Discovery of Sennoside A as a Natural O-GlcNAcase Inhibitor That Ameliorates Parkinson's Disease by Restoring EphrinB2 O-GlcNAcylation.

Down-regulated O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) has been implicated in Parkinson's disease (PD), and restoring O-GlcNAcylation levels in the central nervous system via O-GlcNAcase (OGA) suppression represents a potential therapeutic strategy for PD intervention. Nevertheless, naturally derived OGA inhibitors remain insufficiently explored, and the molecular mechanisms underlying OGA-inhibition-mediated restoration of O-GlcNAcylation to alleviate PD remain poorly defined. Here, a fluorescence-based high-throughput inhibitor screening platform was established and subsequently used to identify sennoside A (SA) as a potent OGA inhibitor from an in-house phytochemical library. As a competitive OGA inhibitor, SA exhibited potent anti-OGA activity both in vitro and in vivo, along with favorable blood-brain barrier permeability and a well-tolerated safety profile. Moreover, SA markedly elevated protein O-GlcNAcylation levels, which in turn attenuated neuroinflammatory response and ameliorated key PD-associated pathological and behavioral deficits. Mechanistically, SA directly inhibits OGA activity in neurons and microglia, predominantly enhancing the O-GlcNAcylation of EphrinB2 at the Ser40/Thr183 sites and stabilizing EphrinB2. This site-specific modification activates the EphrinB2-EphB4 signaling cascade and subsequently blunts mitogen-activated protein kinase-mediated neuroinflammation. Disruption of EphrinB2 O-GlcNAcylation by Ser40A/Thr183A mutation eliminates its neuroprotective function and abolishes the anti-PD effects of SA. Collectively, this work establishes the pharmacological inhibition of OGA as a promising therapeutic strategy for PD and identifies SA as a naturally occurring anti-PD agent that alleviates PD-associated pathological phenotypes by inhibiting OGA-mediated de-O-GlcNAcylation of EphrinB2.