TLR2 Regulates α-Synuclein Phosphorylation and Release in an Enteroendocrine Cell Model

Parkinson's disease (PD) is characterized by the progressive accumulation and prion-like spread of pathological α-synuclein (α-syn). Increasing evidence suggests that α-syn pathology may originate in the gastrointestinal tract before spreading to the central nervous system; however, the intestinal mechanisms that initiate pathological α-syn phosphorylation and intercellular transfer remain poorly understood. Here, we investigated the role of Toll-like receptor 2 (TLR2) in regulating α-syn phosphorylation using intestinal epithelial cell model. We found that pSer129-positive α-syn immunoreactivity and TLR2 expression progressively increased in the mouse jejunum during aging. Although pSer129-positive structures did not substantially colocalize with chromogranin A-positive enteroendocrine cells, we confirmed that TLR2 is expressed in a subset of these cells in vivo. Furthermore, analysis of single-cell transcriptomic data from mouse enteroendocrine cells and human colonic organoids revealed that a small subset of enteroendocrine cells express TLR2. We therefore used the enteroendocrine cell line STC-1 as an in vitro model to investigate TLR2-dependent α-syn phosphorylation in an enteroendocrine-like context. In STC-1 cells, TLR2 enhanced the cellular uptake of extracellular α-syn preformed fibrils (PFFs), promoted the generation of pSer129 α-syn and higher molecular weight α-syn species, and facilitated the extracellular release of factors that increased pSer129 immunoreactivity in recipient cells. Pharmacological inhibition of TLR2 attenuated PFF uptake and pSer129 α-syn generation. Conditioned medium from TLR2-overexpressing STC-1 cells increased pSer129 α-syn immunoreactivity in recipient neuronal cells, suggesting that TLR2 may promote the release of soluble factors capable of inducing α-syn phosphorylation in recipient cells. The molecular identity of the transferred factor(s) and the underlying mechanism remain to be determined. Collectively, our findings identify TLR2 as a regulator of α-syn uptake, phosphorylation, and release in an enteroendocrine cell model. These results support a potential role for aging-associated upregulation of intestinal TLR2 in modulating α-syn phosphorylation, although the in vivo cellular source of pSer129 α-syn and the contribution of enteroendocrine cells require further investigation.