Figure 14. Immunoreactivity for TH in the SNc and in CPu in mice treated with MDMA and metformin

coadministration.

Representative coronal sections of SNc immunostained for TH (a). Mice were treated with vehicle,

metformin (2×200 mg/kg, i.p.), MDMA (4×20 mg/kg, i.p.), and MDMA (4×20 mg/kg, i.p.) plus caffeine

(2×200 mg/kg, i.p.) and sacrificed 48 hours after the last MDMA administration. The graph shows the

number of TH positive neurons in the SNc (b).

Values are expressed as mean ± SEM.*P< 0.05 compared with vehicle; #P< 0.05 compared with MDMA.

Data were statistically compared with one-way ANOVA, followed by Tukey‟s post hoc test.

46

DISCUSSION

The study of neurodegenerative effects induced by neurotoxins, allows understanding the influence

of internal or external factors, such as respectively the age and the simultaneous consumption of

other substances, on the development of neurodegenerative processes.

On these bases, the research work of my thesis was based on the study of new compounds that

many display neuroprotective effects in PD animal models. To this end, firstly I evaluated the

neuroprotective effect of a new MAO-B inhibitor, SZV558, on a chronic MPTPp mouse PD model;

then I studied the influence of the two known neuroprotective drugs, caffeine and metformin, on the

neurodegenerative effect of repeated MDMA administration in mice.

1. The neuroprotective effect of SZV558 in a chronic MPTPp model of PD

The results of the present study, consistent with previous studies, show that chronic administration

of MPTPp in mice induced dopaminergic neurodegeneration as evidenced by a decrease in TH

immunoreactivity in the CPu and SNc, associated with motor and olfactory deficits. Administration

of the new (hetero)arylalkenyl propargylamine compound SZV558, similarly to the well-known

MAO-B inhibitor rasagiline, counteracts these deficits and restores both motor function and normal

DA neuron innervation.

The current PD therapies are largely unsatisfactory since DA-replacement therapy only counteracts

the symptoms of the disease without affecting the course of neurodegeneration. Therefore, the

search for therapies that may delay or stop the disease progression is very active. To pursue this

scope, it is necessary to utilize PD models that closely reproduce the disease neurodegeneration that

is slow and starts several years before the motor symptoms appear.

The model chosen to test SZV558, differently from acute MPTP treatments that induce a rapid

degeneration of dopaminergic nigrostriatal neurons, induces a more progressive degeneration, with

neurons continuing to die after completion of toxin administration (Meredith and Ramedacer 2011).

Moreover, when MPTP is co-administered with probenecid, the degeneration of dopaminergic

neurons takes place over a period of 5–8 weeks, inducing apoptosis. The chronic model utilized in

this study has been well characterized and was validated for utilization in a protocol of drug-

induced neuroprotection (Carta et al. 2013).

In the last decade, the MAO-B inhibitors have been proposed as supplementation of the DA

replacement therapy, but the clinical studies fallen to confirm their protective effects (Ahlskog et al.

2010). Moreover, recent studies showed several adverse effects, such as control disorder (Vitale et

al. 2013). Consequently, researchers focalized the attention on new drugs contain the anti-apoptotic

47

propargyl ring and a high MAO-B selectivity. The SZV558 had showed its neuroprotective activity

in in vitro experiments, and it has confirmed this property in the chronic MPTPp treatment.

In the present study the rescue of neurodegeneration was indicated by the immunohistochemical

results that showed that dopaminergic neurodegeneration, evaluated through TH

immunohistochemistry, was rescued by SZV558. The drug, similarly to rasagiline, completely

counteracted the decrease in TH-positive neurons and terminals observed after chronic MPTPp in

the SNc and CPu, respectively.

However, the beam-walking test showed that MPTPp increases the number of steps and errors,

indicating an impairment of gait. It is interesting to note that the beam-walking test strictly reflects

the deficits that characterize PD, such as slowness of movements, indicated by the increased

number of steps to traverse the length of the beam, together with an unstable gait, indicated by the

number of errors (Fleming et al. 2013). SZV558, similarly to rasagiline, counteracts the slowness of

movement, together with the unstable gait induced by chronic MPTPp. In contrast, no impairment

after chronic MPTPp was observed in the grid test, indicating that limb strength, was not impaired

by the chronic regimen of MPTPp administration and no modifications were produced by either

SZV558 or rasagiline. Not surprisingly, no differences were observed in locomotor activity and

total motility after chronic MPTPp, as adaptive changes in motility are consistently observed when

the DA neuron lesion does not exceed 70–80% (Chia et al. 1996; Meredith and Ramedacer 2011).

Confirming previous studies, MPTPp treatment has a significant impact on olfactory ability.

Olfactory deficits and abnormalities in olfaction-related structures, one of early symptoms of PD,

have been showed years before diagnosis of the disorder and appearance of motor symptoms. The

impairment usually affects all areas of olfaction, including identification and discrimination of

odors and reduced sensitivity (Doty 2012 a, b). SZV558 counteracted the olfactory deficit induced

by chronic MPTPp as measured by the decreasing pellet retrieval time.

Therefore, the results showed a strict association between behavioral and biochemical markers of

dopaminergic neurodegeneration.

Previous results by Baranyi (2016) showed that SZV558 decreased the formation of DAQ, the toxic

DA metabolite well known as an effector molecule of dopaminergic neuro-specific cytoxicity

(Asanuma et al. 2003). The DAQ production has been showed in neurodegenerative disease such as

PD, and in exposure to dopaminergic toxins, such as MPTP and MDMA. In fact, it usually

produced only in case of precedent mitochondrial dysfunction (Baranyi et al. 2016). In particular,

DAQ reduces mitochondrial function and permeability (Berman et al. 1999; Bisaglia et al. 2010; Li

et al. 1997) and inhibits ROS scavenger enzymes (Hauser et al. 2013; Belluzzi et al. 2012), leading

oxidative stress. However, DAQ also inhibits proteasomal activity (Zafar et al. 2006) and

48

inactivates functional proteins of dopaminergic neurons, such as TH (Kuhn et al. 1999; Xu et al.

1998), DAT (Whitehead et al. 2001) and alpha-synuclein (Conway et al. 2001).

All together, these mechanisms may contribute to the protective effects exerted by SZV558 on

MPTP toxicity. In order to verify the real death of dopaminergic neurons in the SNc, Nissl stain was

evaluated. The results of this analysis confirmed that chronic MPTPp causes a degeneration of

dopaminergic cells and that coadministration of SZV558 produces neuroprotection.

The effect of SZV558 lasts for up to 42 hours, consistent with the irreversible inhibition of the

MAO-B by the compounds (Baranyi et al. 2016). Moreover, previous results showed that SZV558

is protective against the depletion of DA even when administered after subchronic MPTP, when

MPTP was already converted to the toxic metabolite MPP+

by MAO-B (Jackson-Lewis and

Przedborski 2007), suggesting that SZV558 protects from progressive dopaminergic degeneration,

not only through irreversible MAO-B inhibition, but through a mechanism independent of the

inhibition of MAO-B enzyme (Baranyi et al. 2016). Finally, to confirm that SZV558 protection is

independent from MAO-B inhibition, even if it was administrated 18 hours before MPTP treatment,

our collaborator Baranyi (2016) detected the MPTP/MPP+ ratio 72 hours after an MPTP acute

treatment. The results did not show any significant difference between the MPTP and MPP+ levels

detected in mice pretreated with SZV558 and mice pretreated with vehicle, confirming that SZV558

has a neuroprotection completely independent by MAO-B inhibition (Baranyi et al. 2016). On these

bases, even in the chronic MPTPp treatment, where the compound was administrated 18 hours

before each MPTPp administration, the SZV558 could have a protective action that is independent

of the inhibition of MAO-B enzyme.

In conclusion, the SZV558 showed a neuropretective efficacy in the MPTPp protocol, a model that

closely reproduces neurodegeneration in PD in which dopaminergic neurodegeneration occurs over

time. PD is a multifactorial disease and several risk factors contribute to the vulnerability of DA

neurons (Sulzer and Surmeirer 2013), therefore, SZV558 by affecting multiple targets, may,

through these differentiated mechanisms have a more favorable action than other MAO-B inhibitors

as neuroprotective agent in the therapy of PD.

The results of this study have been published as part of the article:

“Novel (Hetero)arylalkenyl propargylamine compounds are protective in toxin-induced models of

Parkinson's disease”. Baranyi M, Porceddu PF, Gölöncsér F, Kulcsár S, Otrokocsi L, Kittel Á,

Pinna A, Frau L, Huleatt PB, Khoo ML, Chai CL, Dunkel P, Mátyus P, Morelli M, Sperlágh B.

Mol Neurodegener. 2016 Jan 13;11(1):6.

49

2. The neurotoxic effect of caffeine on repeated MDMA administration in adult and adolescent

mice

The use of MDMA in association with caffeinated energy drinks is very common among

adolescents and young adults. Earlier evidences showed the existence of noxious interactions

between MDMA and caffeine, since as reported by Khairnar and coworkers (2010), caffeine is able

to increase the MDMA-induced neuroinflammation in mice (Khairnar et al. 2010). Moreover recent

results suggest that caffeine is able to potentiate the MDMA neurotoxicity (Frau et al. 2016). As

mentioned above, the effect of caffeine on neurodegenerative processes is controversial and

depends on the dose and/or administration pattern (Ruiz-Medina et al. 2013). Netherless several

studies demonstrated that the acute administration of caffeine during repeated administration of

MDMA, potentiated the neurotoxic MDMA effects. In this regard, previous results showed that

combined treatment of MDMA plus caffeine induced an increased loss of striatal DA fibers

compared with MDMA, only in adolescence, but not in adult mice (Fig. 15) (Frau et al. 2016),

suggesting that the abuse of MDMA during the adolescence could render the neurons more

sensitive to the combined effects of other substances.

To examine in depth the caffeine effects and to understand the age-linked differences in MDMA

neurotoxicity, in collaboration with the group of Dr. Castelli I evaluated, the nNOS expression and,

in collaboration with Dr. Costa I measured the levels of the cytokines IL-1β and TNF-α in CPu of

adult and adolescent mice treated with a combined MDMA plus caffeine.

The nNOS, the enzyme synthesis of NO, is involved in many pathological processes, such as the

neurodegenerative effects of amphetamine-related drugs (Dawson and Dawson 1998; Granado et al.

2008a; Castelli et al. 2014). nNOS has an important role in the integration of dopaminergic and

glutamatergic transmission, since activation of NMDA receptors, following the increase in GLU

release, activates nNOS that produces NO. NO increases DA and GLU release, and, depending on

the level of NO production and the oxidative levels, may interact with H2O2 to produce

peroxynitrite (Itzhak et al. 2006). Moreover, oxidative stress increased by peroxynitrite, promotes

the oxidation of DA in the toxic metabolite DAQ (LaVoie and Hastings 1999a, b) inducing DA

neurotoxicity. The involvement of nNOS expression in the MDMA neurotoxicity was previously

demonstrated by the protective effect of nNOS inhibitor on loss of striatal DA fibers induced by

MDMA (Colado et al. 2001 and 2004) and by the resistance of nNOS knockout mice to MDMA

neurotoxicity (Itzhak et al. 2004). On these bases, the elevation of nNOS by MDMA observed in

adult but not in adolescent mice, confirmed the lower sensitivity of adolescents to MDMA

neurotoxic effect. Accordingly with these data, an increased expression of IL-1β and TNF-α by

50

MDMA in adults has been observed, while in adolescent MDMA-treated mice, cytokines levels

were similar to vehicle group. These results are in line with previous studies showing

proinflammatory cytokines as a marker correlated with dopaminergic neuron degeneration. In fact

elevated levels of cytokines trigger oxidative and nitrosative stress that can induce the

neurodegenerative process (Hemmerle et al. 2012).

The age of consumers is an important factor that may alter the MDMA neurotoxic effects (Hawkins

et al. 1992). In particular, the neurons during the adolescence appear to be particularly vulnerable to

specific drugs abuse (Cadoni et al. 2015; Daza-Losada et al. 2009; Sisk and Zehr 2005; Spear

2000). Netherless, the CPu of adolescent mice showed a lower sensitivity to MDMA

neurodegenerative effect, compared with adults (Reveron et al. 2005; Frau et al. 2016). Moreover,

repeated MDMA administration induces a decrease of striatal TH density in adult, but not in

adolescent mice (Frau et al. 2016). On these bases, I can speculate that low nNOS and cytokines

levels, as observed in adolescent MDMA-treated mice, are not able to lead to process related to

oxidative stress and the resulting neurodegeneration.

Figure 15. Reported current results of striatal neurodegeneration and neuroinflammation in adult and

adolescent mice treated with caffeine alone (2x10mg/kg), MDMA alone (4x20mg/kg) or in combination

(MDMA 4x20mg/kg plus caffeine 2x10mg/kg).

