Decoding the nitration-phosphorylation switch: 7,8-Dihydroxyflavone rebalances TrkB signaling dynamics for retinal neuroprotection.

PURPOSE: To investigate how 7,8-Dihydroxyflavone (7,8-DHF) coordinates competing post-translational modifications of TrkB, specifically examining its role in modulating the redox-sensitive balance between tyrosine nitration and phosphorylation to achieve neuroprotective efficacy.

METHODS

The effects of 7,8-DHF on nitric oxide (NO) inhibition, protein nitration, TrkB activation, and downstream signaling were assessed using a male mouse optic nerve crush (ONC) model and 3D retinal explants. Techniques such as Western blotting, ELISA, immunoprecipitation, immunohistochemistry, metabolomics, and molecular docking were used to examine the biochemical and molecular impacts of 7,8-DHF.

RESULTS

7,8-DHF reduced NO levels and inducible/neuronal nitric oxide synthase (iNOS/nNOS) expression in 3D retinal cultures and ONC mouse models, with molecular docking indicating its binding to iNOS's active site and metabolomics showing downregulation of the arginine biosynthesis pathway. It also decreased TrkB nitration in vivo and in vitro, with the Y816 residue being critical for this modification. 7,8-DHF enhanced TrkB phosphorylation at Y816 and PLCγ1 activation, inhibited by L-arginine and TrkB blockers. Additionally, 7,8-DHF promoted retinal gangion cell survival and axonal regeneration via TrkB signaling, effects reversed by nitration agents or TrkB blockers.

CONCLUSION

7,8-DHF pioneers a new class of nitration-phosphorylation modulators, converting nitrosative damage into neuroregenerative signals by rebalancing TrkB's modification states. This mechanistic insight opens therapeutic avenues for optic nerve disorders.