Tetrahydrobiopterin Supplementation Protects Retinal Ganglion Cells Against NMDA-Induced Excitotoxicity Through nNOS-Coupled Nitric Oxide Signaling

Purpose: Excitotoxic injury triggered by excessive N-methyl-d-aspartate (NMDA) receptor activation represents an important mechanism underlying retinal neurodegeneration in glaucoma. Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS), exhibits neuroprotective potential in multiple neurodegenerative diseases, but its role in retinal ganglion cells (RGCs) under excitotoxic injury remains unclear. This study assessed the neuroprotective effects of BH4 against excitotoxic retinal injury and explored its underlying mechanisms. Methods: Glutamate-treated R28 cells and intravitreal NMDA-injected rat retinas were used as in vitro and in vivo excitotoxic injury models, respectively. BH4 levels were measured after injury. Cell viability, oxidative stress, apoptosis, RGC survival, retinal morphology, visual function, and NOS activity were assessed following BH4 supplementation. Results: BH4 levels were significantly reduced in glutamate-injured R28 cells and NMDA-damaged retinas. BH4 supplementation improved cell survival, reduced oxidative stress, and attenuated apoptosis in injured R28 cells. In vivo, BH4 preserved RGC survival, alleviated retinal damage, and improved visual function after NMDA injury. Mechanistically, BH4 restored neuronal nitric oxide synthase (nNOS) coupling and activity, increased nitric oxide levels, re-established nitroso-redox homeostasis, and suppressed apoptotic signaling. Conclusions: BH4 deficiency contributes to glutamate-induced retinal neurodegeneration by promoting nNOS uncoupling and nitroso-redox imbalance. Restoring BH4 levels protects against excitotoxic RGC damage, suggesting the BH4-nNOS axis as a potential therapeutic target for retinal neurodegeneration.