51

On the other hand, caffeine induces an elevation of nNOS in adolescent but not in adult mice treated

with repeated MDMA administration, showing the potentiation of MDMA effects induced by

caffeine in adolescents (Fig. 15). As shown by Gorska and Gołembiowska (2015), this potentiation

could be explained by an increase of MDMA-stimulated DA release by concomitant administration

of caffeine. In particular, Gorska and Gołembiowska (2015) demonstrated that caffeine potentiates

the DA release induced by MDMA through the antagonism of both A2A and A1 receptors (Gorska

and Gołembiowska 2015). On this basis, the blockade by caffeine of A2A receptors, localized in

striatal glutamatergic terminals and involved in the modulation of GLU release (Ciruela et al. 2006),

could play a role in the nNOS expression. Moreover, the A1 blockade by caffeine might increase

the release of proinflammatory cytokines. In fact, elevated levels of proinflammatory cytokines,

such as IL-1β, are observed in A1 knockout mice (Tsutsui et al. 2004). Another hypothesis that can

explain the elevated cytokines release by combined administration of MDMA and caffeine, is the

ability of caffeine to potentiate the hyperthermia induced by MDMA (McNamara et al. 2006). The

MDMA-induced hyperthermic response is mediated by elevated levels of IL-1β (Green et al. 2004),

consequently it is conceivable that caffeine is able to increase the cytokines release through the

strengthening of hyperthermic mechanisms.

Caffeine alone does not induce any decrease in striatal TH-positive fibers (Fig. 15) (Frau et al.

2016), consequently the only increase in nNOS does not appear to be correlated with any

neurodegenerative process, since the NO may induce oxidative stress only if it interacts with H2O2.

These data allow inferring that the nNOS may lead neurodegeneration only if there is an oxidative

process underway. Therefore, increased nNOS levels together with a neuroinflammatory processes

(demonstrated by elevated levels of IL-1β and TNF-α) which increases oxidative stress, as seen

after MDMA alone and MDMA plus caffeine (Fig. 15), might create a neurotoxic environment

causing dopaminergic neuron degeneration.

In conclusion, the use of caffeine in association with MDMA during adolescence may worsen the

neurotoxicity and neuroinflammation elicited by MDMA, confirming that the adolescence is a

critical phase of life not only for the development of addiction, but also for the possibility of

causing neurotoxicity to dopaminergic neurons.

These results have been published as part of the article: “Influence of caffeine on 3,4-

methylenedioxymethamphetamine-induced dopaminergic neuron degeneration and

neuroinflammation is age-dependent”. Frau L, Costa G, Porceddu PF, Khairnar A, Castelli MP,

Ennas MG, Madeddu C, Wardas J, Morelli M.

J Neurochem. 2016 Jan;136(1):148-62.

52

3. The neuroprotective effect of metformin on neurodegeneration induced by repeated MDMA

administration

The neurotoxicity to dopaminergic neurons induced by MDMA in adult mice has been

demonstrated by several studies, using different drug doses and different administration protocols

(Granado et al. 2008a and 2011; Costa et al. 2013).

In line with previous studies, acute repeated MDMA administration induces a significant loss of

TH-positive neurons and fibers, respectively, in the SNc, and CPu of adult mice (Fig. 15).

The reduction in the number of neurons in the SNc of MDMA-treated mice by about 30% compared

with the vehicle group, was confirmed by cresyl violet staining, showing that MDMA doesn‟t

reduce TH expression, but induces dopaminergic neurodegeneration (Granado et al. 2008a).

Metformin treatment prevented the MDMA-induced decrease in TH-positive neurons and fibers in

the SNc and CPu, respectively, confirmed by the analysis of Nissl-positive neurons that

demonstrated a recovery in the number of neurons in the SNc of mice treated with MDMA plus

metformin by about 30% compared with the MDMA group.

The neuroprotective effect of metformin observed in this study could be particularly useful,

considering the higher incidence of PD reported in amphetamine-related drugs abusers (Brust 2010;

Christine et al. 2010; Callaghan et al. 2012; Curtin et al. 2015) and is in line with the

neuroprotective proprieties of metformin demonstrated in epidemiological and animal models of PD

(Wahlqvist et al. 2012; Patil et al. 2014). In this regard, metformin, attenuating the generation of

ROS and RNS (Chakraborty et al. 2011), and increasing antioxidant enzymes activity (Patil et al.

2014), may counteract the oxidative stress, one of the principal mechanisms involved in

neurodegenerative disease and in MDMA or MPTP neurotoxicity (Fig. 3 and 5) (Meredith and

Kang 2006; Puerta et al. 2010; Górska et al. 2014 and 2015).

The mechanism proposed for the neuroprotection of metformin in the CNS is the activation of

AMPK which is a key regulator of cellular energy metabolism. On the other hand, in drosophila

models of PD, AMPK activation induced by metformin, alleviated dopaminergic dysfunction and

mitochondrial abnormalities (Ng et al. 2012). However, the activation of AMPK exerts a protective

effect against the neurotoxic effects of MPP+ and it has been proposed as a survival factor for

dopaminergic neurons in PD (Choi et al 2010). AMPK has also a potential anti-inflammatory

activity, in fact it is a marker of M2 macrophages which are generally anti-inflammatory (Sag et al.

2008). In this regard, a recent study showed a decreased production of the proinflammatory

cytokine IL-1β and increase in antinflammatory IL-10, induced by metformin in LPS-activated

macrophages, suggesting that this antidiabetic drug may modulate the inflammatory response in

53

microglia (Kelly et al. 2015). Considering the strong correlation between neurodegeneration and

neuroinflammation, the antinflammatory property of metformin has been to take in consideration as

possible neuroprotective mechanism.

Finally, metformin has been reported as promoter of neurogenesis by activating an atypical PKC–

CREB pathway. Moreover, CREB a transcription factor played a major role in neurodevelopment,

synaptic plasticity, and neuroprotection (Sakamoto et al. 2011), promotes the transcription of BDNF

and TH (Piech-Dumas and Tank 1999).

As shown by studies performed in vivo and in vitro, MDMA, altering dopaminergic transmission,

may affect neurogenesis and synaptogenesis (Rice and Barone 2000; Fasano et al. 2008; Goffin et

al. 2010). On this basis, the restorative number of TH-positive neurons in mice treated with MDMA

plus metformin suggests that another mechanism for metformin protection may be the neurogenesis,

which is affected in MDMA-treated animals.

The findings of this study showed that metformin treatment prevents the neurodegenerative effects

of repeated MDMA administration in adult mice, confirming the therapeutic potential of this

antidiabetic medication in the treatment of neurodegenerative processes.

These results and findings have been submitted for publication to the journal Neurotoxicity

Research: “Metformin protects against dopaminergic neurotoxicity induced by 3,4-

methylenedioxymethamphetamine administration”. Porceddu PF, Ishola OI, Morelli M.

54

CONCLUSIONS

These results demonstrated that the dopaminergic neurodegenerative process may be induced or

conditioned by environment stressors or substances which influence, through different ways, the

development of neurodegenerative mechanisms.

In the present study I evaluated the effects of three substances, known as potentially

neuroprotective, in combination with two different neurotoxins that affect the nigrostriatal

dopaminergic system. The SZV558 MAO-B inhibitor and the metformin protected the nigrostriatal

pathway, affected by MPTP- and MDMA- induced neurotoxicity. On the other hand, caffeine,

administrated with MDMA, showed a neurotoxic potential depending on the age of consumers,

confirming the vulnerability of adolescent brain to consumption of drugs and substances that

affected the dopaminergic system.

In conclusion, the study of neurodegenerative processes may be relevant to understand the human

pharmacology, the origin and development of neurodegenerative disease and to predict the

neurotoxic effect of drug abuse.

55

REFERENCES

1. Adams JD Jr, Klaidman LK, Leung AC. MPP+ and MPDP+ induced oxygen radical

formation with mitochondrial enzymes. Free Radic Biol Med. 1993;15(2):181-6.

2. Aguirre N, Barrionuevo M, Ramirez MJ, Del Rio J, Lasheras B. Alpha-lipoic acid prevents

3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity. NeuroReport.

1999;10, 3675–3680.

3. Ahlskog JE, Uitti RJ. Rasagiline, Parkinson neuroprotection, and delayed-start trials: still no

satisfaction? Neurology. 2010;74(14):1143-8.

4. Amato S, Man HY. Bioenergy sensing in the brain: the role of AMP-activated protein

kinase in neuronal metabolism, development and neurological diseases. Cell Cycle

2011;15;10(20):3452-60.

5. Araki E, Forster C, Dubinsky JM, Ross ME, Iadecola C. Cyclooxygenase-2 inhibitor ns-398

protects neuronal cultures from lipopolysaccharide-induced neurotoxicity. Stroke.

2001;32(10):2370-5.

6. Asanuma M, Miyazaki I, Ogawa N. Dopamine- or L-DOPA-induced neurotoxicity: the role

of dopamine quinone formation and tyrosinase in a model of Parkinson's disease. Neurotox

Res. 2003;5(3):165-76.

7. Ascherio A, Zhang SM, Hernán MA, Kawachi I, Colditz GA, Speizer FE, Willett WC.

Prospective study of caffeine consumption and risk of Parkinson's disease in men and

women. Ann Neurol. 2001;50:56-63.

8. Autere J, Moilanen JS, Finnila S, Soininen H, Mannermaa A, Hartikainen P, Hallikainen M,

Majamaa K. Mitochondrial DNA polymorphisms as risk factors for Parkinson‟s disease and

Parkinson‟s disease dementia. Hum Genet. 2004;11:29–35.

9. Bai F, Jones DC, Lau SS, Monks TJ. Serotonergic neurotoxicity of 3,4-(+/-)-

methylenedioxyamphetamine and 3,4-(+/-)-methylenedioxymethamphetamine (Ecstasy) is

potentiated by inhibition of gamma-glutamyl transpeptidase. Chem Res Toxicol. 2001;14,

863–870.

10. Baiguera C, Alghisi M, Pinna A, Bellucci A, De Luca MA, Frau L, Morelli M, Ingrassia R,

Benarese M, Porrini V, Pellitteri M, Bertini G, Fabene PF, Sigala S, Spillantini MG, Liou

HC, Spano PF, Pizzi M. Late-onset Parkinsonism in NFκB/c-Rel-deficient mice. Brain.

2012;135(Pt 9):2750-2765.

11. Bara-Jimenez W, Sherzai A, Dimitrova T, Favit A, Bibbiani F, Gillespie M, Morris MJ,

Mouradian MM, Chase TN. Adenosine A(2A) receptor antagonist treatment of Parkinson‟s

disease. Neurology. 2003;61:293–296.

56

12. Bar-Am O, Amit T, Youdim MB. Aminoindan and hydroxyaminoindan, metabolites of

rasagiline and ladostigil, respectively, exert neuroprotective properties in vitro. J

Neurochem. 2007;103, 500-508.

13. Bar-Am O, Weinreb O, Amit T, Youdim MBH. Regulation of Bcl-2 family proteins,

neurotrophic factors, and APP processing in the neurorescue activity of propargylamine.

FASEB J. 2005;19:1899-1901.

14. Baranyi M, Porceddu PF, Gölöncsér F, Kulcsár S, Otrokocsi L, Kittel Á, Pinna A, Frau L,

Huleatt PB, Khoo ML, Chai CL, Dunkel P, Mátyus P, Morelli M, Sperlágh B. Novel

(Hetero)arylalkenyl propargylamine compounds are protective in toxin-induced models of

Parkinson's disease. Mol Neurodegener. 2016;13;11(1):6.

15. Barcia C, Bautista V, Sánchez-Bahillo A, Fernández-Villalba E, Navarro-Ruis JM, Barreiro

AF, Poza Y Poza M, Herrero MT. Circadian determinations of cortisol, prolactin and

melatonin in chronic methyl-phenyl-tetrahydropyridine-treated monkeys.

Neuroendocrinology. 2003;78(2):118-28.

16. Barcia C, Ros CM, Annese V, Gómez A, Ros-Bernal F, Aguado-Year D, Martínez-Pagán

ME, de Pablos V, Fernandez-Villalba E, Herrero MT. IFN-γ signaling, with the synergistic

contribution of TNF-α, mediates cell specific microglial and astroglial activation in

experimental models of Parkinson's disease. Cell Death Dis. 2011;2:e142.

17. Barcia C, Sànchez Bahillo A, Ferna´ndez-Villalba E, Bautista V, Poza Y, Poza M,

Ferna´ndez-Barreiro A, Hirsch EC, Herrero MT. Evidence of active microglia in substantia

nigra pars compacta of parkinsonian monkeys 1 year after MPTP exposure. Glia.

2004;46:402–409.

