【作者】 张铮；

【导师】 田波；

【作者基本信息】 华中科技大学 ， 神经生物学， 2018， 博士

【摘要】 研究背景:抑郁症（Major depressive disorder,MDD）是一种常见且容易反复的严重精神失调性疾病。抑郁症的发病机制仍未研究清楚,临床上抑郁症的治疗效果仍不容乐观。抑郁症研究中的重要难题是传统动物模型往往不能很好模拟其发病过程,合适的模型对于抑郁症研究至关重要。本研究选用的是近年来发展起来的在抑郁症研究领域最受欢迎的社交失败（Social defeat）刺激动物模型。这种方法不仅能完全模拟抑郁症行为表型,还可引起脑组织在分子水平、细胞水平发生明显异常。另外还可以采用刺激力度较小的微社交失败刺激（Microdefeat）结合其他方法进行相关的分子操控,是进行抑郁症分子机制研究的很好模型。在抑郁症发病过程中组蛋白去乙酰化起着重要作用。sirtuin家族作为第三类组蛋白去乙酰化酶（HDACs）,在精神类疾病中的作用逐渐引起了人们的重视。Sirt2蛋白是中枢神经系统中特异性高表达的蛋白,参与中枢系统多种疾病的发生,然而有关Sirt2蛋白在情感失调疾病的研究非常少。由于目前Sirt2抑制剂的特异性并非很好,Sirt2抑制剂在sirtuin家族成员间可能存在交叉反应,仅有的几项采用Sirt2抑制剂进行抑郁症研究的研究结论也不统一。因此在本研究中我们使用Sirt2基因敲除小鼠并结合社交失败刺激抑郁症模型,研究Sirt2蛋白在抑郁症发生中的作用和分子机制。研究方法:1.采用PCR方法对Sirt2基因敲除小鼠及同窝野生型小鼠进行基因型鉴定,采用Western blotting方法检验Sirt2基因敲除效率。2.采用10天重复Social defeat刺激进行小鼠抑郁症造模。抑郁相关行为学检测采用社交接触实验、糖水偏好实验、强迫游泳实验、高架十字迷宫实验以及旷场实验。3.采用Western blotting方法检测Sirt2总蛋白表达情况、胞浆和胞核中Sirt2蛋白水平、磷酸化Sirt2水平、p25水平以及磷酸化CDK5的水平;采用Immunofluorescence方法检测Sirt2蛋白在胞浆和胞核的分布。4.设计并体外合成肉豆蔻酸修饰的穿膜干扰肽（Sirt2-p）。每天社交失败刺激前对小鼠进行干扰肽或者乱序肽（对照）的腹膜腔注射以特异性干扰CDK5对Sirt2蛋白丝氨酸368和372位点的磷酸化。10天的注射和社交失败刺激结束后对小鼠进行行为学检测及相关分子检测。5.采用化学遗传学方法（cfos-tTA和TRE-hM3D系统）,对VTA区和社交失败刺激相关的神经元进行标记。在VTA区注射病毒rAAV-cfos-tTA-WPRE-pA和rAAV-TRE-tight-hM3D（Gq）-mCherry-WPRE-PA,结合多西环素饮食（DOX）管理,通过对小鼠进行微社交失败刺激（Microdefeat）将hMD（Gq）-mCherry病毒蛋白标记在社交失败刺激下活化的VTA区神经元上。6.对标记了hM3D（Gq）蛋白的小鼠进行化学遗传学操控。实验组腹膜腔注射CNO,对照组注射生理盐水,两小时后进行社交接触行为学实验或者处死小鼠取杏仁核进行相关分子实验。研究结果:1.Sirt2基因敲除小鼠(Sirt2^-/-)和同窝野生型小鼠(Sirt2^+/+)在糖水偏好实验、强迫游泳实验、高架十字迷宫实验和旷场实验中未表现出差异;对其进行10天的慢性社交失败刺激后,再进行社交接触实验、糖水偏好实验、强迫游泳实验、高架十字迷宫实验和旷场实验检测,发现相对于同窝野生型小鼠,Sirt2基因敲除小鼠没有出现明显的社交失败刺激引起的抑郁样行为。2.在普通C57BL/6小鼠上建立社交失败抑郁症模型,以检测抑郁症小鼠Sirt2蛋白表达情况。Western blotting检测发现,和抑郁症关系密切的脑区中Sirt2蛋白总的表达水平并未发生改变。对这些脑区组织进行胞浆胞核蛋白分离提取,Western blotting检测Sirt2蛋白亚细胞定位,发现经过社交失败刺激的小鼠杏仁核区Sirt2蛋白明显向细胞核中聚集。对社交失败刺激小鼠脑组织切片进行Immunofluorescence方法检测,发现杏仁核Sirt2蛋白向胞核分布增多。3.提取小鼠杏仁核区总蛋白,Western blotting方法分析了社交失败刺激对Sirt2蛋白的磷酸化水平以及CDK5的活性的影响。发现和对照组相比,接受社交失败刺激的小鼠杏仁核区磷酸化Sirt2水平升高,杏仁核区p25生成增多以及磷酸化CDK5水平升高。4.设计并合成了肉豆蔻酸修饰的穿膜干扰肽（Sirt2-p）,以特异性干扰CDK5对Sirt2蛋白丝氨酸368和372位点的磷酸化。对小鼠进行行为学检测,发现干扰肽的注射阻断了社交失败刺激引起的小鼠抑郁样行为;抽取杏仁核区胞浆和胞核蛋白进行Western blotting分析,发现相对于乱序肽注射组,干扰肽注射组小鼠没有出现杏仁核区Sirt2蛋白向细胞核中聚集的现象。5.对VTA区在社交失败刺激下活化的神经元进行化学遗传学（cfos-tTA和TRE-hM3D系统）标记,选取小鼠制作脑组织冰冻切片在荧光显微镜下观察病毒蛋白标记效率。结果显示,和对照组相比,微社交失败刺激组（实验组）mCherry信号明显增强且和细胞核重叠良好,说明hMD（Gq）-mCherry病毒蛋白成功表达在了社交失败刺激下活化的VTA区神经元上。6.对标记了hM3D（Gq）蛋白的小鼠进行化学遗传学操控。发现采用CNO激活VTA区在社交失败刺激下活化的神经元可以引起小鼠抑郁样行为;采用Western blotting分析杏仁核区CDK5活性变化,发现注射CNO的小鼠杏仁核区p25水平以及磷酸化CDK5水平均出现升高;抽取小鼠杏仁核区胞浆和胞核蛋白采用Western blotting分析Sirt2蛋白的亚细胞定位,发现注射CNO的小鼠杏仁核区Sirt2蛋白进入细胞核增多。结论:1.Sirt2基因敲除可以阻止Social defeat刺激引起的抑郁样行为。2.CDK5磷酸化Sirt2蛋白丝氨酸368和372位点促使其进入细胞核是Social defeat刺激引起的抑郁症的重要机制。3.特异性抑制上述磷酸化过程可能是抑郁症治疗的新策略。4.杏仁核区CDK5活性的升高及其引起的Sirt2蛋白在细胞核中聚集可能是VTA-杏仁核环路参与小鼠抑郁症发病的重要分子机制。更多还原

