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Summer Research Fellowship Programme of India's Science Academies

Impacts and trials to inhibit the activity of alphasynuclein in Parkinson's disease

Lade Vuha

Pursuing  MSc, Department of Biotechnology, Andhra University, Vishakhapatnam

Dr. Jancy Nixon Abraham

Ramalingaswami Fellow (DBT), Polymer Science and Engineering Division, CSIR-National Chemical Laboratory, Pune 411008

Abstract

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease. It’s characterized by α-synuclein inclusions called Lewy bodies and Lewy neurites. Alpha synuclein is a presynaptic neuronal protein that is linked genetically and neuropathologically to Parkinson’s disease. The molecular mechanism through which α-syn abnormally accumulates and contributes to neurodegeneration remains unknown. The most clinical feature of this disease is the death of dopaminergic neurons in the substantia nigra of the midbrain. The different conformations of the protein such as oligomers, protofibrils, and fibrils are associated with the pathogenesis of Parkinson’s disease. Misfolded proteins form large linear aggregates or amyloids that results in fibrillation. In PD these aggregates are mainly due to α-syn, to inhibit/disassemble the activity of α-syn aggregation, we have synthesized tryptophan endcaped pentadecylphenol derivatives as they are small molecule inhibitors. Small molecule inhibitors can be easily diffuse and effect the targets. To check the inhibitory effects of these molecules in-vitro studies are done. CD (circular dichroism) was carried out to check the inhibitory effect of the synthesized compounds. Further in-vivo studies will be carried out to study the effect of this compounds on inhibition/disassembly of the aggregates in cellular model by using SH SY5Y cell line.

Keywords: fibrillation, inhibitors, aggregation, neurotransmission, lewy bodies

Abbreviations

AbbreviationsFull form
  α-synAlphasynuclein 
 CDCircular Dichroism 
 DNADeoxyribonucleic acid 
 DADopamine 
 FBSFetal bovine serum 
 G Gravitation
 HBSSHanks balanced salt solution 
 KDKilo Dalton 
 LB Lewy body
 LB-broth Luria Bertani broth
 L-CPL Left- circular polarised light
 MMolar 
 µlMicrolitre 
 mlMillilitre 
 nmNanometer 
NFTs  Neurofibrillary Tangles
 PfaParaformaldehyde 
PDParkinson’s disease 
 PBSPhosphate-buffered saline 
R-CPLRight-circular polarised light 
Rpm Rotations per minute 
 SDS Sodium dodecyl sulphate
 PAGEPolyacrylamide gel electrophoresis 
TEM Transmission electron microscope

INTRODUCTION

Parkinson’s disease is a common progressive neurodegenerative condition, second in frequency to Alzheimer disease associated with dementia and show negative impact on quality of life. It belongs to a group of conditions called movement disorders. It is both chronic, meaning it persists over a long period of time, and progressive, meaning its symptoms grows worse over time. As it is a neurodegenerative disorder, nerve cells (neurons) in brain become impaired or die. Patients or victims may begin to notice problems with movement, tremor, stiffness in the limbs, or impaired balance. Not everyone with one or more of these symptoms has PD, as the symptoms appear in other diseases as well.

Neurons of the substantia nigra communicate with neurons of the basal ganglia by liberating the neurotransmitter dopamine (DA). This interaction at the biochemical level is responsible for the fine tuning of organism’s movements. In Parkinson’s disease, neurons of the, substantia nigra progressively degenerate as a result; the amount of dopamine available for neurotransmission is lowered. So, it results in the biochemical imbalance with typical clinical symptoms that include resting tremor, rigidity, bradykinesia, i.e., a gradual slowness of spontaneous movement, and loss of postural reflexes or, in other words, poor balance and motor coordination. 

The incidence of PD increases with advancing age[12]. Onset pf parkinsonian symptoms before the age of 40 years is known as Early-onset Parkinson’s disease. It constitutes about 3-5% cases of PD. Baldereschi M et al., in 2000 observed that, It is twice common in men than women[13]. A protective effect was seen on female sex hormone. The presence of gender-associated genetic mechanisms in exposure to environmental risk factors might explain this male predominance[14]. India, recorded 70 out of 100,000 is one of the world’s lowest incidence of PD. The Parsi community in Mumbai has highest incidences of PD in world about 328 out of 100,000. There is an estimate that, half million people are affected with PD and related disorders in the United States. 