18. Bartels AL, Willemsen AT, Doorduin J, de Vries EF, Dierckx RA, Leenders KL. [11C]-

PK11195 PET: quantification of neuroinflammation and a monitor of anti-inflammatory

treatment in Parkinson‟s disease? Parkinsonism Relat Disord. 2010;16:57–9.

19. Baylen CA, Rosenberg H. A review of the acute subjective effects of MDMA/ecstasy.

Addiction. 2006;101(7):933-47.

20. Becker ML, Visser LE, van Schaik RH, Hofman A, Uitterlinden AG, Stricker BH. OCT1

polymorphism is associated with response and survival time in anti-Parkinsonian drug users.

Neurogenetics. 2011;12(1):79-82.

21. Belluzzi E, Bisaglia M, Lazzarini E, Tabares LC, Beltramini M, Bubacco L. Human SOD2

modification by dopamine quinones affects enzymatic activity by promoting its aggregation:

possible implications for Parkinson's disease. PLoS One. 2012;7(6):e38026.

57

22. Ben-Shachar D, Zuk R, Gazawi H, Ljubuncic P. Dopamine toxicity involves mitochondrial

complex I inhibition: implications to dopamine-related neuropsychiatric disorders. Biochem

Pharmacol. 2004;67:1965-1974

23. Berman SB, Hastings TG. Dopamine oxidation alters mitochondrial respiration and induces

permeability transition in brain mitochondria: implications for Parkinson's disease. J

Neurochem. 1999;73(3):1127–37.

24. Betarbet R, Sherer TB, Greenamyre JT. Animal models of Parkinson's disease. Bioessays.

2002;24(4):308-18.

25. Bezard E, Gross CE, Fournier MC, Dovero S, Bloch B, Jaber M. Absence of MPTP-induced

neuronal death in mice lacking the dopamine transporter. Exp Neurol. 1999;155(2):268-73.

26. Bezard E, Jaber M, Gonon F, Boireau A, Bloch B, Gross CE. Adaptive changes in the

nigrostriatal pathway in response to increased 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine-induced neurodegeneration in the mouse. Eur J Neurosci. 2000;

12(8):2892-2900.

27. Bisaglia M, Soriano ME, Arduini I, MammiS, Bubacco L. Molecular characterization of

dopamine-derived quinones reactivity toward NADH and glutathione: implications for

mitochondrial dysfunction in Parkinson disease. Biochim Biophys Acta. 2010;1802(9):699–

706.

28. Biskup S, and Moore DJ. Detrimental deletions: mitochondria, aging and Parkinson‟s

disease. Bioessays. 2006;28:963–967.

29. Block ML, Hong JS. Microglia and inflammation-mediated neurodegeneration: multiple

triggers with a common mechanism. Prog Neurobiol. 2005;76:77–98.

30. Blum D, Torch S, Lambeng N, Nissou M, Benabid AL, Sadoul R, Verna JM. Molecular

pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution

to the apoptotic theory in Parkinson's disease. Prog Neurobiol, 2001;65:135-172.

31. Boka G, Anglade P, Wallach D, Javoy-Agid F, Agid Y, Hirsch EC. Immunocytochemical

analysis of tumor necrosis factor and its receptors in Parkinson‟s disease. Neurosci Lett.

1994;172:151–154.

32. Brodacki B, Staszewski J, Toczyłowska B, Kozłowska E, Drela N, Chalimoniuk M, Stepien

A. Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFalpha, and INFgamma concentrations

are elevated in patients with atypical and idiopathic parkinsonism. Neurosci Lett.

2008;441:158–162.

58

33. Brodkin J, Malyala A, Nash JF. Effect of acute monoamine depletion on 3,4-

methylenedioxymethamphetamine-induced neurotoxicity. Pharmacol Biochem Behav.

1993;45:647-653.

34. Brust JC. Substance abuse and movement disorders. Mov Disord. 2010;25:2010-2020.

35. Burke WJ, Kumar VB, Pandey N, Panneton WM, Gan Q, Franko MW, O'Dell M, Li SW,

Pan Y, Chung HD, Galvin JE. Aggregation of alpha-synuclein by DOPAL, the monoamine

oxidase metabolite of dopamine. Acta Neuropathol. 2008;115(2):193-203.

36. Cadet JL, Krasnova IN, Jayanthi S, Lyles J. Neurotoxicity of substituted amphetamines:

molecular and cellular mechanisms. Neurotox Res. 2007;11(3-4):183-202.

37. Cadet JL, Thiriet N, Jayanthi S. Involvement of free radicals in MDMA-induced

neurotoxicity in mice. Ann Med Interne (Paris). 2001;152:Suppl 3, IS57-9.

38. Cadoni C, Simola N, Espa E, Fenu S, Di Chiara G. Strain dependence of adolescent

Cannabis influence on heroin reward and mesolimbic dopamine transmission in adult Lewis

and Fischer 344 rats. Addict Biol. 2015;20(1):132-42.

39. Callaghan RC, Cunningham JK, Sajeev G, Kish SJ. Incidence of Parkinson‟s disease among

hospital patients with methamphetamine-use disorders. Mov Disord. 2010;25:2333–2339.

40. Callaghan RC, Cunningham JK, Sykes J, Kish SJ. Increased risk of Parkinson‟s disease in

individuals hospitalized with conditions related to the use of methamphetamine or other

amphetamine-type drugs. Drug Alcohol Depend. 2012;120:35-40.

41. Calne DB, Langston JW, Martin WR, Stoessl AJ, Ruth TJ, Adam MJ, Pate BD, Schulzer M.

Positron emission tomography after MPTP: observations relating to the cause of Parkinson's

disease. Nature. 1985;19-25;317(6034):246-8.

42. Camarasa J, Pubill D, Escubedo E. Association of caffeine to MDMA does not increase

antinociception but potentiates adverse effects of this recreational drug. Brain Res.

2006;1111:72-82.

43. Cannon JR, Greenamyre JT. The role of environmental exposures in neurodegeneration and

neurodegenerative diseases. Toxicol Sci. 2011;124(2):225-50.

44. Capela, J.P., Meisel, A., Abreu, A.R., Branco, P.S., Ferreira, L.M., Lobo, A.M., Remiao, F.,

Bastos ML, Carvalho F. Neurotoxicity of Ecstasy metabolites in rat cortical neurons, and

influence of hyperthermia. J Pharmacol Exp Ther. 2006;316:53–61.

45. Carboni S, Melis F, Pani L, Hadjiconstantinou M, Rossetti ZL. The non-competitive

NMDA-receptor antagonist MK-801 prevents the massive release of glutamate and aspartate

from rat striatum induced by 1-methyl-4-phenylpyridinium (MPP+). Neurosci Lett.

1990;4;117(1-2):129-33.

59

46. Carta AR, Carboni E, Spiga S. The MPTP/probenecid model of progressive Parkinson's

disease. Methods Mol Biol. 2013;964:295-308.

47. Carta AR, Kachroo A, Schintu N, Xu K, Schwarzschild MA, Wardas J, Morelli M.

Inactivation of neuronal forebrain A receptors protects dopaminergic neurons in a mouse

model of Parkinson's disease. J Neurochem. 2009;111(no. 6):1478–1489.

48. Casey BJ, Giedd JN, Thomas KM. Structural and functional brain development and its

relation to cognitive development. Biol Psychol. 2000;54(1-3):241-57.

49. Castagnoli N Jr, Chiba K, Trevor AJ. Potential bioactivation pathways for the neurotoxin 1-

methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Life Sci. 1985;21;36(3):225-30.

50. Castelli MP, Madeddu C, Casti A, Casu A, Casti P, Scherma M, Fattore L, Fadda P, Ennas

MG. Δ9-tetrahydrocannabinol prevents methamphetamine-induced neurotoxicity. PLoS One

2014;9:e98079.

51. Chakraborty A, Chowdhury S, Bhattacharyya M. Effect of metformin on oxidative stress,

nitrosative stress and inflammatory biomarkers in type 2 diabetes patients. Diabetes Res

Clin Pract. 2011;96:53–62.

52. Chan CS, Guzman JN, Ilijic E, Mercer JN, Rick C, Tkatch T, Meredith GE, Surmeier DJ.

„Rejuvenation‟ protects neurons in mouse models of Parkinson‟s disease. Nature. 2007;447,

1081–1086.

53. Chaudhuri KR, Healy DG, Schapira AH. National Institute for Clinical Excellence. Non-

motor symptoms of Parkinson's disease: diagnosis and management. Lancet Neurol.

2006;5(3):235-45.

54. Chen CC, Wang HJ, Shih HC, Sheen LY, Chang CT, Chen RH, Wang TY. Comparison of

the metabolic effects of metformin and troglitazone on fructose-induced insulin resistance in

male Sprague-Dawley rats. J Formos Med Assoc. 2001;100:176–180.

55. Cheng N, Maeda T, Kume T, Kaneko S, Kochiyama H, Akaike A, Goshima Y, Misu Y.

Differential neurotoxicity induced by L-DOPA and dopamine in cultured striatal

neurons. Brain Res. 1996;743:278-283.

56. Chia LG, Ni DR, Cheng LJ, Kuo JS, Cheng FC, Dryhurst G. Effects of 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine and 5,7-dihydroxytryptamine on the locomotor activity and

striatal amines in C57BL/6 mice. Neurosci Lett. 1996;218(1):67–71.

57. Chiba K, Peterson LA, Castagnoli KP, Trevor AJ, Castagnoli N Jr. Studies on the molecular

mechanism of bioactivation of the selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine. Drug Metab Dispos. 1985;13(3):342-7.

60

58. Choi JS, Park C, Jeong JW. AMP-activated protein kinase is activated in Parkinson's disease

models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Biochem Biophys Res

Commun. 2010;1;391(1):147-51.

59. Christine CW, Garwood ER, Schrock LE, Austin DE, McCulloch CE. Parkinsonism in

patients with a history of amphetamine exposure. Mov Disord. 2010;25, 228-231.

60. Ciruela F, Casadó V, Rodrigues RJ, Luján R, Burgueño J, Canals M, Borycz J, Rebola N,

Goldberg SR, Mallol J, Cortés A, Canela EI, López-Giménez JF, Milligan G, Lluis C,

Cunha RA, Ferré S, Franco R. Presynaptic control of striatal glutamatergic

neurotransmission by adenosine A1-A2A receptor heteromers. J Neurosci.

2006;15;26(7):2080-7.

61. Cohen G. Oxidative stress, mitochondrial respiration, and Parkinson's disease. Ann N Y

Acad Sci. 2000;899:112-20.

62. Colado M, Williams J, Green A. The hyperthermic and neurotoxic effects of “ecstasy”

(MDMA) and 3,4 methylenedioxyamphetamine (MDA) in the dark agouti (DA) rat, a model

of the cyp2d6 poor metabolizer phenotype. Br J Pharmacol. 1995;115:1281–1289.

63. Colado MI, Camarero J, Mechan AO, Sanchez V, Esteban B, Elliott JM, Green AR. A study

of the mechanisms involved in the neurotoxic action of 3,4-

methylenedioxymethamphetamine (MDMA, 'ecstasy') on dopamine neurones in mouse

brain. Br J Pharmacol. 2001;134:1711-23.

64. Colado MI, Granados R, O‟Shea E, Esteban B, Green AR. Role of hyperthermia in the

protective action of chlomethiazole against MDMA (“ecstasy”)-induced neurodegeneration,

comparison with the novel NMDA channel blocker AR-R15896AR. Br J Pharmacol.

1998;124:479–484.

65. Colado MI, O'Shea E, Green AR. Acute and long-term effects of MDMA on cerebral

dopamine biochemistry and function. Psychopharmacology (Berl). 2004;173:249-63.

66. Conway KA, Rochet JC, Bieganski RM, Lansbury Jr PT. Kinetic stabilization of the alpha-

synuclein protofibril by a dopamine-alpha-synuclein adduct. Science.

2001;294(5545):1346–9.

67. Costa G, Frau L, Wardas J, Pinna A, Plumitallo A, Morelli M. MPTP-induced dopamine

neuron degeneration and glia activation is potentiated in MDMA-pretreated mice. Mov

Disord. 2013;28:1957-1965.

68. Costa, G., Simola, N., Morelli, M. MDMA administration during adolescence exacerbates

MPTP-induced cognitive impairment and neuroinflammation in the hippocampus and

prefrontal cortex. Psychopharmacology. 2014;231:4007-4018.

61

69. Cozzi NV, Sievert MK, Shulgin AT, Jacob P, Ruoho AE. Inhibition of plasma membrane

monoamine transporters by beta-ketoamphetamines. Eur J Pharmacol. 1999;381:63-9.

70. Cui M, Aras R, Christian WV, Rappold PM, Hatwar M, Panza J, Jackson-Lewis V, Javitch

JA, Ballatori N, Przedborski S, Tieu K. The organic cation transporter-3 is a pivotal

modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad

Sci USA. 2009;12;106(19):8043-8.

