Department of Chemistry

Umeå University
S-901 87 Umeå, Sweden
Degree thesis in Chemistry 15 ECTS-credits
Swedish Master’s level
2006

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

Monoclonal
Control (8) Patient (22)
Ab

Fig1b
Immunoblotting test against the native α-synuclein with
Fig1a
control (6 controls) and patient (6 males & 4 females) sera.
µm
α-synuclein separated by SDS-PAGE and Monoclonal antibodies against the α-synuclein are used as an
stained with coomassie blue internal control.

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.
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).

1.5µm

0µm

0µm 1.5µm
Fig 3a
Fig 3b

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.
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.

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
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.

2.5

µm

0 µm

0 µm 2 µm
Fig 4a
Fig 4b
Tht fluorescence experiments to confirm the
AFM characterization of α-synuclein fibrils used in
cross-β-sheets in the α-sunuclein fibrils. At the
immunoblotting experiments.
time point of 100 hours mature fibrils are
formed. The image clearly displays the α-synuclein fibrils
with a cross section height reaching 9 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.
 Western
blot

Monomeric α-synuclein

Monoclonal Abs

Control (8) Patient (22)

Fig 5a Fig 5b

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).
mAbs Control
PD

Fig 6a Fig: 6b
Native 8-25% gradient gel Immunoblotting test against the α-synuclein fibrils with
control (6 controls) and patient (6 males & 4 females) sera.
Coomassie-staining.
Monoclonal antibodies against the α-synuclein are used as an
internal control.
 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.

Asyn HEWL
Ab
Fig 7b

Asyn HEWL
Fig 7a Ab
Fig 7c

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.
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.
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