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. Results 1. α-Syn was expressed in E. Coli using plasmid Pet11a and purified using MonoQ and Superdex columns. MonoQ Column Superdex Column 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. 3. Syn1 detected a band corresponding to α-syn in small amounts of whole human blood plasma. 4. The sensitivity and specificity of apolipoprotein A1, B and E antibodies were examined on immunoblots. Apo-E Apo-A1 Apo-B WCE C13 C3-4 C1-2 B5-6 B3-4 B1-2 A9 A8 MM α-Syn T2 T0 A15A14A13A12A8A7T1MM α-Syn ng 5 ng 2.5ng0.5ng α-Syn /1001/2001/3001/4001/5001/600R-SynIgG /100 1/125 1/150 1/175 1/200 1/2251/ /1001/1251/1501/ /175 1/150 1/125 1/100 Next Steps 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. 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. 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 α- syn 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. References and Acknowledgements 1. Foulds P, et al (2010) Nature Rev Neurol 6: Tokuda T, et al (2010) Neurology (in press). 3. El-Agnaf OMA, et al. (2006) FASEB J 20: Salem SA, et al (2007) Brain Res. 1170, 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.