71. Curtin K, Fleckenstein AE, Robison RJ, Crookston MJ, Smith KR, Hanson GR.

Methamphetamine/amphetamine abuse and risk of Parkinson's disease in Utah: a

population-based assessment. Drug Alcohol Depend. 2015;146:30-38.

72. Członkowska A, Kohutnicka M, Kurkowska-Jastrzebska I, Członkowski A. Microglial

reaction in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced Parkinson's

disease mice model. Neurodegeneration. 1996;5(2):137-43.

73. Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron.

2003;11;39(6):889-909.

74. Davison D, Parrott AC. Ecstasy (MDMA) in Recreational Users: Self-Reported

Psychological and Physiological Effects. Hum Psychopharmacol. 1997;12:221–226.

75. Dawson TM, Ko HS, Dawson VL. Genetic animal models of Parkinson's disease. Neuron.

2010;10;66(5):646-61.

76. Dawson VL, Dawson TM. Nitric oxide in neurodegeneration. Prog Brain Res.

1998;118:215-29.

77. Daza-Losada M, Rodríguez-Arias M, Aguilar MA, Miñarro J. Acquisition and reinstatement

of MDMA-induced conditioned place preference in mice pre-treated with MDMA or

cocaine during adolescence. Addict Biol. 2009;14(4):447-56.

78. De la Torre R, Farré M, Navarro M, Pacifici R, Zuccaro P, Pichini S. Clinical

pharmacokinetics of amfetamine and related substances monitoring in conventional and

non-conventional matrices. Clin Pharmacokinet. 2004;43:157–185.

79. De la Torre R, Farré M, Roset PN, Lopez CH, Mas M, Ortuño J, Menoyo E, Pizarro N,

Segura J, Cami J. Pharmacology of MDMA in humans. Ann NY Acad Sci. 2000;914:225-

237.

80. Del Zompo M, Piccardi MP, Ruiu S, Quartu M, Gessa GL, Vaccari A. Selective MPP+

uptake into synaptic dopamine vesicles: possible involvement in MPTP neurotoxicity. Br J

Pharmacol. 1993;109(2):411-4.

81. Derlet RW, Tseng JC, Albertson TE. Potentiation of cocaine and d-amphetamine toxicity

with caffeine. Am J Emerg Med. 1992;10:211-216.

62

82. Dervan AG, Meshul CK, Beales M, McBean GJ, Moore C, Totterdell S, Snyder AK,

Meredith GE. Astroglial plasticity and glutamate function in a chronic mouse model of

Parkinson's disease. Exp Neurol. 2004;190(1):145-56.

83. Di Filippo M, Picconi B, Costa C, Bagetta V, Tantucci M, Parnetti L, Calabresi P.

Pathways of neurodegeneration and experimental models of basal ganglia disorders:

downstream effects of mitochondrial inhibition. Eur J Pharmacol. 2006;545:65-72.

84. Doty RL. Olfaction in Parkinson‟s disease and related disorders. Neurobiol Dis.

2012a:46:527–552.

85. Doty RL. Olfactory dysfunction in Parkinson disease. Nat Rev Neurol. 2012b;8:329–339.

86. Dowling GP, McDonough ET, Bost RO. 'Eve' and 'Ecstasy'. A report of five deaths

associated with the use of MDEA and MDMA. JAMA. 1987;27;257(12):1615-7.

87. Downing J. The psychological and physiological effects of MDMA on normal volunteers. J

Psychoactive Drugs. 1986;18:335-40.

88. Drechsel DA, Patel M. Role of reactive oxygen species in the neurotoxicity of

environmental agents implicated in Parkinson's disease. Free Radic Biol Med.

2008;1;44(11):1873-86.

89. Easton N, Fry J, O'Shea E, Watkins A, Kingston S, Marsden CA. Synthesis, in vitro

formation, and behavioural effects of glutathione regioisomers of alpha-methyldopamine

with relevance to MDA and MDMA (ecstasy). Brain Res. 2003;987:144-154.

90. Easton N, Marsden CA. Ecstasy: are animal data consistent between species and can they

translate to humans? J Psychopharmacol 2006;20:194-210.

91. Ekblom J, Jossan SS, Bergström M, Oreland L, Walum E, Aquilonius SM. Monoamine

oxidase-B in astrocytes. Glia. 1993;8(2):122-32.

92. El-Mir MY, Detaille D, R-Villanueva G, Delgado-Esteban M, Guigas B, Attia S, Fontaine

E, Almeida A, Leverve X. Neuroprotective role of antidiabetic drug metformin against

apoptotic cell death in primary cortical neurons. J Mol Neurosci. 2008;34:77–87.

93. Emsley JG, Mitchell BD, Kempermann G, Macklis JD. Adult neurogenesis and repair of the

adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol.

2005;75(5):321-41.

94. Escobedo I, O‟Shea E, Orio L, Sanchez V, Segura M, de la Torre R, Farre M, Green AR,

Colado MI. A comparative study on the acute and long-term effects of MDMA and 3,4-

dihydroxymethamphetamine (HHMA) on brain monoamine levels after i.p. or striatal

administration in mice. Br J Pharmacol. 2005;144:231–241.

63

95. Esteban B, O'Shea E, Camarero J, Sanchez V, Green AR, Colado MI. 3,4-

Methylenedioxymethamphetamine induces monoamine release, but not toxicity, when

administered centrally at a concentration occurring following a peripherally injected

neurotoxic dose. Psychopharmacology. 2001;154:251–260.

96. Esteghamati A, Eskandari D, Mirmiranpour H, Noshad S, Mousavizadeh M, Hedayati M,

Nakhjavani M. Effects of metformin on markers of oxidative stress and antioxidant reserve

in patients with newly diagnosed type 2 diabetes: a randomized clinical trial. Clin Nutr.

2013;32:179–185.

97. Fantegrossi WE, Godlewski T, Karabenick RL, Stephens JM, Ullrich T, Rice KC, Woods

JH. Pharmacological characterization of the effects of 3,4-

methylenedioxymethamphetamine (“ecstasy”) and its enantiomers on lethality, core

temperature, and locomotor activity in singly housed and crowded mice.

Psychopharmacology (Berl). 2003;166:202-211.

98. Farré M, de la Torre R, Mathúna BO, Roset PN, Peiró AM, Torrens M, Ortuño J, Pujadas M,

Camí J. Repeated doses administration of MDMA in humans: pharmacological effects and

pharmacokinetics. Psychopharmacology. 2004;173, 364-375.

99. Fasano C, Poirier A, DesGroseillers L, Trudeau LE. Chronic activation of the D2 dopamine

autoreceptor inhibits synaptogenesis in mesencephalic dopaminergic neurons in vitro. Eur J

Neurosci. 2008;28:1480-1490.

100. Ferré S, Fredholm BB, Morelli M, Popoli P, Fuxe K. Adenosine– dopamine receptor–

receptor interactions as an integrative mechanism in the basal ganglia. Trends Neurosci.

1997;20(10):482–487.

101. Finberg JP, Lamensdorf I, Commissiong JW, Youdim MB. Pharmacology and

neuroprotective properties of rasagiline. J Neural Transm Suppl. 1996;48:95-101.

102. Finberg JP, Takeshima T, Johnston JM, Commissiong JW. Increased survival of

dopaminergic neurons by rasagiline, a monoamine oxidase B inhibitor. Neuroreport.

1998;9;9(4):703-7.

103. Fleming SM, Ekhator OR, Ghisays V. Assessment of sensorimotor function in mouse

models of Parkinson's disease. J Vis Exp. 2013;17;(76).

104. Fleming SM, Zhu C, Fernagut PO, Mehta A, DiCarlo CD, Seaman RL, Chesselet MF.

Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous

infusions of varying doses of rotenone. Exp Neurol. 2004;187:418–429.

105. Fornai F, Lenzi P, Frenzilli G, Gesi M, Ferrucci M, Lazzeri G, Biagioni F, Nigro M, Falleni

A, Giusiani M, Pellegrini A, Blandini F, Ruggieri S, Paparelli A. DNA damage and

64

ubiquitinated neuronal inclusions in the substantia nigra and striatum of mice following

MDMA (ecstasy). Psychopharmacology. 2004;173:353-363.

106. Fornai F, Schlüter OM, Lenzi P, Gesi M, Ruffoli R, Ferrucci M, Lazzeri G, Busceti CL,

Pontarelli F, Battaglia G, Pellegrini A, Nicoletti F, Ruggieri S, Paparelli A, Südhof TC.

Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the

ubiquitin-proteasome system and alpha-synuclein. Proc Natl Acad Sci USA.

2005;102(9):3413-3418.

107. Forno LS, DeLanney LE, Irwin I, Langston JW. Similarities and differences between

MPTP-induced parkinsonsim and Parkinson's disease. Neuropathologic considerations. Adv

Neurol. 1993;60:600-8.

108. Forno LS. Neuropathology of Parkinson's disease. J Neuropathol Exp Neurol.

1996;55(3):259-72.

109. Frau L, Borsini F, Wardas J, Khairnar AS, Schintu N, Morelli M. Neuroprotective and anti-

inflammatory effects of the adenosine A(2A) receptor antagonist ST1535 in a MPTP mouse

model of Parkinson‟s disease. Synapse. 2011;65, 181-188.

110. Frau L, Costa G, Porceddu PF, Khairnar A, Castelli MP, Ennas MG, Madeddu C,Wardas J,

Morelli M. Influence of caffeine on 3,4-methylenedioxymethamphetamine-induced

dopaminergic neuron degeneration and neuroinflammation is age-dependent. J Neurochem.

2016;136(1):148-62.

111. Frau L, Simola N, Plumitallo A, Morelli M. Microglial and astroglial activation by 3,4-

methylenedioxymethamphetamine (MDMA) in mice depends on S(+) enantiomer and is

associated with an increase in body temperature and motility. J Neurochem. 2013;124:69-

78.

112. Fredholm BB, Bättig K, Holmén J, Nehlig A, Zvartau E. Actions of caffeine in the brain

with special reference to factors that contribute to its widespread use. Pharmacol Rev.

1999;51, 83-133.

113. Fritz RR, Abell CW, Patel NT, Gessner W, Brossi A. Metabolism of the neurotoxin in

MPTP by human liver monoamine oxidase B. FEBS Lett. 1985;8;186(2):224-8.

114. Gainetdinov RR, Fumagalli F, Jones SR, Caron MG. Dopamine transporter is required for in

vivo MPTP neurotoxicity: evidence from mice lacking the transporter. J Neurochem.

1997;69(3):1322-5.

115. Garwood ER, Bekele W, McCulloch CE, Christine CW. Amphetamine exposure is elevated

in Parkinson‟s disease. Neurotoxicology. 2006;27, 1003–1006.

65

116. Gerhard A, Pavese N, Hotton G, Turkheimer F, Es M, Hammers A, Eggert K, Oertel W,

Banati RB, Brooks DJ. In vivo imaging of microglial activation with [11C](R)-PK11195

PET in idiopathic Parkinson‟s disease. Neurobiol Dis. 2006;21:404–12.

117. Gibb WR, Lees AJ. The relevance of the Lewy body to the pathogenesis of idiopathic

Parkinson's disease. J Neurol Neurosurg Psychiatry. 1988;51(6):745-52.

118. Goffin D, Ali AB, Rampersaud N, Harkavyi A, Fuchs C, Whitton PS, Nairn AC, Jovanovic

JN. Dopamine-dependent tuning of striatal inhibitory synaptogenesis. J Neurosci.

2010;30:2935–2950.

119. Gołembiowska K, Dziubina A, Kowalska M, Kamińska K. Effect of adenosine A(2A)

receptor antagonists on L-DOPA-induced hydroxyl radical formation in rat striatum.

Neurotox Res. 2009;15(2):155-66.

120. Gołembiowska K, Dziubina A. The effect of adenosine A(2A) receptor antagonists on

hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of

VMAT2. Neurotox Res. 2012;22(2):150-7.

121. Goni-Allo B, O Mathúna B, Segura M, Puerta E, Lasheras B, de la Torre R, Aguirre N. The

relationship between core body temperature and 3,4- methylenedioxymethamphetamine

metabolism in rats: implications for neurotoxicity. Psychopharmacology (Berl).

2008;197(2):263-78.

122. Gordon CJ, Watkinson WP, O'Callaghan JP, Miller DB. Effects of 3,4-

methylenedioxymethamphetamine on autonomic thermoregulatory responses of the rat.

Pharmacol Biochem Behav. 1991;38:339-344.

123. Górska AM, Gołembiowska K. The role of adenosine A1 and A2A receptors in the caffeine

effect on MDMA-induced DA and 5-HT release in the mouse striatum. Neurotox Res.

2015;27:229-45.

