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

Abstract

Monomeric α-synuclein (αSN) 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), αSN 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 αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN 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 αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN 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 αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

“…We hypothesised that aggregated AS binds directly to and stimulates the predominant Ca 2+ pumps in the plasma membrane and endoplasmic reticulum, PMCA and SERCA, because total AS levels remain fairly stable in our cell models and the decrease in cytosolic Ca 2+ is prevented by the aggregation inhibitor ASI-1D. Co-immunoprecipitation experiments followed by proteomic techniques have demonstrated that PMCA from porcine brain binds both monomer and oligomeric AS [20]. Likewise, SERCA was found to bind AS by pull-down experiments; however, pronounced selectivity was seen with strong binding of aggregated AS to SERCA in contrast to very low binding of monomeric AS.…”

Stabilization of α-synuclein oligomers using formaldehyde

“…We successfully cross-linked α-syn oligomers using FA, thereby increasing stability towards SDS and urea denaturation. This oligomer preparation binds neuronal proteins in a conformational specific manner [21, 22], and also bind the two conformational specific antibodies FILA and MJF14. The FA mediated crosslinking did not disturb the overall twisted ribbon-like oligomeric structure, the size of the oligomers, their antigenicity toward FILA and MJF14 or the crosslinked oligomers applicability as calibrators in an α-syn oligomer specific ELISA assay [23].…”

“…Production of α-syn and α-syn oligomers was performed as previously described [19, 21, 32]. In short, purified α-syn was dissolved in PBS to a final concentration of 10mg/mL and incubated on ice for 30 minutes while vortexed regularly.…”

Proteomics; applications in familial Parkinson's disease

“…potentially pathogenic, a‐synuclein (Betzer et al . ). Again, label‐free quantification was used and, interestingly, there were fewer proteins that specifically interacted with monomeric compared to oligomeric a‐synuclein.…”