Inhibition of PKCγ phosphorylation protects against cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion (I/R) injury is a major cause of stroke-related mortality and disability, primarily driven by mitochondrial dysfunction, oxidative stress, and apoptosis. In this study, we identified phosphorylation of PKCγ at the T655 site following cerebral I/R injury using mass spectrometry. Notably, we observed that approximately 5% of total PKCγ translocates to mitochondria following I/R injury, suggesting a direct role in modulating mitochondrial function. We further investigated the functional role of PKCγ both in vitro and in vivo. Our results demonstrate that the regulatory effects of PKCγ on Nrf2 and mitochondrial function depend on its kinase activity, as evidenced by the lack of effect of the kinase-dead G360S mutant. The phospho-mimetic T655D mutant suppressed Nrf2 nuclear translocation, promoted mitochondrial ROS production, fragmentation, and neuronal apoptosis, whereas the dephospho-mimetic T655A mutant exerted the opposite effects. Nuclear/cytoplasmic fractionation and immunofluorescence analyses further confirmed that PKCγ regulates Nrf2 nuclear translocation in both HeLa cells and primary neurons. Knockdown of PKCγ via shRNA in vitro and AAV9-mediated delivery in mice alleviated mitochondrial dysfunction, reduced infarct volume, and improved neurological outcomes. Behavioral assessments further confirmed the neuroprotective effect of PKCγ knockdown in vivo. Collectively, our findings identify T655 phosphorylation as a key mechanism by which PKCγ regulates mitochondrial dysfunction and oxidative stress during cerebral I/R injury, suggesting that targeting this pathway may represent a promising therapeutic strategy for ischemic stroke.