Yang Xuan, Zhang Xinxin, Li Yun, Han Song, Howells David W, Li Shujuan, Li Junfa
Department of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China.
Florey Institute of Neuroscience and Mental Health, Melbourne, Victoria, Australia.
J Neurochem. 2016 May;137(3):446-59. doi: 10.1111/jnc.13538. Epub 2016 Feb 11.
We previously reported that conventional protein kinase C (cPKC)β participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCβ interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCβ, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCβ-mediated CRMP2 phosphorylation via the cPKCβ-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCβ activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCβ modulates CRMP2 phosphorylation and proteolysis, and cPKCβ activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)β-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.
我们之前报道过，传统蛋白激酶C（cPKC）β参与缺氧预处理诱导的针对脑缺血损伤的神经保护作用，并且塌陷反应介导蛋白2（CRMP2）被鉴定为一种cPKCβ相互作用蛋白。在本研究中，我们探究了cPKCβ对CRMP2磷酸化和蛋白水解的调控作用，以及它们在氧糖剥夺（OGD）处理的皮质神经元和大脑中动脉闭塞诱导的缺血性脑卒中小鼠的缺血损伤中的作用。结果表明，cPKCβ通过cPKCβ选择性激活剂12-脱氧佛波醇13-苯乙酸20-乙酸酯（DOPPA）介导CRMP2磷酸化，并且通过钙蛋白酶抑制剂和一种含有TAT肽及CRMP2钙蛋白酶切割位点的融合肽（TAT-CRMP2）抑制钙蛋白酶介导的CRMP2蛋白水解，从而通过抑制培养的皮质神经元中的CRMP2蛋白水解来保护神经元免受OGD诱导的细胞死亡。在皮质神经元中，DOPPA、钙蛋白酶抑制剂和TAT-CRMP2抑制了OGD诱导的CRMP2降解产物的核转位。此外，缺氧预处理以及脑室内注射DOPPA、钙蛋白酶抑制剂和TAT-CRMP2对cPKCβ的激活和CRMP2蛋白水解的抑制，除了减少缺血性脑卒中小鼠梗死体积和梗死周围区域固缩核细胞比例外，还改善了神经功能缺损。这些结果表明，cPKCβ调节CRMP2磷酸化和蛋白水解，并且cPKCβ激活通过磷酸化抑制CRMP2蛋白水解，从而减轻培养的皮质神经元和缺血性脑卒中小鼠大脑中的缺血损伤。局灶性脑缺血诱导大量Ca(2+)内流以激活钙蛋白酶，后者将塌陷反应介导蛋白（CRMP）2切割成降解产物（BDP）。钙蛋白酶抑制剂和TAT-CRMP2对CRMP2切割的抑制减轻了缺血损伤。传统蛋白激酶C（cPKC）β介导的磷酸化可抑制CRMP2蛋白水解，并减轻培养的皮质神经元和缺血性脑卒中诱导小鼠的缺血损伤。
