Department of ChemistryUmeå UniversityS-901 87 Umeå, SwedenDegree thesis in Chemistry 15 ECTS-creditsSwedish Master’s level2006

Autoimmunity against alpha synuclein and its amyloid structures

In the blood sera of Parkinson’s patients

Kumar Swamy Appari

Supervisor: Ludmilla Morozova-Roche Medical Biochemistry and Biophysics

Umeå University, SE-901 87 Umeå, Sweden.

Abstract.

Amyloid structures of α-synuclein, which are involved in Parkinson’s disease pathology, were

characterized by atomic force microscopy and biophysical techniques. Native α-synuclein, its

oligomeric structures and amyloids were subjected to immunoblotting experiments to investigate

the presence of specific autoantibodies in the blood sera of Parkinson’s disease patients.

Pronounced immune response was observed towards monomeric and fibrils of α-synuclein in the

blood sera of Parkinson’s patients compared to age-matched healthy control, but no antibodies

were found against the oligomeric structures in both the control and PD patients. Statistical

analysis of the immune responses towards monomeric alpha-synuclein shown four times larger

mean and median values in patients compared to control group. Further PD patient sera were also

found to contain antibodies against different amyloid structures produced from hen egg white

lysozyme and a beta. These antibodies, which were raised against α-synuclein amyloid fibrils in

Parkinson’s diseased patients, are probably against the conformational epitopes related to cross β-

sheet core that is viewed as a most common feature of amyloid. Future studies can be carried out

based on these investigations to test the capability of these autoantibodies in inhibiting the

fibrillation process, thus paving a path for humoral immune therapy.

Introduction

The second most common neurodegenerative disease after Alzheimer’s disease is the Parkinson’s

disease (PD). The death of the dopaminergic neurons present in the pars compacta region of the

Substantia nigra that produce dopamine, results in a movement disorder, which is a characteristic

feature of Parkinson’s disease (1). However, the causes leading to the death of dopaminergic

neurons is still unknown. By the time the disease symptoms such as bradikinesia, resting tremor,

rigidity and postural disabilities become evident, almost 70-80% of the dopanimergic neurons

have died (2, 3). From this point of view a proper diagnostic tool is very much needed.

Intraneuronal inclusion bodies with amyloid fibrils called lewy bodies, is the most prominent

feature in PD pathology. α-synuclein being the most prominent component of lewy bodies, which

is a pathological hallmark of disease is implicated in the etiology of the PD. Several lines of

evidences support the view that the protein α-synucelin plays an important role in onset of PD

(4). Mutations in α-synuclein and parkin causes familial types of PD. Parkin a known neuronal

ubiquitin ligase is also associated with the Lewy body formation in PD and Lewy body dementia

( 5,6).

α-synuclein is a presynaptic neurotransmitter that plays an essential role in synaptic

transmission and synaptic plasticity (7). It is mostly present in the cytosol as a soluble protein

while some of the protein through its N-terminal is found reversibly bound to the membranes.

Available information strongly suggests the role of α-synuclein in maintaining a presynaptic

vesicle pool in presynaptic nerve terminals (8).

α-synuclein is natively an unfolded protein in soluble form but when bound to lipids it

acquires a folded conformation. The central hydrophobic region of the α-synuclein is prone to

self-association leading to aggregation of the protein. α-synuclein aggregates are stabilized by

acquiring cross-β-pleated configuration which lead to amyloid formation. It is more fibrillogenic

than its counterparts such as β- and γ-synuclein (9, 10). The ability of the wild-type α-synuclein

to aggregate and form fibril implicates that α-synuclein over-expression is a major cause of PD.

Mutants of α-synuclein, A53T and A30P in rare cases of familial early onset PD were observed

in some European families). Environmental factors such as exposure to pesticides and other

chemicals also contribute to oxidative stress and aggregation of α-synuclein (11). Over-

expression of α-synuclein in mouse models and the human population with multiple copies of

functional α-synuclein are the prerequisites for α-synuclein fibrillation and progressive

neurodegeneration (12, 13).

Converging evidences show that the protein aggregates are neurotoxic, in particular

ordered pre-fibrillar oligomers and protofibrills can be responsible for cell death. Understanding

the folding and misfolding mechanisms of the proteins leading to formation of toxic aggregates

may provide rational approaches to therapy (14, 15). Autoimmune response to different amyloid

structures in neurodegenerative diseases like Alzheimer’s disease and PD can be exploited as

marker of protein aggregation and used as a diagnostic feature of the disease (16).

