增强自噬激活p38/MEF2C通路调节突触相关蛋白的表达改善孤独症大鼠的症状
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摘要
目的研究孤独症大鼠前额叶皮质中自噬干预前后p38/肌细胞增强子因子2C(p38/MEF2C)通路调控突触的机制。方法采用Wistar大鼠,孕12.5 d丙戊酸腹腔注射诱导孤独症动物模型,分别用生理盐水或丙戊酸(VPA)处理。生理盐水组产生的后代为对照组, VPA处理组产生的后代随机分为模型组、 5 mg/kg 3-甲基腺嘌呤(3-MA)组、 5 mg/kg雷帕霉素(Rap)自噬增强组,各组处理时间从出生第35天至出生第42天。Western blot法检测大鼠前额叶皮质组织p38、磷酸化的p38(p-p38)、 MEF2C、突触小泡蛋白(SYN)、突触后致密蛋白95(PSD-95)、桥尾蛋白(gephyrin)的蛋白水平,免疫组织化学染色法检测前额叶皮质组织SYN、 PSD-95和gephyrin的表达和分布并进行半定量分析。结果与对照组相比,发育及行为学检测结果显示,模型组发育落后及社交障碍,与模型组相比, Rap组能改善社交障碍, 3-MA组加重社交障碍;与对照组比较,模型组p38、 p-p38、 MEF2C表达下调, SYN、 PSD-95蛋白表达上调, gephyrin表达下调;与模型组比较, Rap组p38、 p-p38、 MEF2C表达上调, SYN、 PSD-95蛋白水平均下调, gephyrin蛋白水平上调,而3-MA组则相反;与对照组相比,模型组大鼠SYN、 PSD-95阳性细胞数增多, gephyrin阳性细胞数减少;与模型组相比, Rap组SYN、 PSD-95阳性细胞数减少, gephyrin阳性细胞数增多, 3-MA组相反。结论孤独症大鼠前额叶皮质中p38/MEF2C信号通路被抑制,通过增强自噬激活p38/MEF2C信号通路可调控突触相关蛋白的表达,改善孤独症行为。
Objective To study the mechanism of p38/myocyte enhancer factor 2C(p38/MEF2C) pathway regulating synapse before and after autophagy intervention in the prefrontal cortex of autistic rats. Methods An animal model of autism was induced by intraperitoneal injection of valproic acid(VPA) at 12.5 days of gestation in Wistar rats. They were treated with normal saline or VPA. The offspring of the saline treatment group served as a control group. The offspring of the VPA treatment group were randomly divided into the model group, 5 mg/kg 3-methyladenine(3-MA) group, and 5 mg/kg rapamycin(Rap) autophagy enhanced group, and the treatment time of each group was from the 35th day of birth to the 42nd day of birth. Western blot analysis was used to detect the protein levels of p38, phosphorylated p38(p-p38), MEF2C, synaptic vesicle protein(SYN), postsynaptic density 95(PSD-95), and gephyrin protein in the prefrontal cortex of rats; immunohistochemical staining was used to detect the expression and distribution of SYN, PSD-95 and gephyrin in the prefrontal cortex, and semi-quantitative analysis was performed then. Results Compared with the control group, the developmental and behavioral test showed that the model group had developmental lag and social disorder. Compared with the model group, the Rap group shoed improved social disorder, and the 3-MA group could aggravate social disorder. Compared with the control group, the expression of p38, p-p38, MEF2C was down-regulated, the expression of SYN and PSD-95 protein was up-regulated, and the expression of gephyrin was down-regulated. Compared with the model group, the expression of p38, p-p38, MEF2C in the Rap group was up-regulated, the levels of SYN and PSD-95 protein were down-regulated, and the level of gephyrin protein was up-regulated, while that in 3-MA group was opposite. Compared with the control group, the number of SYN and PSD-95 positive cells in the model group increased, and the number of gephyrin positive cells decreased. Compared with the model group, the number of SYN and PSD-95 positive cells in the Rap group decreased, and the number of gephyrin-positive cells increased, and the 3-MA group was opposite. Conclusion The p38/MEF2C signaling pathway is inhibited in the prefrontal cortex of rats with autism, which can regulate the expression of synaptic related proteins and improve autistic behavior by enhancing autophagy to activate the p38/MEF2C signaling pathway.
