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Analysis of Dendritic Spine Morphology in Cultured CNS Neurons
Published on: July 13, 2011
PKCε inhibition prevents ischemia‑induced dendritic spine impairment in cultured primary neurons
Chenjie Ge1, Xuefeng Wang2, Yunhong Wang2
1Department of Psychiatry, Huzhou Third Municipal Hospital, The Affiliated Hospital of Huzhou University, Huzhou, Zhejiang 313000, P.R. China.
Abstract:
Brain ischemia is an independent risk factor for Alzheimer's disease (AD); however, the mechanisms underlining ischemic stroke and AD remain unclear. The present study aimed to investigate the function of the ε isoform of protein kinase C (PKCε) in brain ischemia-induced dendritic spine dysfunction to elucidate how brain ischemia causes AD. In the present study, primary hippocampus and cortical neurons were cultured while an oxygen-glucose deprivation (OGD) model was used to simulate brain ischemia. In the OGD cell model, in vitro kinase activity assay was performed to investigate whether the PKCε kinase activity changed after OGD treatment. Confocal microscopy was performed to investigate whether inhibiting PKCε kinase activity protects dendritic spine morphology and function. G-LISA was used to investigate whether small GTPases worked downstream of PKCε. The results showed that PKCε kinase activity was significantly increased following OGD treatment in primary neurons, leading to dendritic spine dysfunction. Pre-treatment with PKCε-inhibiting peptide, which blocks PKCε activity, significantly rescued dendritic spine function following OGD treatment. Furthermore, PKCε could activate Ras homolog gene family member A (RhoA) as a downstream molecule, which mediated OGD-induced dendritic spine morphology changes and caused dendritic spine dysfunction. In conclusion, the present study demonstrated that the PKCε/RhoA signalling pathway is a novel mechanism mediating brain ischemia-induced dendritic spine dysfunction. Developing therapeutic targets for this pathway may protect against and prevent brain ischemia-induced cognitive impairment and AD.