The appropriate cause of PD is unknown. There has been a lot of research into it, but so far, Scientists aren’t sure of the exact cause of PD. In addition to thee, Lewy bodies are linked neuropathologically to PD. Genetic mutations and Environmental factors contribute largely to PD. Both the causes involve the role of α-syn. Certain chemicals used in farming, such as insecticides and herbicides. Some metals and chemicals used in factories, such as manganese, lead, and trichloroethane are found to involve in causing PD. 

About 5-10% of people have PD because of mutation in one of several specific genes. Harbouring the gene may not harm the individual but susceptibility factors put the individual in risk. The risk factors even depend on the age of onset, severity and progression. At least 17 autosomal dominant and autosomal recessive gene mutations have been implicated in development of PD, including SNCA, LRRK2/PARK8, GBA, PRKN, PINK1, DJ1/PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2 and UCHL1.[17] Having a close relative with PD also increases the chances of developing PD. 

PD is characterized by the presence of fibrillar aggregates called Lewy bodies (LB). They are the abnormal aggregates of protein that develop inside nerve cells. Lewy bodies and Lewy neurites are the defining neuropathological markers of PD. In addition to PD Lewy bodies are contributing dementia with Lewy bodies, PD with dementia, and some other disorders. They are also seen in cases of multiple system atrophy, particularly the parkinsonian variant. LB appears as spherical masses that displace other cell components. In Parkinon’s disease, they are found mainly in the substantia nigra which is in the mid-brain, whereas in dementia with lewy body they are more widely distributed throughout the cerebral cortex.  LBDs are mainly characterized pathologically by the progressive accumulation of the presynaptic protein α-synuclein (α-syn), and hence they are often referred to as synucleinopathies. It is composed of the protein α-syn in bulk amounts associated with other proteins, such as ubiquitin and neurofilament protein. Tau proteins may also be present, and LBs may occasionally be surrounded by neurofibrillary tangles. 

It has been identified that α-syn is the major constituent of Lewy bodies. α-syn consists of 140 aminoacid residues is of molecular weight  14 KD. It is highly conserved presynaptic protein that is abundant in various regions of the brain while smaller amounts are also found in the heart, muscle, and other tissues. In the brain it is found mainly at the tips of nerve cells (neurons) in specialized structures called presynaptic terminals. Presynaptic terminals release chemical messengers, called neurotransmitters, from compartments known as synaptic vesicles. The release of neurotransmitters relays signals between neurons and it’s critical for normal brain function.  It is encoded by SNCA gene. There are a number of different alphasynclein conformers that have been associated with the pathogenesis including monomers, oligomers, protofibrils and fibrils. Studies show that fibrils and oligomers are more toxic.[3]

The current research focusses on inhibition of  α-syn fibrillation by using indole derivatives. For this, synthesis of tryptophan endcaped pentadecylphenol derivatives was done. Tryptophan could form hydrogen bonds with the protein and pentadecylphenol could induce steric hindrance and hence inhibit protein fibrillation. Pentadecylphenol is a saturated derivative of cardanol, which is extracted from cashew nut shell liquid. It was found that indole derivatives act as a simple molecular platform for the development of novel fibrillation inhibitors. We have also synthesized tryptophan hexyl tryptophan which acts as a control to study the effect of pentadecylphenol in the former case. 

LITERATURE REVIEW

Literature shows the aggregation of disordered α-syn protein is pathogenically connected with Parkinson’s disease. Therefore, most of the research is on discovering molecules that inhibit the misfolding and aggregation of α-syn. The discovery of these molecules is known to be the best way to treat PD.

Currently, the going on PD management therapy is pharmacological therapy; however these therapies have major limitations in advanced disease. Many disabling features develop in later stages of the disease become troublesome symptoms. The treatment has been dopamine replacement therapy with levodopa, dopamine antagonists, amantidine oxidase type B inhibitors, catechol-O-methyltransferses. Dopamine replacement therapy with levodopa has been the effective since many decades.[16] 

It was reported that appearance of α-syn at pre-synaptic terminals and their associations with synaptic vesicles are observed due to overexpression of α-syn indicating the role of α-syn in the regulation of neurotransmitter release, synaptic function and plasticity.[3] Alpha-synuclein targeted therapies have the potential to slow or stop the progression of PD and other synucleinopathies.[6]

