The interactome of stabilized α-synuclein oligomers and neuronal proteins

While it is generally accepted that alpha-synuclein oligomers (alpha SOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes alpha SOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative alpha SO binding partners in rat primary neurons using DHA-stabilized human alpha SOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the alpha SOs-binding partners. We identify in total 29 proteins associated with DHA-alpha SO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium-potassium pump (Na+/K+ ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified alpha SOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between alpha SOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic alpha SOs.