Identification of Synaptosomal Proteins Binding to Monomeric and Oligomeric α-Synuclein

Monomeric alpha-synuclein (alpha SN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson's disease (PD) and dementia with Lewy body (DLB), alpha SN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the alpha SN gene suggest a gain of function associated to aggregated alpha SN. We have conducted a proteomic screen to identify the alpha SN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human aSN monomers or oligomers as bait. The isolated alpha SN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of alpha SN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer alpha SN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of alpha SN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological alpha SN dependent processes.