It came to know that when monomeric α-syn is incubated at 37 °C, pH 7.4, it forms fibrils with a condition-dependent rate. Agitation, in particular, will greatly accelerate the process. Typically with 70 μM (1 mg/mL) α-syn, fibrillation will be completed within 3 days with agitation, whereas without agitation it will take months.(4) Fibrillar α-syn can be transferred to the cell body of second-order neurons following anterograde transport.(5)

The in vitro kinetics of α-syn fibril formation showed an initial lag phase followed by an exponential growth phase and a final plateau, usually attributed to a nucleation-dependent polymerization. It is also shown that as increasing the concentrations of α-syn the fibrillation rate also increased.[4]

Neurosin plays a significant role in physiological α-syn degradation and also in the pathogenesis of synucleinopathies (Atsushi Iwata, 2003).[15]

Inhibition of fibril formation could be achieved by interaction with short amyloid fragments and cyclic peptides. Interaction with oligopeptides such as KLVFF, LPFFD prevents assembly of Aβ42 into fibrils and induces disassembly of already formed fibers.[8]

Several lines of research suggested that oxidative stress has been implicated in the pathogenesis of Parkinson’s disease. This is another area of controversy that it is not clear whether oxidative stress and mitochondrial dysfunction are causative factors or a result of α-syn aggregation or other pathogenic effects of the disease.

METHODOLOGY

The recombinant human α-syn DNA was purchased from Addgene, and the sequence of the received DNA has been verified. Extracted the DNA from the agar stab and it then transformed into E.coli for storage. DNA isolation is done by inoculating the single bacterial colony from the transformed plate into LB broth and further by mini-prep. To further check the protein expression, a small culture is done and run on SDS-PAGE. To isolate the protein exclusively, anion exchange chromatography was done.

Ion Exchange Chromatography

Ion exchange chromatography is perhaps the most commonly employed chromatographic method for separating proteins (charged molecules). The principle underlying ion exchange chromatography is the reversible exchange of the charged molecules (ions) present in the solution with those electrostatically bound to the insoluble support medium. The molecules therefore, get separated based on their charge. The stationary phase is constituted by the inert polymeric beads that are functionalized with ionisable groups.

As the desired protein for separation (α-syn) is negative charged[4] then anion exchange chromatography is used. Resin involved is positively charged (anion exchange resin). This resin of positive charge binds strongly to the counter ion (negatively charged) from the eluted sample. The strength of the binding between the substance and the resin is based on the strength of the charge. The bound molecules can be eluted by altering the salt concentration of the eluting buffer. Proteins with low negative charge will tend to emerge first, followed by those having a higher charge density.

Anion exchanger: DEAE-cellulose. It is weakly basic with functional diethylaminoethyl group.

Protocol:

  • The bacterial pellet is resuspended in lysis buffer
  • Lyse the cells by sonication(3s pulse on , 1s pulse off)
  • Centrifuge the cell lysate at 12000 rpm/1 hr at 4◦C
  • Collect the supernatant and load it to the DEAE cellulose column. The column has been already equilibrated with buffer A and the protein is eluted by setting a salt gradient of 0-30 % with buffer B of high salt concentration.
  • The protein fractions are selected by observing the absorbance peak at 280nm
  •  The presence of protein is confirmed by SDS-PAGE
  • The protein containing fractions are pulled and dialysed over-night
  • Concentration of the protein is measured by taking nanodrop reading using the absorption co-efficient at 280 nm.

Circular Dichroism

Circular dichroism (CD) spectroscopy is a spectroscopic technique where the CD of molecules is measured over a range of wavelengths. CD spectroscopy is used extensively to study chiral molecules of all types and sizes, but it is in the study of large biological molecules where it finds its most important applications. A primary use is in analysing the secondary structure or conformation of macromolecules, particularly proteins as secondary structure is sensitive to its environment, temperature or pH. It can be used to observe how secondary structure changes with environmental conditions or on interaction with other molecules. Structural, kinetic and thermodynamic information about macromolecules can be derived from circular dichroism spectroscopy.

Measurements carried out in the visible and ultra‐violet region of the electro‐magnetic spectrum monitor electronic transitions, and, if the molecule under study contains chiral chromophores then one CPL state will be absorbed to a greater extent than the other and the CD signal over the corresponding wavelengths will be non‐zero. A circular dichroism signal can be positive or negative, depending on whether L‐CPL is absorbed to a greater extent than R‐ CPL (CD signal positive) or to a lesser extent (CD signal negative). 

