Clinical Diagnosis of Neurodegenerative Diseases by (CSF) Cerebro Spinal Fluid Labelling

Neuropeptide/Neurotransmitter Mapping & Oxidative Stress during

Parkinson’s Disease On / Off states

Parvez, S. H., Collin, C., Qureshi, G.A. & Parvez, S.

CNRS: Neuroendocrine Unit

Institut Alfred Fessard of Neurosciences, French National Research Center,

Bât 5, 91190 Gif Sur Yvette, FranceEmail: hsparvez@yahoo.com & hparvez@lne.cnrs-gif.fr

Hyperactivity & Neuronal System

Human Brain & Specific Functions

Parkinson’s Disease

1) Classical Clinical Features• Resting Tremor :Cogwheel Rigidity Bradykinesia :

Postural Instability

2)Associated Clinical Features• Micrographia : Hypophonia : Hypomimia• Shuffling Gate/Festination : Drooling• Dysphagia : Autonomic Dysfunction• Depression : Dementia

Features of Parkinson’s disease

• Neuropathologic aspects of PD are…– Loss of DA neurons– Formation of Lewy bodiesin substantia nigra (SNpc).

• Degeneration of DA neuron links to the clinical symptoms like akinesia, which is caused by lack of DA. aS/ubi(+/+) Lewy bodies (Spillantini et al., 1998)

Previous Studies

Previous Studies

Previous Studies

Previous Studies

Features of Parkinson’s disease

• Molecular mechanism of dopaminergic (DA) neuron selectivity in formation of inclusion bodies (brainstem type Lewy bodies) is yet unclear (Part1~3, conclusions).

• Mutations of ubiquitin proteasome system (UPS) associated proteins in familial Parkinson’s disease (PD) (Parkin: Kitada et al., 1998; Shimura et al., 2000; DJ-1: Bonifati et al., 2003).

• Failure of proteasome structure and functions become significant in elders (Carrard et al., 2002; Keller et al., 2002) and in PD (McNaught et al., 2001).

modified from Vernace et al., 2007

Lewy bodies in human brainstem were TH positive

Lewy bodies (arrows in A and D) and a Pale body (arrowhead in D) had strong TH immunoreactivity. In the high power images in B, C and E, typical halo and Lewy neurites were shown and all of them were TH immunopositive. Scale bars indicate 20 μm in A, D and 8 μm in B, C, E.

nucleus

Lewy body

Lewy bodies in human brainstem in Parkinson’s disease

Typical Lewy body (A and B) and Pale body (C).Scale bar indicates 8 μm and all the images are same scale.

PET Scan of Normal & Parkinson’s Brain

Blood Brain Barrier

Blood Brain BarrierTransverse to a Capillary

In Vivo Evaluation of Blood Brain barrierby PET 11C Verapamil in primates

Clinical Protocol on PD Patientsduring ON / Off States

PATIENTS

•PD ON GROUP

•18 patients each at different stages of illness

•(Hoehn & Yar Range 2-4)

•And on individual drug combination. These 15 patients were on L-DOPA dose (250 mg/day) and have taken it with combination of other drugs with positive response towards their motor activities.

•PD OFF GROUP

•15 PD patients who have been treated for 6-8 years with the similar drug therapy showed severely off response

•(Hoehn & Yar range -3)

•was included which showed motor fluctuations

CSF SAMPLES & METHODSon PD Patients during ON / Off States

• 10-12 ml cerebrospinal fluid (CSF) was collected from each PD patient and also from healthy controls in sitting position at the L4-L5 levels. The basic CSF analyses such as cell counting by

• phase-contrast microscopy (Siesjo, 1967)• Determination of CSF/Serum/Albumin ratio & CSF/Immunoglobulin• (igG) index (Link et al, 1977)• As well as isoelectric focusing for the detection of oligoclonal IgG

band.• CSF Albumin & IgG were determined using• Hitachi 737 Automatic Analyzer by the method of• Naka Works, Hitachi Ltd, Tokyo, Japan