Objective To study the mechanism of p38/myocyte enhancer factor 2C(p38/MEF2C) pathway regulating synapse before and after autophagy intervention in the prefrontal cortex of autistic rats. Methods An animal model of autism was induced by intraperitoneal injection of valproic acid(VPA) at 12.5 days of gestation in Wistar rats. They were treated with normal saline or VPA. The offspring of the saline treatment group served as a control group. The offspring of the VPA treatment group were randomly divided into the model group, 5 mg/kg 3-methyladenine(3-MA) group, and 5 mg/kg rapamycin(Rap) autophagy enhanced group, and the treatment time of each group was from the 35th day of birth to the 42nd day of birth. Western blot analysis was used to detect the protein levels of p38, phosphorylated p38(p-p38), MEF2C, synaptic vesicle protein(SYN), postsynaptic density 95(PSD-95), and gephyrin protein in the prefrontal cortex of rats; immunohistochemical staining was used to detect the expression and distribution of SYN, PSD-95 and gephyrin in the prefrontal cortex, and semi-quantitative analysis was performed then. Results Compared with the control group, the developmental and behavioral test showed that the model group had developmental lag and social disorder. Compared with the model group, the Rap group shoed improved social disorder, and the 3-MA group could aggravate social disorder. Compared with the control group, the expression of p38, p-p38, MEF2C was down-regulated, the expression of SYN and PSD-95 protein was up-regulated, and the expression of gephyrin was down-regulated. Compared with the model group, the expression of p38, p-p38, MEF2C in the Rap group was up-regulated, the levels of SYN and PSD-95 protein were down-regulated, and the level of gephyrin protein was up-regulated, while that in 3-MA group was opposite. Compared with the control group, the number of SYN and PSD-95 positive cells in the model group increased, and the number of gephyrin positive cells decreased. Compared with the model group, the number of SYN and PSD-95 positive cells in the Rap group decreased, and the number of gephyrin-positive cells increased, and the 3-MA group was opposite. Conclusion The p38/MEF2C signaling pathway is inhibited in the prefrontal cortex of rats with autism, which can regulate the expression of synaptic related proteins and improve autistic behavior by enhancing autophagy to activate the p38/MEF2C signaling pathway.
引文
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[22] Smith K R,Jones K A,Kopeikina K J,et al.Cadherin-10 maintains excitatory/inhibitory ratio through interactions with synaptic proteins[J].J Neurosci,2017,37(46):11127-11139.
[23] Gao R,Penzes P.Common mechanisms of excitatory and inhibitory imbalance in schizophrenia and autism spectrum disorders[J].Curr Mol Med,2015,15(2):146-167.
[24] Lee E,Lee J,Kim E.Excitation/inhibition imbalance in animal models of autism spectrum disorders[J].Biol Psychiatry,2017,81(10):838-847.
[25] Yizhar O,Fenno L E,Prigge M,et al.Neocortical excitation/inhibition balance in information processing and social dysfunction[J].Nature,2011,477(7363):171-178.
[26] Kim H J,Cho M H,Shim W H,et al.Deficient autophagy in microglia impairs synaptic pruning and causes social behavioral defects[J].Mol Psychiatry,2017,22(11):1576-1584.
[27] Guo F,Liu X,Cai H,et al.Autophagy in neurodegenerative diseases:pathogenesis and therapy[J].Brain Pathol,2018,28(1):3-13.
[28] Zhang J,Zhang J X,Zhang Q L.PI3K/AKT/mTOR-mediated autophagy in the development of autism spectrum disorder[J].Brain Res Bull,2016,125:152-158.
[29] Li X G,Du J H,Lu Y,et al.Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling[J].Neural Regen Res,2019,14(4):721-727.
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[31] Tsai N P,Wilkerson J R,Guo W,et al.FMRP-dependent Mdm2 dephosphorylation is required for MEF2-induced synapse elimination[J].Hum Mol Genet,2017,26(2):293-304.
[2] 陈丽萍,吴海涛.突触异常发育在孤独症谱系障碍发生机制中的研究进展[J].中国药理学与毒理学杂志,2017,31(12):19-31.Chen L,Wu H.Advances in the development of synaptic abnormalities in the pathogenesis of autism spectrum disorders[J].Zhongguo Yao Li Xue Yu Du Li Xue Za Zhi,2017,31(12):19-31.
[3] Zhang B,Gokce O,Hale W D,et al.Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses[J].J Exp Med,2018,215(6):1543-1553.
[4] Wang J,Gong J,Li L,et al.Neurexin gene family variants as risk factors for autism spectrum disorder[J].Autism Res,2018,11(1):37-43.
[5] Amal H,Barak B,Bhat V,et al.Shank3 mutation in a mouse model of autism leads to changes in the s-nitroso-proteome and affects key proteins involved in vesicle release and synaptic function[J].Mol Psychiatry,2018 Jul 9.DOI:10.1038/s41380-018-0113-6.[Epub ahead of print].
[6] Jedlicka P,Muellerleile J,Schwarzacher S W.Synaptic plasticity and excitation-inhibition balance in the dentate gyrus:insights from in vivo recordings in neuroligin-1,neuroligin-2,and collybistin knockouts[J/OL].Neural Plast,2018,2018:6015753.DOI:10.1155/2018/6015753.eCollection 2018.
[7] Vijayan V,Verstreken P.Autophagy in the presynaptic compartment in health and disease[J].J Cell Biol,2017,216(7):1895-1906.
[8] Winden K D,Ebrahimi-Fakhari D,Sahin M.Abnormal mTOR activation in autism[J].Annu Rev Neurosci,2018,41:1-23.