As α-synuclein is implicated in the development of PD, study of autoimmune response

against the different forms of this protein can help to develop humoral immune therapy against

PD. Here the presence of immunoglobulin (IgG) antibodies in the human sera of the PD patients

was investigated and controls were made to compare the presence of IgG antibodies against the

different non-aggregated and amyloid forms of α-synuclein.

Materials and Methods

Human subjects. 22 PD patients, with an age range of 38-78 years, with the majority in

their 60s, were recruited from Umeå University Hospital. 10 healthy controls, biologically

unrelated to the patients and of similar age as these, were selected from spouses of patients

attending the outpatient clinic. The medical ethics committee of Umeå University Hospital

approved the study.

Protein samples. A recombinant -synuclein was dissolved in 10 mM sodium phosphate

buffer at pH 7.4. After ca. 40 minutes of dissolving, protein concentrations were determined by

absorbance measurement at 280 nm using the extinction coefficient E1mg/ml= 0.354. To produce

amyloid oligomers and fibrils α-synuclein solutions at a 10 mg/ml concentration were incubated

in 10 mM sodium phosphate (NaP) buffer, pH 7.4 at 37 °C under continuous agitation.

Amyloid assays. The kinetics of α-synuclein amyloid formation was monitored by

thioflavin T (ThT) binding assay using a modification of the method described previously.

Fluorescence of thioflavin T was measured on a Jasco FP-6500 spectrofluorometer (Jasco,

Japan). The dye was excited at 440 nm and emission spectra were recorded between 450-550 nm,

setting the excitation and emission slits at 3 nm.

Atomic force microscopy (AFM). AFM measurements were performed on a PicoPlus

SPM (Molecular Imaging, USA) in a tapping mode using acoustically driven cantilevers as

described previously. A scanner with a 100 µm scan size was used. The cross-section analysis in

the height images was carried out to determine the dimensions of oligomers and fibrils.

IgG Purification. Total IgGs were purified by using Melon Gel Purification kit (Pierce

Biotechnology) according to the manufacturers protocol. Purity of the IgG antibodies were

determined on 15% SDS PAGE gels.

Electrophoresis and Immunoblotting. Aliquots of the fractions were mixed with SDS

sample buffer and applied to 16% Tris-Tricine gels. Prestained molecular weight standards

“SeeBlue” (Invitrogen, USA) was included in each run. For a-synuclein amyloid oligomers and

fibrils we have used native 8-25% gradient gels (Phast gels, GE healthcare, Sweden). Coomassie

blue was used for the staining of the gel.

Immunoblotting was performed by using nitrocellulose membrane according to standard

procedures. Membranes were blocked with 5% milk in Tris buffered saline (TBS) buffer

containing 0.05% of Tween 20 and incubated with each primary antibody. The PD patients and

controls serum were used as primary antibodies. The secondary antibody was a peroxidase-

conjugated anti-human IgG antibody, and immunoreactive protein was detected by using the

enhanced chemiluminescence method (Amersham Biosciences).

Dot blot assay. Recombinant a-synuclein and its amyloid oligomers and fibrils were

dissolved to 3mg/ml concentration in 20mM glycine buffer at pH 2.0. 2 μl of each sample

applied to a nitrocellulose membrane, blocked with 5% non-fat milk in Tris-buffered saline

(TBS) containing 0.05% Tween 20 (TBS-T), at 37 °C for 2 h, washed 3 times for 5 min each with

TBS-T and incubated for overnight at 4 ºC with the samples of the sera from patients and

controls. The serum dilution was 1:5000 in 5% non-fat milk in TBS-T. The membranes were

washed 3 times for 5 min each with TBS-T, then incubated for 1h at 37 °C with horseradish

peroxidase conjugated with anti-human IgG (Sigma, USA) at 1:40.000 dilution in 5% non-fat

milk in TBS-T. The blots were washed 3 times with TBS-T for 15 min each time and the

immunoreactive protein was detected by using the enhanced chemiluminescence substrate kit

(Amersham biosciences, Sweden).

