GlyT1 inhibition promotes post-ischemic neuroprotection in the MCAO model

Glycine transporter type 1 (GlyT1) regulates extracellular glycine levels and modulates N-methyl-D-aspartate receptor (NMDAR) activity, positioning it as a promising target in excitotoxic and ischemic conditions. While previous studies have shown that GlyT1 inhibition prior to injury confers neuroprotection, its therapeutic potential in a post-ischemic context remains unclear. Here, we investigated the neuroprotective effects of NFPS, a selective GlyT1 inhibitor, administered after the induction of permanent middle cerebral artery occlusion (MCAO) in mice. NFPS (1.25, 2.5, or 5.0 mg/kg, i.p.) was administered once daily for three days, beginning 24 h post-ischemia. NFPS treatment significantly reduced infarct volume and improved motor function in a dose-dependent manner. Additionally, NFPS reduced reactive oxygen species, nitric oxide, and lipid peroxidation, alongside adjustments in antioxidant enzymes and glutathione-related activity. Proteomic analysis of cortical tissue from healthy mice treated with NFPS revealed enrichment of pathways related to glutamatergic synapse, Parkinson disease, and dopaminergic synapse pathways, suggesting modulation of synaptic plasticity and metabolic resilience. Western blot analysis confirmed an increase in GluN2A and a decrease in GluN2B expression, consistent with a shift toward prosurvival NMDAR signaling. Collectively, these findings demonstrate that post-ischemic GlyT1 inhibition with NFPS confers robust neuroprotection through coordinated regulation of excitotoxicity and oxidative stress, supporting its potential as a therapeutic strategy for ischemic stroke.