Patients n Female Age CSF-Albumin CSF-IgG IgG-Index CSF-Glucose

Controls 16 7 63 ± 7 218 ± 17 34 ± 4 0.43 ± 0.01 1.02 ±0.13

PD (On) 18 6 72 ±12 332 ± 42* 51 ± 7 ** 0.44 ± 0.01 1.06 ± 0.12

PD (Off) 18 7 74 ± 14 313 ± 37* 46 ± 6 ** 0.42 ± 0.01 0.99 ± 0.09

________________________________*p< 0.05, ** p< 0.01

CSF Data on Clinical Markers on the Healthy Controls & Parkinson’s Patients in both On & Off Groups

Direct and Indirect Pathways in Basal Ganglia

• GABA • Glutamate• Dopamine• Acetylcholine• Adenosine• Neuropeptide

Controls PD (ON) PD (OFF)

**

***

100

200

0

Pm

ol/l

GABA

Controls PD (ON) PD (OFF)

**

***

10

20

0

µm

ol/l

GLU

****

30

ASP

Controls PD (ON) PD (OFF)

**

100

200

0

Pm

ol/l

DOPACHVA

DA

300

***

*

Controls PD (ON) PD (OFF)

**

100

200

0

Pm

ol/l

HMPG

NA

*

*

Controls PD (ON) PD (OFF)

**

1000

2000

0

Pm

ol/l

TRP3000

*

Controls PD (ON) PD (OFF)

***

2

8

0

Pm

ol/l

5HT

*

4

6

10

12

14

Controls PD (ON) PD (OFF)

*

**

10

0

pmol

/lNPY

20

30

50

40

Controls PD (ON) PD (OFF)

**

10

20

0

pmol

/l

CCK-8s

*

***

30

CCK-4

CSF NeuropeptidesSubstance-P during PD-on & PD-off

pmol

/l

0

1

2

3

4

5

Controls PD (ON) PD (OFF)

Substance P***

NO

2

Glu

1

2

3

4

5

00 10 20 30

Relation between nitrite and glutamic acid in PD On and Off

PD (Off)

PD (On)

Controls

5 -

HT

30

40

50

60

70

00 3 4

PD (Off)

PD (On)

Controls

Substance P

5

80

NA

100

180

801 3 4

PD (Off)

PD (On)

Controls

CCK - 4

72 5 6

120

140

160

200

220

240

HC

1

2

00 1

Cobalamin deficiency and its dynamic impact on neurotoxicity and oxidative stress

PD (Off)

PD (On)

Controls

2 3 4

Cerebro Spinal Fluid (CSF)Neuropeptidesduring Cerebro vascular Dementia(CVD)

•Parameter Normal Humans CVD

•NPY (pmol/l) 29.2 +1.2 23.1+1.3*

•CCK-4 (pmol/l) 5.2 +0.3 3.1+0.6

•CCK-8 (pmol/l) 20.8 +1.1 12.3+0.8**

•Sub.P (pmol/l) 3.1 +0.6 5.2+0.7*

•*P<0.05 **p<0.01 Methods: CSF venipuncture of L4-L5 - Sub.P RIA by SP2 antisera:Sakurada et al 1985•NYP RIA(Theodrsson-Norheim:1985 Limit 0.2 to 0.5 pmol/l)•CCK-4 & CCK-8 (Qureshi et al: 1993) LC-MS/FAB Limit 0.1 p.mol/l

Synthesis of NO

Arginine NADPH NADP+

N-w-Hydroxyarginine

ARGININE SYTHASE

Citrulline + NO

Nitric acid

Free Radicals as Markers of Oxidative stress

a) Most of the free radicals are unstable species and can extract an electron from neighbouring molecuiles leading to oxidative stress

b) Oxygen-centered free radicals are the main radicals formed in cells such as OH; O2; H2O2, NO.

c) Free radical causes Protein, DNA, Lipid damage

Controls PD (ON) PD (OFF)