Circular dichroism spectra are measured using a circular dichroism spectrometer. CD spectrometers measure alternately the absorption of L‐ and R‐CPL, usually at a frequency of 50 kHz, and then calculate the circular dichroism signal. Secondary structure prediction is only part of the power of circular dichroism spectroscopy. Changes in circular dichroism spectra are very good proxies for changes in the structure of a molecule. Couple this with the facts that (i) spectra can be recorded in minutes and (ii) single wavelength kinetics can be recorded from milliseconds onwards, CD is a particularly powerful tool to follow dynamic changes in protein structure. For instance changes induced by changing temperature, pH, ligands, or denaturants are all commonly used.

A powerful application of circular dichroism is to compare two macromolecules, or the same molecule under different conditions, and determine if they have a similar structure, therefore to observe change in the structure(1). To check the inhibitory effect of Tryptophan pentadecylphenol Tryptophan, fibrillar samples of α-syn are prepared in different concentrations. CD checked under different wavelengths below 260nm (far-UV) is plotted on X-axis and molar ellipticity (L-CPL and R-CPL) on Y-axis. It was found that fibrils formation is steadily inhibited by increasing concentration.

Transmission Electron Microscopy

Image formation in the TEM relies on scattering of beam electrons by atoms in a sample. Atoms of relatively large molecular weights will be more electron-dense, and tend to scatter electrons by larger angles, and these are stopped by the microscope’s objective aperture. Because these electrons fail to reach the image plane, it leads to corresponding dark regions developing in the image. Scattered and un-scattered electrons collectively give rise to an image containing varying degrees of contrast (grey levels). Information from samples is seen as a 2-D projection image along the Z direction, therefore conventional TEM cannot allow you to determine if a feature is located at the top, middle or bottom of a sample as all this information with be imaged at the same time on a single plane.

Optimal results are obtained when samples do not exceed 100 nm thickness. In thicker samples, multiple collisions between illuminating electrons and sample atoms inevitably lead to heating damage (as energy is shared/transferred to the sample) as well as lower image quality because a) the information from too many stacked atomic ‘layers’ is seen superimposed, and b) because of these (inelastic) collisions emerging electrons have a large spread of electrons energies (chromatic aberration).

Another fundamental requirement for TEM is that samples must be completely dry before imaging. ‘Wet’ samples (either water or other solvents and oil) will degrade the high-vacuum environment inside the column, not only causing undesirable contamination of the microscope, but also leading to artefacts in the sample (contamination and changes in sample structure).

For Transmission electron microscopy (TEM), 2 μL of solution was placed on carbon-coated, 200-mesh copper grid. Then staining must be done with uranylacetate (electron dense stain) with an incubation time of 3 minutes for better contrast.

Cell Culture Experiments

 What is Cell culture 

  • Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in their respective favourable artificial environment. 
  • The cells can be derived from a cell line or cell strain that has already been established.

Primary cell culture

  • Primary cells are the cells that are taken directly from the tissue and processed to establish them under optimized culture conditions.
  • They are more similar to the in vivo state and exhibit normal physiology 
  • They have a limited lifespan and will stop diving after a certain number of divisions.
  • They can be more difficult to culture and maintain, than a normal continuous cell line.

Confluency

  • It is the measure of the number of the cells in a cell culture dish or a flask, and refers to the coverage of the dish or the flask by the cells.

Cell line

  • After the first subculture, the primary culture becomes known as cell line or subclone. They are derived from primary cultures have a limited life span.

Finite cell lines

  • Cell lines which divide only a limited number of times before losing their ability to proliferate are known as finite.

Continuous cell lines

  • Cell lines which divide indefinitely, it may be a cancerous cell line or cell lines which undergoes transformation by spontaneously or can be chemically or induced by virus can also become a continuous cell line. 

Adherent cell culture

  • These are anchorage-dependent cells, are grown in culture medium that are attached to the bottom of a tissue culture flask.

Suspension cell culture

  • Cells that are grown in a free-floating culture medium. Cell aggregates are suspended in a liquid agitated medium.

Passaging

  • Passaging is nothing but subculturing. Culture made by transferring some cells from a previous culture to fresh growth medium.

Passage number

  • It is the number of times a cell culture has been subcultured, and knowing the passage number can make or break an experiment.

