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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
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。
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 coimmunoprecipitation, 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.
［1］Stemer A, Lyden P. Evolution of the thrombolytic treatment window for acute ischemic stroke ［J］. Curr Neurol Neurosci Rep, 2010, 10(1): 29-33.
［2］Marsh JD, Keyrouz SG. Stroke prevention and treatment ［J］. J Am Coll Cardiol, 2010, 56(9): 683-691.
［3］Gao C, Cai Y, Zhang X, et al. Ischemic preconditioning mediates neuroprotection against ischemia in mouse hippocampal CA1 neurons by inducing autophagy ［J］. PLoS One, 2015, 10(9): e0137146.
［4］Parmar J, Jones NM. Hypoxic preconditioning can reduce injury-induced inflammatory processes in the neonatal rat brain ［J］. Int J Dev Neurosci, 2015, 43: 35-42.
［5］Poinsatte K, Selvaraj UM, Ortega SB, et al. Quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice ［J］. J Vis Exp, 2015, (99): e52675.
［6］Zhang N, Yin Y, Han S, et al. Hypoxic preconditioning induced neuroprotection against cerebral ischemic injuries and its cPKCgamma-mediated molecular mechanism ［J］. Neurochem Int, 2011, 58 (6): 684-692.
［7］Nikolaev M, Heggelund P. Functions of synapsins in corticothalamic facilitation: important roles of synapsin I ［J］. J Physiol,2015,593(19):4499-4510.
［8］Brenes O, Giachello CN, Corradi AM, et al. Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release ［J］.J Neurosci Res, 2015, 93(10):1492-1506.
［9］Cesca F, Baldelli P, Valtorta F, et al. The synapsins: key actors of synapse function and plasticity ［J］. Prog Neurobiol, 2010, 91(4): 313-348.
［10］Giachello CN, Fiumara F, Giacomini C, et al. MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity ［J］. J Cell Sci, 2010, 123(Pt 6): 881-893.
［11］Jovanovic JN, Benfenati F, Siow YL, et al. Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions ［J］. Proc Natl Acad Sci USA,1996, 93(8): 3679-3683.
［12］Matsubara M, Kusubata M, Ishiguroi K, et al. Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions ［J］.J Biol Chem,1996, 271(35): 21108-21113.
［13］Li JF, Zhang XX, Zhong J, et al. Endogenous cPKC gamma protein levels affect cerebral infarction volume and neurological outcome of mice with ischemic stroke ［J］. Basic ＆ Clinical Medicine, 2013, 33(11): 1452-1459.(in Chinese)
李洁霏, 章欣欣, 钟洁, 等. 脑内源性cPKCgamma 水平影响缺血性卒中小鼠脑梗死体积和神经功能损伤 ［J］. 基础医学与临床, 2013, 33(11): 1452-1459.
［14］Zhang XX, Yang X, Ma W, et al. Role of conventional protein kinase Cγ and its interacting protein 14-3-3γ in oxygen-glucose deprivation-induced ischemic injury of primary cultured mouse cortical neurons ［J］.Acta Anatomica Sinica, 2015, 46(2): 145-150. (in Chinese)
章欣欣, 杨璇, 马维, 等. 经典型蛋白激酶Cγ及其相互作用蛋白14-3-3γ 在原代培养小鼠脑皮层神经元氧-糖剥夺缺血损伤中的作用 ［J］.解剖学报, 2015, 46(2): 145-150.
［15］Bolay H, Gürsoy-Ozdemir Y, Sara Y, et al. Persistent defect in transmitter release and synapsin phosphorylation in cerebral cortex after transient moderate ischemic injury ［J］. Stroke, 2002, 33(5): 1369-1375.
［16］Jung YJ, Park SJ, Park JS, et al. Glucose/oxygen deprivation induces the alteration of synapsin I and phosphosynapsin ［J］. Brain Res, 2004, 996(1): 47-54.
［17］Yin Y, Huang P, Han Z, et al. Collagen nanofibers facilitated presynaptic maturation in differentiated neurons from spinal-cord-derived neural stem cells through MAPK/ERK1/2-Synapsin I signaling pathway ［J］. Biomacromolecules, 2014, 15(7): 2449-2460.
［18］Vara H, Onofri F, Benfenati F, et al. ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I ［J］. Proc Natl Acad Sci USA, 2009, 106(24): 9872-9877.
［19］Severin SE, Moskvitina EL. Polyphosphoinositides as activators of PKC-dependent synapsin I phosphorylation ［J］. FEBS Lett, 1991, 282(2): 217-219.
［20］Liao MH, Xiang YC, Huang JY, et al. The disturbance of hippocampal CaMKⅡ/PKA/PKC phosphorylation in early experimental diabetes mellitus ［J］. CNS Neurosci Ther, 2013, 19(5): 329-336
［21］Stein ES, Itsekson-Hayosh Z, Aronovich A, et al. Thrombin induces ischemic LTP (iLTP): implications for synaptic plasticity in the acute phase of ischemic stroke ［J］. Sci Rep, 2015, 5: 7912.
［22］Leitmeyer K, Glutz A, Radojevic V, et al. Inhibition of mTOR by Rapamycin results in auditory hair cell damage and decreased spiral ganglion neuron outgrowth and neurite formation in vitro ［J］. Biomed Res Int, 2015, 2015: 925890.
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
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