Mechanisms of alpha-synuclein-dependent neuronal deathrole of n-terminal acetylation and identification of protein interactors

Resumen

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. We investigate the role of a co/post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry (MS) analysis. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Considering the previous results, we hypothesized that protein-protein interactions (PPIs) favored by increased aSyn protein levels, pathological mutations or N-terminal acetylation modulate aSyn toxicity. To identify aSyn-dependent PPIs we used a novel assay based on a promiscuous biotin ligase (BioID2). BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by MS analysis. Biotynilation pulldown experiments and MS analysis were performed with BioID2-tagged aSyn versions (WT, E46K and N-terminal mutants D2P and D2A) to identify the aSyn interactome. With this methodology we detected a specific protein interactor for our most toxic aSyn variant, E46K. Additionally, we found specific interactors for WT and E46K aSyn. Among them, we identified the protein 14-3-3 epsilon. In vitro studies show that it interacts with aSyn and thus validates our approach to detect aSyn protein interactors. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity. Therefore, our work identifies the modulation of aSyn N-terminal acetylation as a potential therapeutic target. Moreover, the BioID2 methodology constitutes a powerful and sensitive approach that has allowed the detection of specific and relevant aSyn protein interactors. Future efforts should be oriented to modulate aSyn N-terminal acetylation and aSyn-dependent protein interactions revealed in this work as potential new therapeutic approaches to treat synucleinopathies.