**

***

1

0

µm

ol/l

NO2

2

3

4

CSF Levels of Homocysteine & Vitamin B12in Controls, PD/ON & PD/OFF Stages

Patients Homocysteineµmol/l

Vitamin B12µmol/l

Controls 1.1 +0.1 0.08+0.006

PD (On) 1.56+0.130.06+0.005*

PD (Off) 1.89+0.210.041+0.003**

*P> 0.05 ** p>0.01

Cerebro Spinal Fluid (CSF) Markers of Neurotoxicity

CSF venipuncture of L4-L5 (10 ml): Vit B12(Ikeda et al.1984): Nitrite(Hayland et al 1992 by HPLC)Homocysteine (Sakura et al. 1985): Albumin & IgG by Hitachi Autoanalyzer (Link et al 1977)

Parameter Normal Humans Cerebro Vascular Dementia

Vitamin B-12 (nmol/l) 80+11 54+8**

NO2 (µmol/l) 1.02+0.1 2.4+0.1***Homocysteine (µmol/l) 1.04+0.06 3.1+0.16***

Albumin (mg/l) 218+17 313+31*

IgG (mg/l) 34+4 46+7*

Neuropeptides & Neuroprotective Role in Health & Disease

-Neuropeptides are mostly Neurohormonal secretions

-Neuropeptides triger & activate central/peripheral feedbacs

-Neuropeptide Concentration decreases significantly during diseases such as PD, AD, CVD but in

-Autoimmune diseases such as MS Neuropeprtide Concentration rises

-Such a discrimination shows that the patients of established Autoimmune Diseases try to activate their Immune response by producing higher levels of Neuropeptides for better resistance to neuropathologies.

-Neuropeptide activation of Adrenal Cortex produces Hyperglycaemic Pool of Epinephrine, Norepinephrine & also Cortisol etc which is ready to mobilize energy reserves to confront and adjust againstNeuronal Injury & start of Neurological Disease.

Dynamic Levels of METAL IONS in Cerebrospinal Fluid (CSF) during Parkinson’s Disease : On/OFF Phenomena

• The Role of Iron

• Copper

• Zinc

• And their Effects

• In On/Off Parkinson’s Patients

• On l-dopa therapy

Levels of Iron, Copper & Zinc in Healthy Controls (HC) & Parkinson’s Patients with

On and Off Status on l-dopa Therapy

• Metal HC PD(On) PD (On/Off)

• N° of Patients 21 17 19

• IRON serum(mg/ml) 1.16 1.02 1.02

• IRON CSF (ug/l) 237 345 397

• COPPER serum (mg/ml)) 1.22 1.16 1.27

• COPPER CSF (ug/l) 132 119 109

•

• ZINC serum(mg/ml) 0.89 0.91 0.79

• ZINC CSF (ug/l) 161 117 96

Metal Ions & Neurodegeneration

B R A I NHigh Lipid Content Metal Ion Catalyzed

Formation of Reactive Oxygen Species & Oxidative Damage by H2O2

DNA adduct formation & Protein Damage or Transformation of PrP(sc) to Patho PrP27-30

Iron Induced overproduction of OH radical in Striatum & Brain Stem in Iron Loaded Mice

Iron Loaded Mice are highly prone to MPTP induced ParkinsonismIron induces Neuronal Apoptosis not directly but through its triggering action to

produce highly toxic free radicals : Any change in Iron homeostasis can lead to neurnal injury with a start of

neurodegenerative disease

a) Increase in Oxidative stress due to accumulation of free radicals

b) Increase neurotoxicity in the form of elevated Homocysteine and Glutamic acid

c) Deficiency of Vitamin B12

d) Change in enzyme activities

e) Decrease of number of receptors as can be seen in PD

Mechanism of Neurodegeneration

All these events one way or other contribute in degeneration of brain function

THANK YOU AGAIN

For your kind attention

Parvez, S. H., Collin, C., Qureshi, G.A. & Parvez, S.