124. Graham DG. Oxidative pathways for catecholamines in the genesis of neuromelanin and

cytotoxic quinones. Mol Pharmacol. 1978;14(4):633-43.

125. Granado N, Ares-Santos S, Moratalla R. D1 but not D4 dopamine receptors are critical for

MDMA-induced neurotoxicity in mice. Neurotox Res. 2014;25:100-109.

126. Granado N, Ares-Santos S, Oliva I, O'Shea E, Martin ED, Colado MI, Moratalla R.

Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity

induced by methamphetamine or MDMA. Neurobiol Dis. 2011;42:391–403.

127. Granado N, Escobedo I, O'Shea E, Colado I, Moratalla R. Early loss of dopaminergic

terminals in striosomes after MDMA administration to mice. Synapse. 2008b;62:80-84.

66

128. Granado N, O'Shea E, Bove J, Vila M, Colado MI, Moratalla R. Persistent MDMA-induced

dopaminergic neurotoxicity in the striatum and substantia nigra of mice. J Neurochem.

2008a;107:1102-1112.

129. Green AR, Mechan AO, Elliott JM, O‟Shea E, Colado MI. The pharmacology and clinical

pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, „ecstasy‟). Pharmacol

Rev. 2003;55, 463-508.

130. Green AR, O'shea E, Colado MI. A review of the mechanisms involved in the acute MDMA

(ecstasy)-induced hyperthermic response. Eur J Pharmacol. 2004;500:3-13.

131. Greenamyre JT, Sherer TB, Betarbet R, Panov AV. Complex I and Parkinson's disease.

IUBMB Life. 2001;52(3-5):135-41.

132. Greer G, Tolbert R. Subjective reports of the effects of MDMA in a clinical setting. J

Psychoactive Drugs. 1986;18:319-327.

133. Grondin R, Bedard PJ, Hadj Tahar A, Gregoire L, Mori A, Kase H. Antiparkinsonian effect

of a new selective adenosine A2A receptor antagonist in MPTP-treated monkeys.

Neurology. 1999;52:1673–1677.

134. Gudelsky GA, Yamamoto BK. Actions of 3,4-methylenedioxymethamphetamine (MDMA)

on cerebral dopaminergic, serotonergic and cholinergic neurons. Pharmacol Biochem Behav.

2008;90:198-207.

135. Gudelsky, G.A. Effect of ascorbate and cysteine on the 3,4-

methylenedioxymethamphetamine- induced depletion of brain serotonin. J Neural Transm.

1996;102:1397–1404.

136. Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT,

Surmeier DJ. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated

by DJ-1. Nature. 2010;468:696–700.

137. Haddad PM, Strickland P, Anderson I, Deakin JF, Dursun SM. Effects of MDMA (ecstasy)

use and abstention on serotonin neurons. Lancet. 2002;4;359(9317):1616-7.

138. Hald A, Lotharius J. Oxidative stress and inflammation in Parkinson's disease: is there a

causal link? Exp Neurol. 2005;193(2):279-90.

139. Hall AP, Henry JA. Acute toxic effects of 'Ecstasy' (MDMA) and related compounds:

overview of pathophysiology and clinical management. Br J Anaesth. 2006;96, 678-685.

140. Hamre K, Tharp R, Poon K, Xiong X, Smeyne RJ. Differential strain susceptibility

following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration acts in an

autosomal dominant fashion: quantitative analysis in seven strains of Mus musculus. Brain

Res. 1999;15;828(1-2):91-103.

67

141. Hauptmann N, Grimsby J, Shih JC, Cadenas E. The metabolism of tyramine by monoamine

oxidase A/B causes oxidative damage to mitochondrial DNA. Arch Biochem Biophys.

1996;335(2):295-304.

142. Hauser DN, Dukes AA, Mortimer AD, Hastings TG. Dopamine quinone modifies and

decreases the abundance of the mitochondrial selenoprotein glutathione peroxidase 4. Free

Radic Biol Med. 2013;65:419–27.

143. Hauser RA, Shulman LM, Trugman JM, Roberts JW, Mori A, Ballerini R, Sussman NM,

Istradefylline 6002-US-013 Study Group. Study of istradefylline in patients with

Parkinson‟s disease on levodopa with motor fluctuations. Mov Disord. 2008;23:2177–2185.

144. Hawkins JD, Catalano RF, Miller JY. Risk and protective factors for alcohol and other drug

problems in adolescence and early adulthood: implications for substance abuse prevention.

Psychol Bull. 1992;112(1):64-105.

145. Heikkila RE, Manzino L, Cabbat FS, Duvoisin RC. Protection against the dopaminergic

neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by monoamine oxidase

inhibitors. Nature. 1984;311:467-469.

146. Heikkila RE, Sieber BA, Manzino L, Sonsalla PK. Some features of the nigrostriatal

dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the

mouse. Mol Chem Neuropathol. 1989;10(3):171-83.

147. Heikkila RE, Youngster SK, Manzino L, Cabbat FS, Duvoisin RC. Effects of 1-methyl-4-

phenyl-1,2,5,6-tetrahydropyridine and related compounds on the uptake of [3H]3,4-

dihydroxyphenylethylamine and [3H]5-hydroxytryptamine in neostriatal synaptosomal

preparations. J Neurochem. 1985;44(1):310-3.

148. Hemmerle AM, Herman JP, Seroogy KB. Stress, depression and Parkinson‟s disease. Exp

Neurol. 2012;233:79–86.

149. Herndon JM, Cholanians AB, Lau SS, Monks TJ. Glial cell response to 3,4-(+/-)-

methylenedioxymethamphetamine and its metabolites. Toxicol Sci. 2014;138:130-138.

150. Hiramatsu M, Kumagai Y, Unger SE, Cho AK. Metabolism of

methylenedioxymethamphetamine: formation of dihydroxymethamphetamine and a quinone

identified as its glutathione adduct. J Pharmacol Exp Ther. 1990;254(2):521-7.

151. Hirsch EC, Breidert T, Rousselet E, Hunot S, Hartmann A, Michel PP. The role of glial

reaction and inflammation in Parkinson‟s disease. Ann NY Acad Sci. 2003;991:214–228.

152. Hirsch EC, Hunot S. Nitric oxide, glial cells and neuronal degeneration in parkinsonism.

Trends Pharmacol Sci. 2000;21(5):163-5.

68

153. Hirsch EC, Vyas S, Hunot S. Neuroinflammation in Parkinson‟s disease. Parkinsonism

Relat Disord. 2012;18:S210–S2.

154. Hu SC, Chang FW, Sung YJ, Hsu WM, Lee EH. Neurotoxic effects of 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine in the substantia nigra and the locus coeruleus in BALB/c mice. J

Pharmacol Exp Ther. 1991;259(3):1379-87.

155. Huang QY, Wei C, Yu L, Coelho JE, Shen HY, Kalda A, Linden J, Chen JF. Adenosine

A2A receptors in bone marrow-derived cells but not in forebrain neurons are important

contributors to 3-nitropropionic acid-induced striatal damage as revealed by celltype-

selective inactivation. J Neurosci. 2006;44:11371–11378.

156. Huleatt PB, Khoo ML, Chua YY, Tan TW, Liew RS, Balogh B Deme R, Gölöncsér F,

Magyar K, Sheela DP, Ho HK, Sperlágh B, Mátyus P, Chai CL. Novel

arylalkenylpropargylamines as neuroprotective, potent, and selective monoamine oxidase B

inhibitors for the treatment of Parkinson's disease. Journal of medicinal chemistry.

2015;58(3):1400-19.

157. Hurley LL, Tizabi Y. Neuroinflammation, neurodegeneration, and depression. Neurotox

Res. 2013;23(2):131-44.

158. Ikeda K, Kurokawa M, Aoyama S, Kuwana Y. Neuroprotection by adenosine A2A receptor

blockade in experimental models of Parkinson's disease. J Neurochem. 2002;80:262–70.

159. Iravani MM, Syed E, Jackson MJ, Johnston LC, Smith LA, Jenner P. A modified MPTP

treatment regime produces reproducible partial nigrostriatal lesions in common marmosets.

Eur J Neurosci. 2005;21:841-854.

160. Itzhak Y, Ali SF, Achat CN, Anderson KL. Relevance of MDMA ("ecstasy")-induced

neurotoxicity to long-lasting psychomotor stimulation in mice. Psychopharmacology.

2003;166:241-248.

161. Itzhak Y, Ali SF. Role of nitrergic system in behavioral and neurotoxic effects of

amphetamine analogs. Pharmacol Ther. 2006;109(1-2):246-62.

162. Itzhak Y, Anderson KL, Ali SF. Differential response of nNOS knockout mice to MDMA

("ecstasy")- and methamphetamine-induced psychomotor sensitization and neurotoxicity.

Ann NY Acad Sci. 2004;1025:119-28.

163. Izco M, Gutierrez-Lopez MD, Marchant I, O'Shea E, Colado MI. Administration of

neurotoxic doses of MDMA reduces sensitivity to ethanol and increases GAT-1

immunoreactivity in mice striatum. Psychopharmacology. 2010;207:671-679.

164. Jackson-Lewis V, Przedborski S. Protocol for the MPTP mouse model of Parkinson's

disease. Nat Protoc. 2007;2(1):141–51.

69

165. Jakowec MW, Petzinger GM. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned model

of parkinson's disease, with emphasis on mice and nonhuman primates. Comp Med.

2004;54(5):497-513.

166. Javitch JA, Snyder SH. Uptake of MPP(+) by dopamine neurons explains selectivity or

parkinsonism-inducing neurotoxin, MPTP. Eur J Pharmacol. 1984;13:455-456.

167. Jayanthi S, Ladenheim B, Andrews AM, Cadet JL. Overexpression of human copper/zinc

superoxide dismutase in transgenic mice attenuates oxidative stress caused by

methylenedioxymethamphetamine (Ecstasy). Neuroscience. 1999;91:1379–1387.

168. Jennekens FG. A short history of the notion of neurodegenerative disease. J Hist Neurosci.

2014;23(1):85-94.

169. Kanda T, Jackson MJ, Smith LA, Pearce RK, Nakamura J, Kase H, Kuwana Y, Jenner P.

Combined use of the adenosine A(2A) antagonist KW-6002 with L-DOPA or with selective

D1 or D2 dopamine agonists increases antiparkinsonian activity but not dyskinesia in

MPTP-treated monkeys. Exp Neurol. 2000;162:321–327.

170. Kelly B, Tannahill GM, Murphy MP, O'Neill LA. Metformin inhibits the production of

reactive oxygen species from nadh:ubiquinone oxidoreductase to limit induction of

interleukin-1β (IL-1β) and boosts interleukin-10 (IL-10) in lipopolysaccharide (LPS)-

activated macrophages. J Biol Chem. 2015;290(33):20348-59.

171. Khairnar A, Plumitallo A, Frau L, Schintu N, Morelli M. Caffeine enhances astroglia and

microglia reactivity induced by 3,4-methylenedioxymethamphetamine ('ecstasy') in mouse

brain. Neurotox Res. 2010;17:435-439.

172. Kindlundh-Högberg AM, Schiöth HB, Svenningsson P. Repeated intermittent MDMA

binges reduce DAT density in mice and SERT density in rats in reward regions of the

adolescent brain. Neurotoxicology. 2007;28:1158-1169.

173. Kirkpatrick MG, Lee R, Wardle MC, Jacob S, de Wit H. Effects of MDMA and Intranasal

oxytocin on social and emotional processing. Neuropsychopharmacology. 2014;39:1654-

1663.

174. Kish SJ, Furukawa Y, Ang L, Vorce SP, Kalasinsky KS. Striatal serotonin is depleted in

brain of a human MDMA (Ecstasy) user. Neurology. 2000;55(2):294-296.

175. Klaidman LK, Adams JD Jr, Leung AC, Kim SS, Cadenas E. Redox cycling of MPP+:

evidence for a new mechanism involving hydride transfer with xanthine oxidase, aldehyde

dehydrogenase, and lipoamide dehydrogenase. Free Radic Biol Med. 1993;15(2):169-79.

176. Kohutnicka M, Lewandowska E, Kurkowska-Jastrzebska I, Członkowski A, Członkowska

A. Microglial and astrocytic involvement in a murine model of Parkinson's disease induced

70

by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Immunopharmacology.

1998;39(3):167-80.

177. Kopin IJ. Toxins and Parkinson's disease: MPTP parkinsonism in humans and animals. Adv

Neurol. 1987;45:137-44.

178. Kuhn DM, Arthur Jr RE, Thomas DM, Elferink LA. Tyrosine hydroxylase is inactivated by

catechol-quinones and converted to a redox-cycling quinoprotein: possible relevance to

Parkinson's disease. J Neurochem. 1999;73(3):1309–17.

