经典型蛋白激酶Cγ对突触蛋白-Ⅰ磷酸化调节及其在脑缺血损伤中的作用

doi:10.16098/j.issn.0529-1356.2016.02.001

解剖学报 ›› 2016, Vol. ›› Issue (2): 145-151.doi: 10.16098/j.issn.0529-1356.2016.02.001

• 神经生物学 • 下一篇

经典型蛋白激酶Cγ对突触蛋白-Ⅰ磷酸化调节及其在脑缺血损伤中的作用

朱弘倚¹ 张楠² 于津泠¹魏海萍¹ 韩松¹李俊发^1*

1.首都医科大学神经生物学系，北京脑重大疾病研究院，北京 100069; 2.首都医科大学人体解剖学与组织学胚胎学系，北京 100069

收稿日期:2015-09-15 修回日期:2015-11-03 出版日期:2016-04-06 发布日期:2016-04-06
通讯作者: 李俊发 E-mail:junfali@ccmu.edu.cn
基金资助:
特定 microRNA 和 PKC 亚型特异性信号蛋白对脑卒中小鼠神经保护作用及其调节机制;蛋白激酶Cgamma在缺血性脑卒中鼠脑皮层神经元缺血损伤中作用及其分子机制研究;缺血性脑卒中内源性神经保护机制和干预措施及生物分子标记物研究;人类离体缺血性脑卒中：一种用于候选药物筛选和更新PKC亚型特异性信号通路的测试平台;蛋白激酶Cγ对缺血性卒中脑保护作用及其机制研究

Regulation of conventional protein kinase Cγ on synapsin-Ⅰ phosphorylation and their role in cerebral ischemic injury

ZHU Hong-yi¹ ZHANG Nan² YU Jin-ling¹ WEI Hai-ping¹ HAN Song¹ LI Jun-fa^1*

1. Department of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, China; 2.Department of Anatomy,Histology and Embryology, Capital Medical University, Beijing 100069, China

Received:2015-09-15 Revised:2015-11-03 Online:2016-04-06 Published:2016-04-06
Contact: LI Jun-fa E-mail:junfali@ccmu.edu.cn

摘要/Abstract

摘要：

目的探讨经典型蛋白激酶Cγ（cPKCγ）对突触蛋白-Ⅰ磷酸化水平的影响及其在小鼠原代脑皮层神经元氧-糖剥夺(OGD)缺血损伤中作用。方法借助cPKCγ 基因敲除 (cPKCγ -/-) 小鼠，利用小鼠大脑中动脉阻塞（MCAO）在体缺血模型和原代脑皮层神经元OGD 离体细胞缺血模型，采用免疫共沉淀（Co-IP）、蛋白免疫印迹和免疫荧光等生物化学技术，验证cPKCγ与突触蛋白-Ⅰ的相互作用，探讨cPKCγ 对突触蛋白-Ⅰ磷酸化水平的调节及小鼠原代脑皮层神经元OGD 损伤后对神经元形态的影响。结果免疫共沉淀结果证明，正常及缺血小鼠脑皮层组织中cPKCγ 与突触蛋白-Ⅰ 均存在相互作用；对突触蛋白-Ⅰ 5个可能的丝氨酸磷酸化位点进行筛选发现，只有Ser549 和Ser553位点的磷酸化水平在OGD和cPKCγ 敲除前后有明显变化；进一步统计分析得出，野生型(cPKCγ+/+)小鼠原代脑皮层神经元中，OGD 处理使突触蛋白-Ⅰ Ser549 和 Ser553 位点的磷酸化水平显著降低(每组n=5，与野生型常氧组相比P<0.05)，cPKCγ 基因敲除可显著降低上述两个位点的磷酸化水平(每组n=5，与野生型常氧组相比P<0.05)，而OGD 处理后降低趋势更明显(每组n=5，与野生型 OGD 组相比P<0.05)；除此之外，OGD处理可使cPKCγ+/+ 和cPKCγ-/- 脑皮层神经元突起的长度和数目均显著降低(每组n=8，与野生型常氧组相比，P<0.05)，cPKCγ-/- 组降低现象更加显著(每组n=8，与野生型 OGD 组相比，P<0.05)。结论缺血/低氧损伤情况下，cPKCγ 可能通过调节突触蛋白-Ⅰ Ser549 和 Ser553 磷酸化水平影响神经元突起的形态，从而保护神经元免受缺血／低氧损伤。

Abstract:

Objective To explore the regulatory effect of conventional protein kinase Cγ (cPKCγ) on synapsin-Ⅰ phosphorylation level and their role in oxygen-glucose deprivation (OGD) induced ischemic injury in primary cultured cortical neurons of mice. Methods By using the middle cerebral artery occlusion (MCAO) mouse model in vivo and OGD-induced ischemic model of primary cultured cortical neurons in vitro, we examined the interaction and regulation of cPKCγ on synapsin-Ⅰ phosphorylation, and the effect of cPKCγ on synaptic morphology after OGD injury through coimmunoprecipitation, Western blotting and immunofluorescence with the help of cPKCγ knockout (cPKCγ -/-) mice. Results cPKCγ interacted with synapsin-Ⅰ in both the intact and injured cerebral cortex of mice. Five possible serine phosphorylation cites were screened and results showed that only Ser549 and Ser553 were obviously changed after OGD and cPKCγ knockout. The statistic analysis further revealed that OGD insults significantly reduced phosphorylation of synapsin-Ⅰ at Ser549 and Ser553( n=5 per group，P<0.05, compared with cPKCγ +/+ normoxia group). The knockout of cPKCγ decreased synapsin-Ⅰ phosphorylation (n=5 per group, P<0.05, compared with cPKCγ +/+ normoxia group), while OGD further extended the decrease (n=5 per group，P<0.05, compared with cPKCγ +/+ OGD group). In addition, OGD treatment also decreased the length and number of neurites of primary cultured cortical neurons(n=8 per group, P<0.05, compared with cPKCγ +/+ normoxia group), and cPKCγ -/- group worsened the damage of synaptic morphology induced by OGD (n=8 per group, P<0.05, compared with cPKCγ +/+ OGD group). Conclusion cPKCγ may influence the morphology of neurite growth through regulating synapsin-Ⅰ phosphorylation at Ser549 and Ser553, thereby protect the cortical neurons against ischemic/hypoxic injury.

朱弘倚张楠于津泠魏海萍韩松李俊发*. 经典型蛋白激酶Cγ对突触蛋白-Ⅰ磷酸化调节及其在脑缺血损伤中的作用[J]. 解剖学报, 2016, (2): 145-151.

ZHU Hong-yi ZHANG Nan YU Jin-ling WEI Hai-ping HAN Song LI Jun-fa*. Regulation of conventional protein kinase Cγ on synapsin-Ⅰ phosphorylation and their role in cerebral ischemic injury[J]. AAS, 2016, (2): 145-151.

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经典型蛋白激酶Cγ对突触蛋白-Ⅰ磷酸化调节及其在脑缺血损伤中的作用

Regulation of conventional protein kinase Cγ on synapsin-Ⅰ phosphorylation and their role in cerebral ischemic injury

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