Transcript Detection of α-Synuclein in human plasma and its

Detection of α-Synuclein in human plasma
and its interaction with different apolipoproteins
By
Fatemeh Nouri Emamzadeh (supervisor David Allsop)
Division of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University
Introduction
Human α-synuclein (α-syn) is a small, soluble protein of 140
amino acid residues and 14.5 KDa molecular mass that is
encoded by the SNCA gene. α-Syn is predominantly a
neuronal protein that is involved in synaptic plasticity,
maintenance of the synaptic vesicle pool, neuronal
differentiation, and regulation of dopamine biosynthesis.
This protein can be considered as a hallmark of Parkinson’s
disease (PD) because it is the most abundant protein in
Lewy bodies. Moreover, duplication, triplication and
mutation of the SNCA gene can all cause an inherited form
of PD. In the nerve cells of PD patients, soluble α-syn
aggregates into insoluble fibrils, causing neuronal cell
damage and death. PD is characterised by major nerve cell
death in the substantia nigra of the brain, often with the
loss of more than 80% of dopamine-producing neurones.
Next Steps
Results
1. Separation of different lipoproteins (VLDL, LDL,
HDL and VHDL) from plasma by ultracentrifugation
and detection of apolipoprotein fractions by using
specific antibodies for ApoB, ApoE and ApoAI (and
possibly other apolipoproteins) by immunoblotting.
1. α-Syn was expressed in E. Coli using plasmid
Pet11a and purified using MonoQ and Superdex
columns.
MonoQ Column
WCE
C13
C3-4
C1-2
B5-6 B3-4
B1-2
A9
A8
Superdex Column
MM
T2
T1
A15
T0
A14
A13
A12
A8
A7
MM
30
30
20
20
α-Syn
15
α-Syn
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2. Syn1 was found to be the best antibody for
detection of α-syn on immunoblots, with detection
down to 0.5ng of the protein.
5 ng
10 ng
2.5ng
0.5ng
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2. Detection of possible interaction of α-syn with
these various lipoprotein fractions from plasma
by carrying out immunoblotting using Syn1
antibody.
3. Immunoprecipitation of α-syn from plasma by
using an anti-α-syn antibody to capture the entire
protein/lipid complex and then determine which
apoplipoproteins and lipoprotein fractions are
involved.
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α-Syn
Purpose
At present, when a clinical diagnosis of PD is made, based on
symptoms such as shaking, stiff muscles and slow movement,
serious damage has already been done to nerve cells of the
substantia nigra. The diagnosis of PD in its earlier stages,
before this irreversible damage, would be of enormous benefit
for future treatment strategies designed to slow or halt the
progression of PD.
α-Syn is present in cerebrospinal fluid (CSF) and blood
plasma and is under investigation as a biomarker for PD [1].
Recently, investigators have shown elevated levels of both αsyn oligomers and the ratio between oligomeric and total α-syn
in the CSF of PD patients [2]. Elevated α-syn oligomers have
also been reported in the blood plasma of PD patients [3].
α-Syn is known to interact with lipids [4], and the initial aim
of this project is to elucidate any interactions between α-syn
and lipoproteins (VLDL, LDL, HDL, VHDL) in blood plasma, so
that we can better appreciate how it is transported in the
blood, how it might cross the blood-brain barrier, and how we
might further develop α-syn as a reliable biomarker for PD.
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3. Syn1 detected a band corresponding to α-syn in
small amounts of whole human blood plasma.
1/100
1/200
1/300
1/400
1/500
1/600
R-Syn
IgG
60
50
40
30
20
References and Acknowledgements
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4. The sensitivity and specificity of apolipoprotein
A1, B and E antibodies were examined on
immunoblots.
Apo-E
Apo-A1
1/100
1/175
1/150
1/125
1/125
1/150
Apo-B
1/175
60
1/100
110
1/100
50
40
260
80
60
50
40
30
160
110
30
20
20
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1. Foulds P, et al (2010) Nature Rev Neurol 6: 359-361.
2. Tokuda T, et al (2010) Neurology (in press).
3. El-Agnaf OMA, et al. (2006) FASEB J 20: 419-425
4. Salem SA, et al (2007) Brain Res. 1170, 103-111.
80
1/125
1/150
1/175
1/200
1/225
1/250
This work is supported by an EC Framework 7 Marie Curie Fellowship
Training Network Grant (NEURASYNC) on ‘α-Synuclein-Related Brain
Diseases ‘. We also wish to thank Dr. Takashi Kasai for help with the
project, Prof. David Mann (Manchester) for blood plasma samples,
and Dr. Fiona Benson for the Pet11a expression system.