S. Huebinger, O. Bannach, A. Funke, D. Willbold, E. Birkmann

Introduction: Parkinson's Disease (PD) is one of the most common neurodegenerative diseases in humans, and the most common one associated with motor deficiencies [1]. It mainly affects the elderly with a peak age of onset at around 60 years [2], although familiar forms are known that show an earlier onset of disease. The symptoms are caused by the degeneration of dopaminergic neurons in the brain, which are accompanied by the misfolding and aggregation of the protein α-synuclein. Aims: We established a method called Surface-FIDA (Surface-Fluorescence Intensity Distribution Assay) [3] with the aim to identify and distinguish the monomeric form of α-synuclein from the aggregated form, specifically amyloid fibrils. Method: The α-synuclein was recombinantly expressed in E. coli and afterwards purified. Fibrils were formed in vitro from this purified material. In the Surface-FIDA measurement, α-synuclein is bound to a glass surface via capture antibodies. The synuclein is then labeled with two different detection antibodies which are conjugated with different fluorescent dyes. The fluorescence distribution on the surface can be detected by laser scanning microscopy. Aggregates can be identified as a burst in fluorescence because of their ability to bind many detection antibodies at the same time. Results: First results show significant differences in fluorescence intensity between samples containing aggregated α-synuclein and those with the monomeric form or negative controls. The fluorescence intensity of a measured surface is dependent on the amount of α-synuclein present in the sample and whether it is in monomeric or fibrilar form. Conclusion: With the Surface-FIDA method we are able to specifically detect aggregates from recombinant α-synuclein to differentiate them from monomers. Therefore we present the proof of principle that Surface-FIDA has the potential to become a diagnostic tool for PD using aggregated α-synuclein as a biomarker. [1] Farrer, MJ. (2006) Genetics of Parkinson disease: paradigm shifts and future prospects. Nat. Rev. Genet. 7, 306-18 [2] Fahn, S. (2003) Description of Parkinson's disease as a clinical syndrome. Ann. NY Acad. Sci. 991, 1-14 [3] Birkmann et al. (2007) Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Vet. Microbiol. 123, 294-304

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Fluorescence Microscopy