179. Kumagai Y, Lin LY, Hiratsuka A, Narimatsu S, Suzuki T, Yamada H, Oguri K, Yoshimura

H, Cho AK. Participation of cytochrome P450-2B and -2D isozymes in the

demethylenation of methylenedioxymethamphetamine enantiomers by rats. Mol Pharmacol.

1994;45(2):359-65.

180. Kurkowska-Jastrzebska I, Wrońska A, Kohutnicka M, Członkowski A, Członkowska A.

The inflammatory reaction following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

intoxication in mouse. Exp Neurol. 1999;156(1):50-61.

181. Kurosaki R, Muramatsu Y, Kato H, Araki T. Biochemical, behavioral and

immunohistochemical alterations in MPTP-treated mouse model of Parkinson's disease.

Pharmacol Biochem Behav. 2004;78(1):143-53.

182. Kuwayama K, Inoue H, Kanamori T, Tsujikawa K, Miyaguchi H, Iwata Y, Miyauchi S,

Kamo N, Kishi T. Interactions between 3,4-methylenedioxymethamphetamine,

methamphetamine, ketamine, and caffeine in human intestinal Caco-2 cells and in oral

administration to rats. Forensic Sci Int. 2007;170(2-3):183-8.

183. Łabuzek K, Liber S, Gabryel B, Okopien B. Metformin has adenosine-monophosphate

activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary

microglial cultures. Pharmacol Rep. 2010a;62:827–848.

184. Łabuzek K, Suchy D, Gabryel B, Bielecka A, Liber S, Okopień B. Quantification of

metformin by the HPLC method in brain regions, cerebrospinal fluid and plasma of rats

treated with lipopolysaccharide. Pharmacol Rep. 2010a;62(5):956-65.

185. Langston JW, Ballard P, Tetrud JW, Irwin I. Chronic Parkinsonism in humans due to a

product of meperidine-analog synthesis. Science. 1983;219(4587):979-80.

186. Langston JW, Ballard P. Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP): implications for treatment and the pathogenesis of Parkinson's

disease. Can J Neurol Sci. 1984 Feb;11(1Suppl):160-5.

187. Langston JW, Ballard PA Jr. Parkinson's disease in a chemist working with 1-methyl-4-

phenyl-1,2,5,6-tetrahydropyridine. N Engl J Med. 1983;309(5):310.

71

188. Langston JW, Forno LS, Tetrud J, Reeves AG, Kaplan JA, Karluk D. 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. 1999;46(4):598-605.

189. Langston JW. Parkinson's disease: current view. Am Fam Physician. 1987;35(3):201-6.

190. LaVoie MJ, Hastings TG. Dopamine quinone formation and protein modification associated

with the striatal neurotoxicity of methamphetamine: evidence against a role for extracellular

dopamine. J Neurosci. 1999;19(4):1484-91.

191. LaVoie MJ, Hastings TG. Peroxynitrite- and nitrite-induced oxidation of dopamine:

implications for nitric oxide in dopaminergic cell loss. J Neurochem. 1999;73(6):2546-54.

192. Lees AJ, Hardy J, Revesz T. Parkinson's disease. Lancet. 2009;373(9680):2055-66.

193. Leonardi ET, Azmitia EC. MDMA (ecstasy) inhibition of MAO type A and type B:

comparisons with fenfluramine and fluoxetine (Prozac). Neuropsychopharmacology.

1994;10:231-238.

194. LeWitt PA, Guttman M, Tetrud JW, Tuite PJ, Mori A, Chaikin P, Sussman NM;6002-US-005

Study Group. Adenosine A2A receptor antagonist istradefylline(KW-6002) reduces "off"

time in Parkinson's disease: a double-blind, randomized, multicenter clinical trial (6002-US-

005). Ann Neurol. 2008;63(3):295-302.

195. Li H, Dryhurst G. Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-

3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid (DHBT-1): a putative

nigral endotoxin of relevance to Parkinson's disease. J Neurochem. 1997;69(4):1530–41.

196. Liberatore GT, Jackson-Lewis V, Vukosavic S, Mandir AS, Vila M, McAuliffe WG,

Dawson VL, Dawson TM, Przedborski S. Inducible nitric oxide synthase stimulates

dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med.

1999;5(12):1403-9.

197. Lim HK, Foltz RL. In vivo and in vitro metabolism of 3,4-

(methylenedioxy)methamphetamine in the rat: identification of metabolites using an ion trap

detector. Chem Res Toxicol. 1988;1(6):370-8.

198. Lopez-Rodriguez AB, Llorente-Berzal A, Garcia-Segura LM, Viveros MP. Sex-dependent

long-term effects of adolescent exposure to THC and/or MDMA on neuroinflammation and

serotoninergic and cannabinoid systems in rats. Br J Pharmacol. 2014;171:1435-1447.

199. Luellen BA, Miller DB, Chisnell AC, Murphy DL, O'Callaghan JP, Andrews AM. Neuronal

and astroglial responses to the serotonin and norepinephrine neurotoxin: 1-methyl-4-(2'-

aminophenyl)-1,2,3,6-tetrahydropyridine. J Pharmacol Exp Ther. 2003;307(3):923-31.

72

200. Luo Y, Umegaki H, Wang X, Abe R, Roth GS. Dopamine induces apoptosis through an

oxidationinvolved SAPK/JNK activation pathway. J Biol Chem. 1998;273:3756-3764.

201. Lv Y, Zhang Z, Hou L, Zhang L, Zhang J, Wang Y, Liu C, Xu P, Liu L, Gai X, Lu T. Phytic

acid attenuates inflammatory responses and the levels of NF-κB and p-ERK in MPTP-

induced Parkinson's disease model of mice. Neurosci Lett. 2015;597:132-6.

202. Ma TC, Buescher JL, Oatis B, Funk JA, Nash AJ, Carrier RL, Hoyt KR. Metformin therapy

in a transgenic mouse model of Huntington's disease. Neurosci Lett. 2007;411(2):98-103.

203. Malberg J.E., Seiden L.S. Small changes in ambient temperature cause large changes in 3,4-

methylenedioxymethamphetamine (MDMA)-induced serotonin neurotoxicity and core body

temperature in the rat. J Neurosci. 1998;18:5086–5094.

204. Mallajosyula JK, Kaur D, Chinta SJ, Rajagopalan S, Rane A, Nicholls DG, Di Monte DA,

Macarthur H, Andersen JK. MAO-B elevation in mouse brain astrocytes results in

Parkinson's pathology. PLoS One. 2008;3(2):e1616.

205. Mandel SA, Sagi Y, Amit T. Rasagiline Promotes Regeneration of Substantia Nigra

Dopaminergic Neurons in Post-MPTP-induced Parkinsonism via Activation of Tyrosine

Kinase Receptor Signaling Pathway. Neurochem Res. 2007;32:1694–1699.

206. Marchitti SA, Deitrich RA, Vasiliou V. Neurotoxicity and metabolism of the catecholamine-

derived 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde: the role

of aldehyde dehydrogenase. Pharmacol Rev. 2007;59(2):125-50.

207. Marey-Semper I, Gelman M, Lévi-Strauss M. A selective toxicity toward cultured

mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic

metabolism impairment and NMDA receptor activation. J Neurosci. 1995;15(9):5912-8.

208. Marey-Semper I, Gelman M, Lévi-Strauss M. The high sensitivity to rotenone of striatal

dopamine uptake suggests the existence of a constitutive metabolic deficiency in

dopaminergic neurons from the substantia nigra. Eur J Neurosci. 1993;5(8):1029-34.

209. Maruyama W, Akao Y, Carrillo MC, Kitani K, Youdium MB, Naoi M. Neuroprotection by

propargylamines in Parkinson's disease: suppression of apoptosis and induction of

prosurvival genes. Neurotoxicol Teratol. 2002;24(5):675-82.

210. Maruyama W, Nitta A, Shamoto-Nagai M, Hirata Y, Akao Y, Yodim M, Furukawa S,

Nabeshima T, Naoi M. N-Propargyl-1 (R)-aminoindan, rasagiline, increases glial cell line-

derived neurotrophic factor (GDNF) in neuroblastoma SH-SY5Y cells through activation of

NF-kappaB transcription factor. Neurochem Int. 2004;44(6):393-400.

211. McCann, UD, Eligulashvili, V, Mertl, M, Murphy, DL, Ricaurte, GA. Altered

neuroendocrine and behavioral responses to m-chlorophenylpiperazine in 3,4-

73

methylenedioxymethamphetamine (MDMA) users. Psychopharmacology (Berl).

1999;147(1):56-65.

212. McCann, UD, Ridenour, A, Shaham, Y, Ricaurte, GA. Serotonin neurotoxicity after (+/-

)3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy"): a controlled study in humans.

Neuropsychopharmacology. 1994;10(2):129-138.

213. McGeer PL, Schwab C, Parent A, Doudet D. Presence of reactive microglia in monkey

substantia nigra years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration.

Ann Neurol. 2003;54(5):599-604.

214. McNamara R, Kerans A, O‟Neill B, Harkin A. Caffeine promotes hyperthermia and

serotonergic loss following co-administration of the substituted amphetamines, MDMA

(„Ecstasy‟) and MDA („Love‟). Neuropharmacology. 2006;50:69-80.

215. McNamara R, Maginn M, Harkin A. Caffeine induces a profound and persistent tachycardia

in response to MDMA („ecstasy‟) administration. Eur J Pharmacol. 2007;555:194-198.

216. Mechan AO, O'Shea E, Elliott JM, Colado MI, Green AR. A neurotoxic dose of 3,4-

methylenedioxymethamphetamine (MDMA; ecstasy) to rats results in a long-term defect in

thermoregulation. Psychopharmacology (Berl). 2001;155(4):413-8.

217. Meredith GE, Kang UJ. Behavioral models of Parkinson's disease in rodents: a new look at

an old problem. Mov Disord. 2006;21(10):1595-1606.

218. Meredith GE, Rademacher DJ. MPTP mouse models of Parkinson's disease: an update. J

Parkinsons Dis. 2011;1(1):19-33.

219. Meredith GE, Totterdell S, Petroske E, Santa Cruz K, Callison RC Jr, Lau YS. Lysosomal

malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of

Parkinson's disease. Brain Res. 2002;956(1):156-65.

220. Miller DB and O‟Callaghan JP. The role of temperature, stress, and other factors in the

neurotoxicity of the substituted amphetamines 3,4-methylenedioxymethamphetamine and

fenfluramine. Mol Neurobiol. 1995;11:177-192.

221. Milusheva E, Baranyi M, Kormos E, Hracsko Z, Sylvester Vizi E, Sperlagh B. The effect of

antiparkinsonian drugs on oxidative stress induced pathological [3H]dopamine efflux after

in vitro rotenone exposure in rat striatal slices. Neuropharmacology. 2010;58(4-5):816-25.

222. Mizuno Y, Sone N, Saitoh T. Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and

1-methyl-4-phenylpyridinium ion on activities of the enzymes in the electron transport

system in mouse brain. J Neurochem. 1987;48(6):1787-93.

74

223. Mogi M, Harada M, Kondo T, Riederer P, Inagaki H, Minami M, Nagatsu T. Interleukin-1β,

interleukin-6, epidermal growth factor and transforming growth factor-α are elevated in the

brain from parkinsonian patients. Neurosci Lett. 1994;180:147–150.

224. Mohamed WM, Ben Hamida S, Cassel JC, de Vasconcelos AP, Jones BC. MDMA:

interactions with other psychoactive drugs. Pharmacol Biochem Behav. 2011;99(4):759-74.

225. Moratalla R, Khairnar A, Simola N, Granado N, García-Montes JR, Porceddu PF, Tizabi Y,

Costa G, Morelli M. Amphetamine-related drugs neurotoxicity in humans and in

experimental animals: Main mechanisms. Prog Neurobiol. 2015. pii: S0301-0082(15)00100-

8.

226. Morelli M and Simola N. Methylxanthines and drug dependence: a focus on interactions

with substances of abuse. Handb Exp Pharmacol. 2011:483-507.

227. Morelli M, Carta AR, Kachroo A, Schwarzschild MA. Pathophysiological roles for purines:

adenosine, caffeine and urate. Prog Brain Res. 2010;183:183-208.

228. Morelli M. Adenosine A2A antagonists: potential preventive and palliative treatment for

Parkinson‟s disease. Exp Neurol. 2003;184:20–23.

229. Mori A, Shindou T. Modulation of GABAergic transmission in the striatopallidal

system by adenosine A2A receptors: a potential mechanism for the antiparkinsonian

effects of A2A antagonists. Neurology. 2003;61(11 Suppl 6):S44–S48.