Seeding

  • Seeding simply means to spread a defined amount (volume or cell number) of a cell suspension into a new flask or onto a plate etc.

Secondary Cell Culture Experiments

For secondary cell culture we have used SH-SY5Y cell line. It is a human derived cell line. The original cell line, called SK-N-SH, from which it was subcloned was isolated from a bone marrow biopsy of a four-year-old girl child with neuroblastoma. We chosen this particular cell line because they express dopaminergic markers. As it is a cancerous cell line, the cells are robust and will grow in most widely used tissue culture media.

Passaging

Many adherent cell cultures will cease proliferating once they become confluent, and some will die if they are left in confluent state for too long. Adherent cell cultures need to be routinely passaged, that is, once they are confluent, and have no room for expansion then a fraction of the cells need to be transferred to the new cell culture vessel. It enables the further propagation of the cell line.

Protocol:

  • Remove and discard the spent cell culture media from the culture vessel.
  • Wash cells using a HBSS. Gently add wash solution to the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer.
  •  Remove and discard the wash solution from the culture vessel.
  • Add 0.25% warm trypsin as much as volume that covers all the cells for the dissociation of the cells from the surface and also to get like individual cells. Tap the culture dish for complete coverage of the cell layer. Incubate for 3 minutes at room temperature.
  • Now transfer the cells to a falcon and centrifuge at 200×g for 5 minutes at 1000 rpm.
  • Resuspend the cell pellet in the growth medium and remove a sample of 10µl for cell counting to check the viability of cells.

Cell counting

It is often necessary to count cells before seeding. Cell counting can be done by using Automatic cell counter or haemocytometer. A haemocytometer (also called as Neubauer’s chamber) contains 2 chambers. Each chamber is ruled into 9 major squares (1mm squares).

Neubauer-Chamber

    How to count:

    • Clean the surface of the hemocytometer with 70% ethanol, take care not to scratch the surface of the central area. Dry it 
    • Clean the coverslip, wet the edges very slightly, lay it over the grooves and central area of the hemocytometer.
    • It is important that the coverslip is properly attached to obtain the correct chamber depth. The appearance of Newton’s rings (bright and dark rings caused by interference in the air between the coverslip and the glass surface of the hemocytometer) will confirm that the coverslip is attached properly.
    • Using a pipette, immediately transfer volume of 10 µl to the edge of one side of the coverslip to fill one chamber of the hemocytometer. Do the same thing for the second chamber.
    • The cell distribution should be homogenous in both chambers. The cell suspension is drawn under the coverslip and into the chamber by capillary action.
    • The cell suspension should just fill the chamber. Blot off any surplus fluid without disturbing the sample underneath the coverslip.
    • Place the Neubauer chamber on the microscope stage. Using the 10x objective, focus both onto the grid pattern and the cell particles.
    • Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares. Count all the cells in the four 1 mm corner squares. Count only viable cells by keeping a rule to include cells touching upper portion and left border.
    • View the cells under a microscope at 100x magnification. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares. Count all the cells in the four 1 mm corner squares. If there are too many or too few cells to count, repeat the procedure, either concentrating or diluting the original suspension as appropriate.
    • The cell count is found to be 280 cells by doing the average of the four corners count. To determine the cell count in ml it should be multiplied by 104. Therefore the cell count is 2.8×106 cell/ml.

    MTT Assay is done to check the viability of cells

    • Plate 10,000 cells per well in a 96-well plate at certain dilution of the cells that we got in cell count.
    • After 24 hours add fibrils to each well.
    • Then after 24 hours remove the medium and wash cells with PBS.
    • Add MTT reagent (5mg/ml) and incubate for 3 hrs. 
    • If there is any crystal formation then add DMSO to dissolve the crystals.

    Labelling

    The visualization of the cell and tracking of protein in the cells is mandatory to identify, map and analyse the actions of our protein and its influence in the cell.  The attachment of a reporter group (label) is often required for the detection and characterization of our protein.

