Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma

Background: Neuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-alpha) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-alpha induces retinal ganglion cell (RGC) hyperexcitability and injury. Methods: Whole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ currents in RGCs. Both intravitreal injection of TNF-alpha and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-alpha effects on RGCs. Results: Intravitreal injection of soluble TNF-alpha significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-alpha receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-alpha-induced effects, suggesting that TNF-alpha may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-alpha-injected retinas, Na+ current densities were upregulated. Perfusing TNF-alpha in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-alpha selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-alpha upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-kappa B inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-alpha-induced RGC apoptosis. Conclusions: TNF-alpha/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na+ channels, thus contributing to RGC apoptosis in glaucoma.