230. Morton J. Ecstasy: pharmacology and neurotoxicity. Curr Opin Pharmacol. 2005;5:79–86.

231. Ng CH, Guan MS, Koh C, Ouyang X, Yu F, Tan EK, O‟Neill SP, Zhang X, Chung J, Lim

KL. AMP kinase activation mitigates dopaminergic dysfunction and mitochondrial

abnormalities in Drosophila models of Parkinson‟s disease. J Neurosci. 2012;32(41):14311–

14317.

232. Nicklas WJ, Youngster SK, Kindt MV, Heikkila RE. MPTP, MPP+ and mitochondrial

function. Life Sci. 1987;40(8):721-9.

233. Nisijima K, Kuboshima K, Shioda K, Yoshino T, Iwamura T, Kato S. Memantine attenuates

3,4-methylenedioxymethamphetamine-induced hyperthermia in rats. Neurosci Lett.

2012;531(2):198-203.

234. Novikova L, Garris BL, Garris DR, Lau YS. Early signs of neuronal apoptosis in the

substantia nigra pars compacta of the progressive neurodegenerative mouse 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease. Neuroscience.

2006;140(1):67-76.

75

235. Obeso JA, Rodriguez-Oroz MC, Goetz CG, Marin C, Kordower JH, Rodriguez M, Hirsch

EC, Farrer M, Schapira AH, Halliday G. Missing pieces in the Parkinson‟s disease puzzle.

Nat Med. 2010;16:653–661.

236. O'Callaghan JP, Miller DB. Neurotoxicity profiles of substituted amphetamines in the

C57BL/6J mouse. J Pharmacol Exp Ther. 1994;270(2):741-51.

237. Olanow CW, Rascol O. The delayed-start study in Parkinson disease: can't satisfy everyone.

Neurology. 2010;74(14):1149-1150.

238. Olanow CW. The pathogenesis of cell death in Parkinson's disease--2007. Mov Disord.

2007;22 Suppl 17:S335-42.

239. Orio L, O‟Shea E, Sanchez V, Pradillo JM, Escobedo I, Camarero J, Moro MA, Green AR,

Colado MI. 3,4-Methylenedioxymethamphetamine (MDMA) increases IL-1h levels and

activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5-

HT depletion. J Neurochem. 2004;89,1445 – 1453.

240. Panneton WM, Kumar VB, Gan Q, Burke WJ, Galvin JE. The neurotoxicity of DOPAL:

behavioral and stereological evidence for its role in Parkinson disease pathogenesis. PLoS

One. 2010;5(12):e15251.

241. Paris JM, Cunningham K. Lack of serotonin neurotoxicity after intraraphe microinjection of

(+)-3,4-methylenedioxymethamphetamine (MDMA). Brain Res Bull. 1992;28, 115–119.

242. Parrott AC, Rodgers J, Buchanan T, Scholey AB, Heffernan T, Ling J. The reality of

psychomotor problems, and the possibility of Parkinson‟s disorder, in some recreational

ecstasy/MDMA users: a rejoinder to Sumnall et al. (2003). Psychopharmacology. 2004;171,

231–233.

243. Patil SP, Jain PD, Ghumatkar PJ, Tambe R, Sathaye S. Neuroprotective effect of metformin

in MPTP-induced Parkinson's disease in mice. Neuroscience. 2014;277:747-54.

244. Pavlovic D, Kocic R, Kocic G, Jevtovic T, Radenkovic S, Mikic D, Stojanovic´ M,

Djordjevic´ PB. Effect of four-week metformin treatment on plasma and erythrocyte

antioxidative defense enzymes in newly diagnosed obese patients with type 2 diabetes.

Diabetes Obes Metab. 2000;2:251–256.

245. Perriello G, Misericordia P, Volpi E, Santucci A, Santucci C, Santucci C, Ferrannini E,

Ventura MM, Santeusanio F, Brunetti P, Bolli GB. Acute antihyperglycemic mechanisms of

metformin in NIDDM. Evidence for suppression of lipid oxidation and hepatic glucose

production. Diabetes. 1994;43:920–928.

76

246. Petroske E, Meredith GE, Callen S, Totterdell S, Lau YS. Mouse model of Parkinsonism: a

comparison between subacute MPTP and chronic MPTP/probenecid treatment.

Neuroscience. 2001;106(3):589-601.

247. Piech-Dumas KM, Tank AW. CREB mediates the cAMP-responsiveness of the tyrosine

hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell

lines. Brain Res Mol Brain Res. 1999;70(2):219-30.

248. Pieri M, Christian HC, Wilkins RJ, Boyd CA, and Meredith D. The apical (hPepT1) and

basolateral peptide transport systems of Caco-2 cells are regulated by AMP-activated

protein kinase. Am J Physiol Gastrointest Liver Physio. 2010;l299:G136 –G143.

249. Pierri M, Vaudano E, Sager T, Englund U. KW-6002 protects from MPTP induced

dopaminergic toxicity in the mouse. Neuropharmacology. 2005;48:517–524.

250. Piper BJ, Meyer JS. Memory deficit and reduced anxiety in young adult rats given repeated

intermittent MDMA treatment during the periadolescent period. Pharmacol Biochem Behav.

2004;79:723–731.

251. Potts MB, Lim DA. An old drug for new ideas: metformin promotes adult neurogenesis and

spatial memory formation. Cell Stem Cell. 2012;11(1):5-6.

252. Pringsheim T, Jette N, Frolkis A, Steeves TD. The prevalence of Parkinson‟s disease: a

systematic review and meta-analysis. Mov Disord. 2014;29:1583–90.

253. Przedborski S, Kostic V, Jackson-Lewis V, Naini AB, Simonetti S, Fahn S, Carlson E,

Epstein CJ, Cadet JL. Transgenic mice with increased Cu/Zn-superoxide dismutase activity

are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. J

Neurosci. 1992;12(5):1658-67.

254. Przedborski S, Vila M, Jackson-Lewis V. Neurodegeneration: what is it and where are we? J

Clin Invest. 2003;111(1):3-10.

255. Przedborski S, Vila M. MPTP: a review of its mechanisms of neurotoxicity. Clinical

Neuroscience Research. 2001;1:407–418.

256. Puerta E, Hervias I, Goñi-Allo B, Zhang SF, Jordán J, Starkov AA, Aguirre N.

Methylenedioxymethamphetamine inhibits mitochondrial complex I activity in mice: a

possible mechanism underlying neurotoxicity. Br J Pharmacol. 2010;160(2):233-45.

257. Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT. Systemic LPS

causes chronic neuroinflammation and progressive neurodegeneration. Glia. 2007;55:453–

462.

77

258. Ramsay RR, Singer TP. Energy-dependent uptake of N-methyl-4-phenylpyridinium, the

neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, by mitochondria. J

Biol Chem. 1986;261(17):7585-7.

259. Reissig CJ, Strain EC, Griffiths RR. Caffeinated energy drinks – a growing problem. Drug

Alcohol Depend. 2009;99:1-10.

260. Reneman L, Booij J, De BK, Reitsma JB, de Wolff FA, Gunning WB, den Heeten GJ, van

den BW. Effects of dose, sex, and long-term abstention from use on toxic effects of MDMA

(ecstasy) on brain serotonin neurons. Lancet. 2001b;358:1864–1869.

261. Reneman L, Booij J, Majoie CBL, van den Brink W, den Heeten GJ. Investigating the

potential neurotoxicity of ecstasy (MDMA): an imaging approach. Hum Psychopharmacol.

2001a;16:579–588.

262. Reneman L, Booij J, Schmand B, van den Brink W, Gunning B. Memory disturbances in

"Ecstasy" users are correlated with an altered brain serotonin neurotransmission.

Psychopharmacology (Berl). 2000;148(3):322-324.

263. Reveron ME, Monks TJ, Duvauchelle CL. Age-dependent (+)MDMA-mediated

neurotoxicity in mice. Neurotoxicology. 2005;26:1031-40.

264. Reznichenko L, Kalfon L, Amit T, Youdim MBH, Mandel SA. Low Dosage of Rasagiline

and Epigallocatechin Gallate Synergistically Restored the Nigrostriatal Axis in MPTP-

Induced Parkinsonism. Neurodegenerative Dis. 2010;7:219-231.

265. Ricaurte GA, DeLanney LE, Irwin I, Langston JW. Toxic effects of MDMA on central

serotonergic neurons in the primate: importance of route and frequency of drug

administration. Brain Res. 1988;446:165-8.

266. Ricaurte GA, Finnegan KT, Irwin I, Langston JW. Aminergic metabolites in cerebrospinal

fluid of humans previously exposed to MDMA: preliminary observations. Ann NY Acad

Sci. 1990;600:699-708.

267. Ricaurte GA, Langston JW, Delanney LE, Irwin I, Peroutka SJ, Forno LS. Fate of

nigrostriatal neurons in young mature mice given 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine: a neurochemical and morphological reassessment. Brain Res.

1986;376(1):117-24.

268. Rice D. and Barone S. Critical periods of vulnerability for the nervous system: evidence

from humans and animal models. Environ Health Perspect. 2000;108:511–533.

269. Richardson JR, Caudle WM, Guillot TS, Watson JL, Nakamaru-Ogiso E, Seo BB, Sherer

TB, Greenamyre JT, Yagi T, Matsuno-Yagi A, Miller GW. Obligatory role for complex I

78

inhibition in the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP). Toxicol Sci. 2007;95(1):196-204.

270. Ross GW, Abbott RD, Petrovitch H, Morens DM, Grandinetti A, Tung KH, Tanner CM,

Masaki KH, Blanchette PL, Curb JD, Popper JS, White L. Association of coffee and

caffeine intake with the risk of Parkinson disease. JAMA. 2000;283:2674–2679.

271. Rudnick G, Wall SC. p-chloroamphetamine induces serotonin release through serotonin

transporters. Biochemistry. 1992;31:6710-6718.

272. Ruiz-Medina J, Pinto-Xavier A, Rodríguez-Arias M, Miñarro J, Valverde O. Influence of

chronic caffeine on MDMA-induced behavioral and neuroinflammatory response in mice.

Psychopharmacology (Berl). 2013;226(2):433-44.

273. Sag D, Carling D, Stout RD, Suttles J. Adenosine 5'-monophosphate-activated protein

kinase promotes macrophage polarization to an anti-inflammatory functional phenotype. J

Immunol. 2008;181(12):8633-41.

274. Sagi Y, Mandel S, Amit T, Youdim MB. Activation of tyrosine kinase receptor signaling

pathway by rasagiline facilitates neurorescue and restoration of nigrostriatal dopamine

neurons in post-MPTP-induced parkinsonism. Neurobiol Dis. 2007;25:35-44.

275. Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK. A

Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from

inflammation-induced death. Cell. 2009;137:47–59.

276. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity

and protection. J Neurochem. 2011;116:1–9.

277. Sakar Y, Meddah B, Faouzi MA, Cherrah Y, Bado A, Ducroc R. Metformininduced

regulation of the intestinal D-glucose transporters. J Physiol Pharmacol. 2010;61:301–307.

278. Sanchez V, Camarero J, O'Shea E, Green AR, Colado MI. Differential effect of dietary

selenium on the long-term neurotoxicity induced by MDMA in mice and rats.

Neuropharmacology. 2003;44:449-461.

279. Sattler R, Tymianski M. Molecular mechanisms of calcium-dependent excitotoxicity. J Mol

Med (Berl). 2000;78(1):3-13.

280. Sattler R, Tymianski M. Molecular mechanisms of glutamate receptor-mediated excitotoxic

neuronal cell death. Mol Neurobiol. 2001;24(1-3):107-29.

281. Saxena S, Caroni P. Selective neuronal vulnerability in neurodegenerative diseases: from

stressor thresholds to degeneration. Neuron. 2011;71(1):35-48.

282. Schifano F, Di FL, Forza G, Minicuci N, Bricolo R. MDMA („ecstasy‟) consumption in the

context of polydrug abuse: a report on 150 patients. Drug Alcohol Depend. 1998;52:85–90.

79

283. Schintu N, Frau L, Ibba M, Garau A, Carboni E, Carta AR. Progressive dopaminergic

degeneration in the chronic MPTPp mouse model of Parkinson's disease. Neurotox Res.

2009;16(2):127-139.

284. Schwarzschild MA, Agnati L, Fuxe K, Chen JF, Morelli M. Targeting adenosine A2A

receptors in Parkinson‟s disease. Trends Neurosci. 2006;29:647–654.

285. Schwarzschild MA, Xu K, Oztas E, Petzer JP, Castagnoli K, Castagnoli NJr, Chen JF.

Neuroprotection by caffeine and more specific A2A receptor antagonists in animal models

of Parkinson's disease. Neurology. 2003;61:S55-61.

286. Sedelis M, Hofele K, Auburger GW, Morgan S, Huston JP, Schwarting RK. MPTP

susceptibility in the mouse: behavioral, neurochemical, and histological analysis of gender

and strain differences. Behav Genet. 2000;30(3):171-82.