Results

Autoimmune response towards native α-synuclein

Recombinant α-synuclein was separated using 16% Tris-Tricine gels (Fig1a). The 16 kDa

protein was subjected to immunoblot analysis using peripheral blood serum obtained from 22

patients suffering from PD and 8 healthy age-matched controls. It was observed that control

serum from the healthy people without any history of PD does not contain any IgG antibodies

against the native α-synuclein. However, the Parkinson’s patient serum demonstrated immune

reaction against native α-synuclein. This is a clear indication of increased level of IgG antibodies

against the α-synuclein in PD patient.

Immune responses observed in the immunoblots were quantified using scion image

software and the results are summarized in box-plots (Fig2). The median and the median values

for immune response in PD patients shows four-fold increase compared to the healthy control

group indicating that the level of antibodies raised against native α-synuclein in PD patients

group was four times greater than control group.

Fig1a

µm

Fig1b

α-synuclein separated by SDS-PAGE and stained with coomassie blue

Immunoblotting test against the native α-synuclein with control (6 controls) and patient (6 males & 4 females) sera.

Monoclonal antibodies against the α-synuclein are used as an internal control.

Control (8)Monoclonal Ab

Patient (22)

Fig 2: Box-plots showing immune response to α-synuclein in the blood sera of control and patient groups

- The boxes include from 25% to 75% of all values of immune responses.

- Central squares indicate the mean value for each group.

- The line drawn across the box shows median value for each group.

- The whiskers indicate the distribution from 5% to 95%,

- The dots correspond to remaining 10% of the immune responses within the groups.

- Mean increase of the immune response towards α-synuclein by 4 folds compared to control group.

- Median value is also increase by 4 folds compared to control group.

Characterization of α-synuclein amyloid structures by ThT fluorescence & AFM

The kinetics of α-synuclein amyloid formation during incubation in 10 mM sodium

phosphate buffer at pH 7.4 and 37 °C under constant agitation was measured by ThT binding

assay. ThT binds specifically to the cross- - sheet-containing amyloid structures which gives rise

to fluorescence (17).

The increase of ThT fluorescence indicates the appearance and accumulation of pre-

fibrillar structures (Fig 3a). AFM characterization of the oligomeric structures displays different

sizes of oligomers with height ranging from 2-3 nm (Fig 3b).

A small amount (2-5%) of the pre-fibrillar structures produced after 180 hrs of incubation

was used as a seed for the freshly prepared α-synuclein in the same conditions. The seeded fresh

α-synuclein was further incubated at same conditions which were mentioned in materials and

methods. After 48 hrs of incubation the formation of maximum amount of matured α-sunuclein

fibrils was observed (Fig 4a). Structural changes of amyloid assemblies were monitored in

Fig 3a

Thioflavin T fluorescence was measured every 20 hours to study the kinetics of formation of oligomeric structures with cross- β -sheet conformations. At the time point of 180 hours mature oligomers are formed.

Fig 3b

AFM image of oligomeric structures produced from α-sunuclein at time point of 180 hours. The cross section height of the structures ranges from 2-3 nm. The image clearly shows the formation of oligomeric structures of α-synuclein.

1.5µm

0µm 1.5µm

0µm

parallel with ThT fluorescence experiments by AFM. The AFM image of α-synuclein amyloid

fibrils is presented in Figure 4b. In the cross section analysis on AFM, the height of the α-

synuclein oligomers was observed as 1-2 nm and the fibrils height as 8-10 nm.

Autoimmune response to α-synuclein amyloid oligomers and fibrils

Blood sera from PD patients as well as from healthy controls were screened for the

presence of immune response towards α-synuclein oligomers and fibrils. Oligomeric structures of

α-synuclein were separated on native PhastGel gradient 4-20% Tris-Cl gel and stained with

Coomassie blue, different sizes of α-synuclein oligomers were observed (Fig 5a). The PD patient

and control sera were subjected to immunoblot analysis to detect auto-antibodies against different

sizes of oligomeric structures produced in in vitro conditions (Fig 5b). No antibodies were found

in both the control and PD patients groups against the α-synuclein oligomers.

Tht fluorescence experiments to confirm the cross-β-sheets in the α-sunuclein fibrils. At the time point of 100 hours mature fibrils are formed.

0 µm 2 µm

2.5

µm

0 µm

AFM characterization of α-synuclein fibrils used in immunoblotting experiments.

