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Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia
Published on: July 31, 2017
Inhibition of PKCγ phosphorylation protects against cerebral ischemia-reperfusion injury
Chenchen Li1, Jinlun Chen1, Xiangbin Ouyang1
1Department of Neurology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China; Clinical Medical Research Center for Stroke Prevention and Treatment of Hunan Province, Department of Neurology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Abstract:
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.
Insights
Phosphorylation of PKCγ at T655 drives mitochondrial dysfunction and neuronal apoptosis in cerebral ischemia-reperfusion injury. Targeting this PKCγ pathway offers a potential therapeutic strategy for ischemic stroke.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Cerebral ischemia-reperfusion (I/R) injury is a leading cause of stroke, characterized by mitochondrial dysfunction, oxidative stress, and apoptosis.
- Protein Kinase C gamma (PKCγ) has been implicated in various cellular processes, but its specific role in cerebral I/R injury remains unclear.
Purpose of the Study:
- To investigate the role of PKCγ phosphorylation at the T655 site in cerebral I/R injury.
- To elucidate the functional impact of PKCγ on mitochondrial function, oxidative stress, and neuronal apoptosis.
- To evaluate the therapeutic potential of targeting the PKCγ pathway in ischemic stroke.
Main Methods:
- Mass spectrometry to identify PKCγ phosphorylation sites.
- In vitro and in vivo studies using PKCγ mutants and knockdown models.
- Mitochondrial function assays, ROS production measurements, and apoptosis assessments.
- Nuclear/cytoplasmic fractionation and immunofluorescence analyses.
- In vivo stroke models with behavioral assessments.
Main Results:
- PKCγ is phosphorylated at T655 and translocates to mitochondria following cerebral I/R injury.
- PKCγ kinase activity is crucial for its regulatory effects on Nrf2 and mitochondrial function.
- T655 phosphorylation promotes mitochondrial dysfunction, oxidative stress, and neuronal apoptosis.
- PKCγ knockdown alleviates I/R injury, reduces infarct volume, and improves neurological outcomes.
- PKCγ regulates Nrf2 nuclear translocation in neuronal cells.
Conclusions:
- T655 phosphorylation of PKCγ is a key mechanism mediating mitochondrial dysfunction and oxidative stress in cerebral I/R injury.
- Targeting the PKCγ T655 phosphorylation pathway presents a promising therapeutic strategy for ischemic stroke.
