Multiomic Analysis Reveals Molecular Pathways Associated with Intestinal Aggregation of α-Synuclein.

Aggregates of the protein α-synuclein may initially form in the gut before propagating to the brain in Parkinson's disease (PD). Indeed, our prior work supports that enteroendocrine cells, specialized intestinal epithelial cells, could play a key role in the development of this disease. Enteroendocrine cells natively express α-synuclein and form synapses with enteric neurons as well as the vagus nerve. Severing the vagus nerve reduces the load of α-synuclein aggregates in the brain, suggesting that this nerve is a conduit for gut-to-brain spread. Enteroendocrine cells line the gut lumen; as such, they are in constant contact with metabolites of the gut microbiota. We previously found that when enteroendocrine cells are exposed to nitrite─a potent oxidant produced by gut bacterial Enterobacteriaceae─a biochemical pathway is initiated that results in α-synuclein aggregation. Here, we detail the cellular and molecular mechanisms involved. First, we holistically profiled nitrite-exposed enteroendocrine cells through untargeted proteomics. Next, we performed targeted analyses that specifically probed the mechanistic role of dopamine, as our prior findings suggested that dopamine is critical for nitrite-induced α-synuclein aggregation. In dopamine-free HeLa cells treated with nitrite, α-synuclein aggregation was indeed suppressed. Proteomic signatures in dopamine-free cells treated with nitrite were distinct from those in nitrite-treated enteroendocrine cells, highlighting pathways relevant to intestinal development of PD. Intriguingly, we observed that enteroendocrine cells maintain viability upon exposure to nitrite and in the presence of α-synuclein aggregates. This cellular robustness suggests that these cells may be a reservoir of toxic α-synuclein aggregates. As a possible antidote, our findings show that benserazide and α-methyl tyrosine─chemical inhibitors of dopamine biosynthesis─limited aggregation. Curious about mechanisms of disease etiology outside of α-synuclein aggregation, we also profiled the enteroendocrine cell lipidome─an emerging area of interest in PD research─to motivate future targeted studies delineating the roles of dysregulated lipid metabolism in disease onset. Overall, these studies lay a foundation for mechanistically informed therapeutic targets to prevent the intestinal formation of α-synuclein aggregates before they spread to the brain.