The image clearly displays the α-synuclein fibrils with a cross section height reaching 9 nm.

Fig 4b Fig 4a

Fig: 5a) Oligomers of α-synuclein separated on 4-20% Native Gel (Coomassie blue staining).

5b) immunoblotting test against the oligomeric structures.

Surprisingly, in the immunoblot detection we have observed a specific response towards α-

synuclein amyloid fibrils in both the group of PD patients and healthy controls (Fig 6b). We have

also purified the total IgGs from the pooled blood serum of both the group of PD patients and

controls to eliminate the possibility of unspecific reaction (Fig 7a).

Fig: 6b

mAbs Control

PD

Immunoblotting test against the α-synuclein fibrils with control (6 controls) and patient (6 males & 4 females) sera.

Monoclonal antibodies against the α-synuclein are used as an internal control.

Fig 6aNative 8-25% gradient gel

Coomassie-staining.

Monomeric α-synuclein

Fig 5a Fig 5b

Western blot

Monoclonal Abs

Control (8) Patient (22)

The purified total IgGs of both groups of PD patients and controls displayed similar

response pattern towards the amyloid fibrils of α-synuclein, Hen Egg White Lysozyme and A

beta peptide (Fig 7a, 7b). These results suggest that the antibodies towards amyloid fibrils most

likely recognize a common amyloid conformational epitope on the fibrils of different protein

origin (29). There was no immune reaction towards α-synuclein oligomers detected by

immunoblot analysis in any group of PD patients and healthy individuals.

Fig: 7a) Total IgG purified from pooled sera of PD patients and control groups separated on SDS with commasie staining.

7b) Immunoblotting test against the α-synuclein, Hen Egg White Lysozyme and Abeta fibrils with pooled control sera.

7c) Immunoblotting test against the α-synuclein fibrils, Hen Egg White Lysozyme and Abeta with pooled patient sera.

Asyn HEWL Ab

Asyn HEWL Ab

Fig 7c

Fig 7b

Fig 7a

Discussion

PD arises from the loss of the dopaminergic neurons in the substantia nigra, Sporadic PD

is the second most common neurodegenerative disease and the most common age-related

moment disorder (18).α-synuclein was first implicated in the pathogenesis of neurodegenerative

diseases when two peptide fragments of α-synuclein termed non-Aβ component were co purified

with amyloid plaque cores isolated from Alzheimer’s disease (19). Further the discovery of two

pathogenic missense mutations in the α-synuclein gene in rare kindred with autosomal

Parkinson’s disease (PD) stimulated interest in understanding the contribution of α-synuclein in

neurodegenerative diseases (20).

α-synuclein is abundant in the intraneuronal deposits called Lewy bodies and considered

as the hallmark of the PD neuropathology (21). α-synuclein is a natively unfolded protein, with

little or no ordered structure under physiological conditions, fibril formation involve the partial

folding of α-synuclein into the highly fibrillation-prone pre-molten globule like conformation,

which represents a key intermediate on the fibrillation pathway (22). α-synuclein can also form

several morphologically different types of aggregates, oligomers, amorphous aggregates, and

amyloid like fibrils. (23,24). Aggregation of α-synuclein leading to dopaminergic neuronal cell

death may be exerted by specific population of α-synuclein aggregates and/or mediated via

various routes involved in different cellular processes (25).

More recently the local inflammatory and immune responses are identified as the key

factors that exacerbate the neurodegenerative process in PD, which involve local tissue microglia,

infiltrating peripheral monocytes and leucocytes. A profound increase in the microglia

proliferation is found in the Substantianigra (SN), Striatum of PD patients (26). The

consequences of the initial immune activation in the affected regions of the PD brain are the

increase in the local permeabilisation of the blood-brain barrier. The increased permeability of

blood-brain barrier further leads to infiltration by monocytes and/or leucocytes, which is believed

to be critical step in the development of autoimmune reaction (27). Eventually the death of

neurons or damage of axons and synaptic terminals could result in the release of various soluble

or aggregated forms of a-synuclein into the extracellular space and, in accord with this scenario;

monomeric and oligomeric forms of α-synuclein have been found in the CSF and plasma of PD

patients (28).

In the current study immmunoblotting experiments were carried out on different forms of

α-synuclein from monomeric to fibrillar structures to investigate the presence of auto antibodies

raised against these structures in PD patient and control sera.