[9] Comins C,Simpson G R,Rogers W,et al.Synergistic antitumour effects of rapamycin and oncolytic reovirus[J].Cancer Gene Ther,2018,25(5/6):148-160.
[10] Paoletti E.mTOR inhibition and cardiovascular diseases:cardiac hypertrophy[J].Transplantation,2018,102(2S Suppl 1):S41-S43.
[11] 秦利燕,戴旭芳.雷帕霉素对自闭症大鼠病症行为的改善作用[J].第三军医大学学报,2015,37(5):420-424.Qin L,Dai X.Role of rapamycin in improvement of autistic rats[J].Di San Jun Yi Da Xue Xue Bao,2015,37(5):420-424.
[12] Kong N R,Davis M,Chai L,et al.MEF2C and EBF1 co-regulate B cell-specific transcription[J/OL].PLoS Genet,2016,12(2):e1005845.DOI:10.1371/journal.pgen.1005845.eCollection 2016 Feb.
[13] Harrington A J,Raissi A,Rajkovich K,et al.MEF2C regulates cortical inhibitory and excitatory synapses and behaviors relevant to neurodevelopmental disorders[J/OL].Elife,2016,5:e20059.DOI:10.7554/eLife.20059.
[14] Schneider T,Przew?ocki R.Behavioral alterations in rats prenatally exposed to valproic acid:animal model of autism[J].Neuropsychopharmacology,2005,30(1):80-89.
[15] Yan Y,Zhang Y X,Fang W F,et al.Roles of immunohistochemical staining in diagnosing pulmonary squamous cell carcinoma[J].Asian Pac J Cancer Prev,2015,16(2):551-557.
[16] Wang R,Tan J,Guo J,et al.Aberrant development and synaptic transmission of cerebellar cortex in a VPA induced mouse autism model[J/OL].Front Cell Neurosci,2018,12:500.DOI:10.3389/fncel.2018.00500.eCollection2018.
[17] Nicolini C,Fahnestock M.The valproic acid-induced rodent model of autism[J].Exp Neurol,2018,299(Pt A):217-227.
[18] Chen J,Lei L,Tian L,et al.Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA):an aquatic model for autism[J].Neurotoxicol Teratol,2018,66:8-16.
[19] Daghsni M,Rima M,Fajloun Z,et al.Autism throughout genetics:Perusal of the implication of ion channels[J/OL].Brain Behav,2018,8(8):e00978.DOI:10.1002/brb3.978.Epub 2018 Jun 22.
[20] 陈运华,勾云,周波,等.孤独症大鼠前额叶皮质突触相关蛋白的表达[J].中国儿童保健杂志,2017,25(5):470-473.Chen Y,Gou Y,Zhou B,et al.Expression of synaptic related proteins in prefrontal cortex of autistic rats[J].Zhongguo Er Tong Bao Jian Za Zhi,2017,25(5):470-473.
[21] Codagnone M G,Podestá M F,Uccelli N A,et al.Differential local connectivity and neuroinflammation profiles in the medial prefrontal cortex and hippocampus in the valproic acid rat model of autism[J].Dev Neurosci,2015,37(3):215-231.
[22] Smith K R,Jones K A,Kopeikina K J,et al.Cadherin-10 maintains excitatory/inhibitory ratio through interactions with synaptic proteins[J].J Neurosci,2017,37(46):11127-11139.
[23] Gao R,Penzes P.Common mechanisms of excitatory and inhibitory imbalance in schizophrenia and autism spectrum disorders[J].Curr Mol Med,2015,15(2):146-167.
[24] Lee E,Lee J,Kim E.Excitation/inhibition imbalance in animal models of autism spectrum disorders[J].Biol Psychiatry,2017,81(10):838-847.
[25] Yizhar O,Fenno L E,Prigge M,et al.Neocortical excitation/inhibition balance in information processing and social dysfunction[J].Nature,2011,477(7363):171-178.
[26] Kim H J,Cho M H,Shim W H,et al.Deficient autophagy in microglia impairs synaptic pruning and causes social behavioral defects[J].Mol Psychiatry,2017,22(11):1576-1584.
[27] Guo F,Liu X,Cai H,et al.Autophagy in neurodegenerative diseases:pathogenesis and therapy[J].Brain Pathol,2018,28(1):3-13.
[28] Zhang J,Zhang J X,Zhang Q L.PI3K/AKT/mTOR-mediated autophagy in the development of autism spectrum disorder[J].Brain Res Bull,2016,125:152-158.
[29] Li X G,Du J H,Lu Y,et al.Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling[J].Neural Regen Res,2019,14(4):721-727.
[30] Westmark C J.FMRP:a triple threat to PSD-95[J/OL].Front Cell Neurosci,2013,7:57.DOI:10.3389/fncel.2013.00057.eCollection 2013.
[31] Tsai N P,Wilkerson J R,Guo W,et al.FMRP-dependent Mdm2 dephosphorylation is required for MEF2-induced synapse elimination[J].Hum Mol Genet,2017,26(2):293-304.