In Situ Peroxidase Labeling and Mass-Spectrometry Connects Alpha-Synuclein Directly to Endocytic Trafficking and mRNA Metabolism in Neurons

Synucleinopathies, including Parkinson's disease (PD), are associated with the misfolding and mistrafficking of alpha-synuclein (alpha-syn). Here, using an ascorbate peroxidase (APEX)-based labeling method combined with mass spectrometry, we defined a network of proteins in the immediate vicinity of alpha-syn in living neurons to shed light on alpha-syn function. This approach identified 225 proteins, including synaptic proteins, proteins involved in endocytic vesicle trafficking, the retromer complex, phosphatases and mRNA binding proteins. Many were in complexes with alpha-syn, and some were encoded by genes known to be risk factors for PD and other neurodegenerative diseases. Endocytic trafficking and mRNA translation proteins within this spatial alpha-syn map overlapped with genetic modifiers of alpha-syn toxicity, developed in an accompanying study (Khurana et al., this issue of Cell Systems). Our data suggest that perturbation of these particular pathways is directly related to the spatial localization of alpha-syn within the cell. These approaches provide new avenues to systematically examine protein function and pathology in living cells.