The immunoblot experiments showed that the antibodies developed in the PD patients

towards amyloid fibrils are not specific to α-synuclein fibrils alone. We have observed that these

antibodies towards amyloid fibrils are also recognizing the different origin of amyloid fibrils such

as hen egg white lysozyme and A-beta peptide. The IgG antibodies developed in the PD patients

against α-synuclein fibrils could most likely recognizing the common conformational epitopes on

different fibril species. The existence of a major conformational epitope present in many amyloid

fibril composed of diverse protein sequences was suggested earlier (29). The PD patient serum as

well as the control serum in our study did not show any immune response against the oligomeric

structures of α-synuclein. Immune response towards α-synuclein fibrils was observed in both the

control and patients. The experimental results also demonstrated that there is a specific humoral

immune response against the α-synuclein in Parkinson’s disease. 22 PD patients and 8 healthy

age matched control sera were tested for immune response against monomeric native α-

synuclein, most of the PD patients demonstrated a clear immune reaction to the native α-

synuclein, whereas the control sera did not show any immune response to α-synuclein.

Statistical analysis after the quantification of the immune responses in each group

displayed a four fold increase in the mean and median values for the immune responses in

patients over the control group. This indicates that the level of auto antibodies developed against

α-synuclein in patients is four times higher than those in the control group.

Fibrils with cross-β conformations develop further during PD. Understanding the

mechanism behind the formation of the amyloid aggregates from α-synuclein could help in

establishing the cause of PD (14, 15). Progressive development of autoimmunity against the

amyloids from α-synucelin could accompany development of PD. The study of development of

progressive autoimmunity to the α-synuclein fibrils in the PD can establish a method for humoral

immune therapy against the disease.

Further studies are to be carried out to investigate the capacity of the purified IgG

antibodies from both the control and patient sera in inhibiting the process of α-synuclein

fibrillation and disintegration of the already formed fibrils in in-vitro conditions. This could help

in understanding the progressive autoimmune response against the different forms of α-synuclein

during PD at different stages of amyloid formation. This might lead ultimately to the

development of a proper method of humoral immune therapy for Parkinson’s disease.

References

1. Fearnley J. M and Lees A. J. Ageing and Parkinson’s disease: substantia nigra regional selectivity. Brain, 1991, 114, 2283-2301.

2. Tinazzi M, Vesco C. D, Fincati E, Ottaviani S, Nicola, Smania, Moretto G, Fiaschi A, Martino D and Defazio G. Pain and motor complications in Parkinson's, J. Neurol. Neurosurg. Psychiatry, Mar 2006, July 1, doi: 10, 1136.

3. Youdim M.B.H and Riederer.P. Understanding Parkinson's disease. Scientific American, 1997, 276, 52-59.

4. Dawson T. M. and Dawson V.L. Molecular Pathways of Neurodegeneration in Parkinson's disease. Science, 2003,302, 819 - 822.

5. Michael G. S, Matthew P.F, Gai W .P, Medina M, Sharma N, Forno L, Ochiishi T, Shimura H, Sharon R, Hattori N, Langston J. W, Mizuno Y, Hyman B.T, Selkoe D. J & Kosik K.S. Parkin Localizes to the Lewy Bodies of Parkinson Disease and Dementia with Lewy Bodies. American Journal of Pathology, 2002,160, 1655-1667.

6. Kingsbury A.E, Susan E.D, Hardev S, Sarah E, Andrew J. L, Oliver J.F.F. Alteration in α -synuclein mRNA expression in Parkinson's disease. Moment Disorders, 2003, 19, 162-170.

7. Liu S, Ninan I, Antonova I, Battaglia F, Trinchese F, Narasanna A, Kolodilov N, Dauer W, Hawkins R.D and Arancio O. Alpha-Synuclein produces a long-lasting increase in neurotransmitter release. The EMBO Journal, 2004, 23, 4506–4516.

8. Murphy D. D, Rueter S.M, Trojanowski J.Q and Lee V.M.-Y. Synucleins Are Developmentally Expressed, and a-Synuclein Regulates the Size of the Presynaptic Vesicular Pool in Primary Hippocampal Neurons. The Journal of Neuroscience, 2000, 20(9):3214–3220 9. Uversky, V. N & Fink, A. L. Conformational Constraints for Amyloid Fibrillation: The Importance of Being Unfolded. Biochim Biophys Acta, 2004, 1698, 131-153.