    Protocol:

    • Seed the cells on sterile glass coverslips. Poly-d-lysine is used for proper adhesion of cells on the coverslips.
    • Fixation:  Aspirate the culture media from the dish and gently wash with PBS at room temperature. 
    • 4% paraformaldehyde is added for fixation and incubates for 10 minutes at room temperature.
    • Give 2 PBS washes for 10 minutes each as pfa is toxic.
    • Permeabilization: Give PBST wash and incubate for 10 minutes. This makes the cell membrane permeable.
    • Blocking: Blocking buffer is added and kept on the shaker for uniform dispersing and incubated for 10 minutes.
    • Antibody incubation: Primary ab is anti-alphasyn-antibody syn211 Alexa flour (Fluoroscent labelled primary antibody) raised from mouse is added in 1:2500 dilution with PBS buffer and then incubated for over-night at 4◦C
    • Next day take out from incubation.
    • Give 2 PBST washes and 1 time PBS wash with time interval of 10 minutes.
    • Secondary antibody incubation: Add secondary ab raised from anti mouse. Allow 3 hrs incubation. CY3 is the flurophore.
    • Give 2 PBS washes with an interval of 10 minutes.
    • DAPI staining is done to stain the nuclei
    • Invert the coverslip onto a glass slide with a drop of mounting media
    • Carefully remove the excess mounting media if necessary and seal as required with nail polish.
    • Examine under fluorescence microscope.
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      RESULTS AND DISCUSSION

      As our target is to inhibit the α-syn fibrillation, inhibitors that are tryptophan pentadecylphenol tryptophan synthesis was done. Testing of these inhibitors on fibrils should be taken place. Firstly TEM studies are done for the conformation of fibrils and then to check the effect of these inhibitors, CD experiments were carried.

        Circular Dichroism

        CD of α-syn fibrils with tryptophan pentadecylphenol tryptophan at 10µM concentration show disassembly of fibrils as comparing to the standard peak of β-pleated sheets. The X-axis shows the increasing wavelengths under UV and Y-axis shows molar ellipticity. From the peak of 50µM concentration of  α-syn inhibition is identified as much more than10µM concentration. The red color peak describes about 50µM fibrils. They are in β-pleated sheets secondary structures.

        TEM Imaging

          These studies revealed the formation of fibrillar structures at a definite concentration of Alphasynuclein. It is subjected to agitation and incubated at 37◦C.

          ACKNOWLEDGEMENTS

          I would like to express my sincere thanks and heartfelt gratitude to Dr. Jancy Nixon Abhraham, Polymer Science and Engineering Division, CSIR–NCL for providing me an excellent platform for development. I am thankful her valuable guidance and timely discussions, which always inspired me.

          I am thankful to Dr. Asha S.K for her quick and immediate works, ultimate care and for allowing me to work in her lab.

          I am indebted to IASc-INSA-NASI for giving me such a great opportunity, for exploring me to research environment. 

          I would also like to thank my fellow trainee Swastik Bisvas and project assistant Mrunal karle  for being with me all time helpful, supportive throughout my project and for giving me lifetime memories.

          I am extremely grateful to my mentor Lisni and Priya for being supportive and pointing out my mistakes as shown great patience during my project. I am thankful for that.

          I am also thankful to all my seniors, Shibam pal, Srikanth Nikam, Pooja B. Fulare, Sarath S. Kamath, Moumitha Roy, Aryan A. Wavhal, Ganesh N. Kamble  and help during the whole span of my work. They were very friendly and I am thankful for the lab treat. It has been my privilege to work with these extremely talented, interactive great minds. 

          I also thank my friends and roommates who gave me wonderful company throughout the stay in Pune. Huge thanks to my classmates and dearest friends for their all time support. 

          I would thank my parents for sending me to a far place for the first time. I  will be debtful to my cousin without whom this trip won’t be fruitful.

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          11) MORTIMER.M. Parkinson’s Disease, the Dopaminergic Neuron and Gammahydroxybutyrate. Springer(2018).

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          13) BALDERESCHI M. Parkinson’s disease and parkinsonism in a longitudinal study: two-fold higher incidence in men. ILSA Working Group Italian Longitudinal Study on Aging. Neurology. 2000 Nov 14;55(9):1358-63.

          14 ) VAN DEN EEDEN SK. Incidence of Parkinson’s disease: variation by age, gender, and race/ethnicity. Am J Epidemiol 2003 Jun 1; 157(11): 1015-22.

          15) ATUSHI IWATA. Alpha-synuclein degradation by serine protease neurosin: implication for pathogenesis of synucleinopathies, Human Molecular Genetics, Issue 20, 15 october, pages 2625-2635. 

          16) NANCY L DIAZ & CHERYL H WATERS  Current strategies in the treatment of parkinson’s disease and a personalized approach to management. Pages 1781-1789.

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