287. Sedelis M, Schwarting RK, Huston JP. Behavioral phenotyping of the MPTP mouse model

of Parkinson's disease. Behav Brain Res. 2001;125(1-2):109-25.

288. Semple DM, Ebmeier KP, Glabus MF, O‟Carroll RE, Johnstone EC. Reduced in vivo

binding to the serotonin transporter in the cerebral cortex of MDMA („ecstasy‟) users. Br J

Psychiatry. 1999;175:63–69.

289. Shankaran M, Gudelsky GA. A neurotoxic regimen of MDMA suppresses behavioral,

thermal and neurochemical responses to subsequent MDMA administration.

Psychopharmacology. 1999;147:66–72.

290. Shankaran M, Yamamoto BK, Gudelsky GA. Ascorbic acid prevents 3,4-

methylenedioxymethamphetamine (MDMA)- induced hydroxyl radical formation and the

behavioral and neurochemical consequences of the depletion of brain 5-HT. Synapse.

2001;40:55–64.

291. Shiotsuki H, Yoshimi K, Shimo Y, Funayama M, Takamatsu Y, Ikeda K, Takahashi R,

Kitazawa S, Hattori N. A rotarod test for evaluation of motor skill learning. J Neurosci

Methods. 2010;189(2):180-185.

292. Shulgin A. “History of MDMA.” 1990. In: Perutka S,ed: Ecstasy: The Clinical,

Pharmacological and Neurotoxicological Effects of the Drug MDMA. Norwell, Mass:

Klewer Academic Publishing

293. Sian J, Dexter DT, Lees AJ, Daniel S, Jenner P, Marsden CD. Glutathione-related enzymes

in brain in Parkinson's disease. Ann Neurol. 1994;36(3):356-61.

294. Simola N, Tronci E, Pinna A, Morelli M. Subchronic-intermittent caffeine amplifies the

motor effects of amphetamine in rats. Amino Acids. 2006;31:359-363.

80

295. Sisk CL, Zehr JL. Pubertal hormones organize the adolescent brain and behavior. Front

Neuroendocrinol. 2005;26(3-4):163-74.

296. Smith TS, Bennett JP Jr. Mitochondrial toxins in models of neurodegenerative diseases. I: In

vivo brain hydroxyl radical production during systemic MPTP treatment or following

microdialysis infusion of methylpyridinium or azide ions. Brain Res. 1997;765(2):183-8.

297. Snow BJ, Vingerhoets FJ, Langston JW, Tetrud JW, Sossi V, Calne DB. Pattern of

dopaminergic loss in the striatum of humans with MPTP induced parkinsonism. J Neurol

Neurosurg Psychiatry. 2000;68(3):313-6.

298. Sonsalla PK, Heikkila RE. The influence of dose and dosing interval on MPTP-induced

dopaminergic neurotoxicity in mice. Eur J Pharmacol. 1986;129(3):339-45.

299. Spear LP. The adolescent brain and age-related behavioral manifestations. Neurosci

Biobehav Rev. 2000;24(4):417-63.

300. Spragu JE, Everman SL, Nichols DE. An integrated hypothesis for the serotonergic axonal

loss induced by 3,4- methylenedioxymethamphetamine. Neurotoxicology. 1998;19:427–

441.

301. Stacy M, Silver D, Mendis T, Sutton J, Mori A, Chaikin P, Sussman NM. A 12-week,

placebo-controlled study (6002-US-006) of istradefylline in Parkinson disease. Neurology.

2008;71:953.

302. Steele TD, Nichols DE, Yim GK. Stereochemical effects of 3,4-

methylenedioxymethamphetamine (MDMA) and related amphetamine derivatives on

inhibition of uptake of [3H]monoamines into synaptosomes from different regions of rat

brain. Biochem Pharmacol. 1987;36:2297-2303.

303. Stoll G, Jander S. The role of microglia and macrophages in the pathophysiology of the

CNS. Prog Neurobiol. 1999;58:233–247.

304. Stumvoll M, Nurjhan N, Perriello G, Dailey G, Gerich JE. Metabolic effects of metformin in

non-insulin dependant diabetes mellitus. N Engl J Med. 1995;333: 550–554.

305. Sulzer D, Surmeier DJ. Neuronal vulnerability, pathogenesis, and Parkinson's disease. Mov

Disord. 2013;28(6):715–24.

306. Suzuki K, Mizuno Y, Yoshida M. Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP)-like compounds on mitochondrial respiration. Adv Neurol. 1990;53:215-8.

307. Tanaka T, Kai S, Matsuyama T, Adachi T, Fukuda K, Hirota K. General anesthetics inhibit

LPS-induced IL-1beta expression in glial cells. PLoS One. 2013;8:e82930.

81

308. Tansey MG, McCoy MK, Frank-Cannon TC. Neuroinflammatory mechanisms in

Parkinson‟s disease: Potential environmental triggers, pathways, and targets for early

therapeutic intervention. Exp Neurol. 2007;208:1–25.

309. Teixeira-Gomes A, Costa VM, Feio-Azevedo R, Bastos Mde L, Carvalho F, Capela JP. The

neurotoxicity of amphetamines during the adolescent period. Int J Dev Neurosci.

2015;41:44-62.

310. Thomas DM, Dowgiert J, Geddes TJ, Francescutti-Verbeem D, Liu X Kuhn DM. Microglial

activation is a pharmacologically specific marker for the neurotoxic amphetamines.

Neurosci Lett. 2004;367:349-54.

311. Tillerson JL, Caudle WM, Reverón ME, Miller GW. Detection of behavioral impairments

correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine. Exp Neurol. 2002;178(1):80-90.

312. Todd G, Noyes C, Flavel SC, Della Vedova CB, Spyropoulos P. Illicit stimulant use is

associated with abnormal substantia nigra morphology in humans. PloS One.

2013;8:e56438.

313. Touriño C, Zimmer A, Valverde O. THC Prevents MDMA Neurotoxicity in Mice. PLoS

One. 2010;5(2):e9143.

314. Tsutsui S, Schnermann J, Noorbakhsh F, Henry S, Yong VW, Winston BW, Warren K,

Power C. A1 adenosine receptor upregulation and activation attenuates neuroinflammation

and demyelination in a model of multiple sclerosis. J Neurosci. 2004;24(6):1521-9.

315. Vanattou-Saïfoudine N, Gossen A, Harkin A. A role for adenosine A(1) receptor blockade

in the ability of caffeine to promote MDMA "Ecstasy"-induced striatal dopamine release.

Eur J Pharmacol. 2011;650, 220-228.

316. Vanattou-Saïfoudine N, McNamara R, Harkin A. Caffeine provokes adverse interactions

with 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') and related

psychostimulants: mechanisms and mediators. Br J Pharmacol. 2012;167:946-959.

317. Vanattou-Saïfoudine N, McNamara R, Harkin A. Mechanisms mediating the ability of

caffeine to influence MDMA ('Ecstasy')-induced hyperthermia in rats. Br J Pharmacol.

2010;160:860-77.

318. Verrico, C.D., Mille,r G.M., Madras, B.K. MDMA (Ecstasy) and human dopamine,

norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity

and treatment. Psychopharmacology. 2007;189:489-503.

82

319. Vingerhoets FJ, Snow BJ, Tetrud JW, Langston JW, Schulzer M, Calne DB. Positron

emission tomographic evidence for progression of human MPTP-induced dopaminergic

lesions. Ann Neurol. 1994;36(5):765-70.

320. Vitale C, Santangelo G, Erro R, Errico D, Manganelli F, Improta I et al. Impulse control

disorders induced by rasagiline as adjunctive therapy for Parkinson's disease: report of 2

cases. Parkinsonism Relat Disord. 2013;19(4):483-4.

321. Vollenweider FX, Gamma A, Liechti M, Huber T. Psychological and cardiovascular effects

and short-term sequelae of MDMA ("ecstasy") in MDMA-naïve healthy volunteers.

Neuropsychopharmacology. 1998;19:241-251.

322. Wahlqvist ML, Lee MS, Hsu CC, Chuang SY, Lee JT, Tsai HN. Metformin-inclusive

sulfonylurea therapy reduces the risk of Parkinson's disease occurring with Type 2 diabetes

in a Taiwanese population cohort. Parkinsonism Relat Disord. 2012;18(6):753-8.

323. Wang J, Gallagher D, DeVito LM, Cancino GI, Tsui D, He L, Keller GM, Frankland PW,

Kaplan DR, Miller FD. Metformin activates an atypical PKC-CBP pathway to promote

neurogenesis and enhance spatial memory formation. Cell Stem Cell. 2012;11:23–35.

324. Watson L and Beck J. "New age seekers: MDMA use as an adjunct to spiritual

pursuit." Journal of Psychoactive Drugs. 1991;23(3): 261-270.

325. Weinreb O, Amit T, Bar-Am O, Youdim MB. Rasagiline: a novel anti-Parkinsonian

monoamine oxidase-B inhibitor with neuroprotective activity. Prog Neurobiol. 2010;92:330-

344.

326. Weinreb O, Bar-Am O, Amit T, Chillag-Talmor O, Youdim MB. Neuroprotection via pro-

survival protein kinase C isoforms associated with Bcl-2 family members. FASEB J.

2004;18(12):1471-3.

327. Wersinger C, Chen J, Sidhu A. Bimodal induction of dopamine-mediated striatal

neurotoxicity is mediated through both activation of D1 dopamine receptors and

autoxidation. Mol Cell Neurosci. 2004;25:124-137.

328. Whitehead RE, Ferrer JV, Javitch JA, Justice JB. Reaction of oxidized dopamine with

endogenous cysteine residues in the human dopamine transporter. J Neurochem.

2001;76(4):1242–51.

329. Wimalasena DS, Perera RP, Heyen BJ, Balasooriya IS, Wimalasena K. Vesicular

monoamine transporter substrate/inhibitor activity of MPTP/MPP+ derivatives: a structure-

activity study. J Med Chem. 2008;51(4):760-8.

330. Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK,

Ischiropoulos H, Przedborski S. Blockade of microglial activation is neuroprotective in the

83

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J

Neurosci. 2002;22:1763–1771.

331. Wu RM, Murphy DL, Chiueh CC. Suppression of hydroxyl radical formation and protection

of nigral neurons by l-deprenyl (selegiline). Ann NY Acad Sci. 1996;786:379-390.

332. Xu Y, Stokes AH, Roskoski Jr R, Vrana KE. Dopamine, in the presence of tyrosinase,

covalently modifies and inactivates tyrosine hydroxylase. J Neurosci Res. 1998;54(5):691–

7.

333. Yamamoto BK, Moszczynska A, Gudelsky GA. Amphetamine toxicities: classical and

emerging mechanisms. Ann NY Acad Sci. 2010;1187:101-121.

334. Yazdani U, German DC, Liang CL, Manzino L, Sonsalla PK, Zeevalk GD. Rat model of

Parkinson's disease: chronic central delivery of 1-methyl-4-phenylpyridinium (MPP+). Exp

Neurol. 2006;200(1):172-83.

335. Youdim MB, Edmondson D, Tipton KF. The therapeutic potential of monoamineoxidase

inhibitors. Nat Rev Neurosci. 2006;7(4):295-309.

336. Youdim MB, Lavie L. Selective MAO-A and B inhibitors, radical scavengers and nitric

oxide synthase inhibitors in Parkinson's disease. Life Sci. 1994;55(25-26):2077-2082.

337. Youdim MB, Riederer PF. A review of the mechanisms and role of monoamine oxidase

inhibitors in Parkinson's disease. Neurology. 2004;63:S32-5.

338. Youdim MB, Weinstock M. Molecular basis of neuroprotective activities of rasagiline and

the anti-Alzheimer drug TV3326 [(N-propargyl-(3R)aminoindan-5-YL)-ethyl methyl

carbamate). Cell Mol Neurobiol. 2001;21:555-573.

339. Zafar KS, Siegel D, Ross D. A potential role for cyclized quinones derived from dopamine,

DOPA, and 3,4-dihydroxyphenylacetic acid in proteasomal inhibition. Mol Pharmacol.

2006;70(3):1079–86.

340. Zarrindast MR, Tabatabai SA. Involvement of dopamine receptor subtypes in mouse

thermoregulation. Psychopharmacology. 1992;107:341-346.

341. Zhang X, Lee TH, Xiong X, Chen Q, Davidson C, Wetsel WC, Ellinwood EH.

Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit and

GAD67 expression. Biochem Biophys Res Commun. 2006;351:300-305.

342. Zheng H, Gal S, Weiner LM, Bar-Am O, Warshawsky A, Fridkin M, et al. Novel

multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for

neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid

peroxide formation and monoamine oxidase inhibition. J Neurochem. 2005;95(1):68-78.

84

343. Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease: a mechanism of

pathogenic and therapeutic significance. Ann NY Acad Sci. 2008;1147:93-104.