10. Biere A.L, Wood S.J, Wypych J, Steavenson S, Jiang Y, Anafi D, Jacobsen F.W, Jarosinski M.A, Wu G-M, Louis J-C, Martin F, O L. Narhi and Martin Citron. Parkinson’s Disease-associated α-Synuclein is More Fibrillogenic than β- and γ-Synuclein and Cannot Cross-seed Its Homologs. J. Biol. Chem. 2000, 275, 34574-34579.

11. Steece-Collier K, Maries E and Kordower J.H. Etiology of Parkinson's disease: Genetics and environment revisited. PNA S, 2002, 99, 13972-13974.

12. Chandra S, Gallardo G, Fernández-Chacón R, Schlüter O and Südhof T. Cell, 2005, 123, Issue 3, 383-396.

13. Farrer M, Kachergus J, Forno L, Lincoln S, Wang DS, Hulihan M, Maraganore D, Gwinn-Hardy K, Wszolek Z, Dickson D and Langston JW. Comparison of kindreds with parkinsonism and alpha-synuclein genomic multiplications. Ann Neurol, 2004, 55: 174-179.

14. Caughey B., Lansbury P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci, 2003, 99, 13972-13974.

15. Dobson C. M. Protein folding and misfolding. Nature, 2003, 426, 884-890.

16. Grudena M.A, Davudovab T.B, Malisˇauskasf. M, Zamotinf V.V, Sewelle R.D.E, Voskresenskayac N.I, Kostanyand I.A, Sherstneva V.V, Morozova-Rochef L.A. Autoimmune Responses to Amyloid Structures of Aß(25–35) Peptide and Human Lysozyme in the Serum of Patients with Progressive Alzheimer’s Disease. Dement Geriatr Cogn Disord, 2004, 18:165–171..

17, Levine H. Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci, 1993, 2, 404-410.

18. Galvin, J. E., Lee, V. M., Schmidt, M. L., Tu, P. H., Iwatsubo, T., and Trojanowski, J. Q. () Adv. Neurol. 1999, 80, 313–324.

19. Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Yoshimoto M, Otero DA, Kondo J, Ihara Y, Saitoh T.. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. PNAS, 1993, 90:11282–11286.

20. Polymeropoulos M.H,, Lavedan C., Leroy E., Ide S.E., Dehejia A., Dutra A., Pike B., Root H., Rubenstein J., Boyer R., Stenroos E.S., Chandrasekharappa S., Athanassiadou A., Papapetropoulos T., Johnson W.G., Lazzarini A.M., Duvoisin R.C., Di Iorio G., Golbe L.I., Nussbaum R.L. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science, 1997, 276:2045–2047.

21. Goedert M. Familial Parkinson’s disease. The awakening of a-synuclein. Nature, 1997,388, 232–233.

22. Uversky V. N., Li J. and Fink A. L. Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J. Biol. Chem. 2001a, 276, 10737–10744.

23. Uversky V. N., Li J., Souillac P., Millett I. S., Doniach S., Jakes R., Goedert M. and Fink A. L. Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alphasynuclein assembly by beta- and gamma-synucleins. J. Biol. Chem. 2002d, 277, 11970–11978.

24. Uversky V. N. A protein-chameleon: conformational plasticity of alpha-synuclein, a disordered protein involved in neurodegenerative disorders. J. Biomol. Struct. Dyn. 2003, 21, 211–234.

25. Lundvig D., Lindersson E. and Jensen P. H. Pathogenic effects of alpha-synuclein aggregation. Brain Res. Mol. Brain Res. 2005, 134, 3–17.

26. Hunot S. and Hirsch E. C. Neuroinflammatory processes in Parkinson’s disease. Ann. Neurol. 2003, 53, S49–S60.

27. Racke M. K., Ratts R. B., Arredondo L., Perrin P. J. and Lovett-Racke A. The role of costimulation in autoimmune demyelination. J. Neuroimmunol. 2000, 107, 205–215.

28. El-Agnaf O. M. A., Salem S. A., Paleologou K. E. et al. Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. FASEB J. 2006, 20, 419–425.

29. Brian O’Nuallain and Ronald.W. Conformational Abs recognizing a generic amyloid fibril epitope. PNAS, 2002, 99, 1485-1490.