【Abstract】 Background:Major depressive disorder（MDD）,simply known as depression,is a common,severe and recurrent disorder worldwide.However,MDD research continues to face the major challenge that traditional animal models often do not have multiple validities,which may explain the paucity of novel therapeutic interventions targeting the molecular mechanisms of MDD.A newly-developed mouse model that utilizes social defeat stress（SDS）has shown perfect etiological,predictive,discriminative and face validity for depression studies.Repeated exposure to SDS causes a robust depression-like phenotype and high degree of molecular plasticity,providing a potential means to detect the neuropathological mechanisms underlying affective illnesses,such as depression.Histone deacetylases（HDACs）reportedly play an essential role in the mechanisms underlying depression.As class III HDACs,sirtuins have garnered attention for their function in psychiatric disorders.Among all sirtuins the expression of Sirt2 was the strongest in the brain and Sirt2 has been associated with many nervous system disorders.However,there is a paucity of literature on the role of Sirt2 in affective illnesses.The role of Sirt2 in depression remains unclear.The few previous research on the role of Sirt2 in depression is contradictory.These conflicting data suggest that the use of inhibitors may not be appropriate for Sirt2-based research because of the crosstalk among sirtuin subfamily members.Therefore,we employed Sirt2 knockout mice to explore the role of Sirt2 in SDS-induced depression mouse model.Methods:1.PCR was used to identify the genotype of Sirt2 knockout mice and the wildtype littermates.Western blotting was used to analyze the expression of Sirt2 protein in Sirt2^-/-mice.2.10-day repeated social defeat stress was adopted to establish the mouse model of MDD.Social interaction test,sucrose preference test,forced swimming test,elevated plus maze test and open field test were performed to assess the behaviors associated with depression.3.Western blotting was performed for the analysis of total Sirt2 protein level,cytoplasmic and nuclear Sirt2 protein levels,phosphorylated Sirt2 level,p25 level and phosphorylated CDK5 level.Immunofluorescence was used to assess the distribution of Sirt2 protein in nucleus and cytoplasm.4.A myristoylated membrane-permeabilising peptide（Sirt2-p）was developed from a12-amino acid fragment of the Sirt2 protein to specifically interfere with the phosphorylation of Sirt2 by CDK5 at serine residues 368 and 372.Sirt2-p or scrambled peptide（control）were intraperitoneally injected once a day before each SDS session.After the 10-day repeated SDS was completed,behavior tests and molecular experiments were performed.5.Then the VTA was targeted with viruses of rAAV-cfos-tTA-WPRE-pA and rAAV-TRE-tight-hM3D（Gq）-mCherry-WPRE-PA to selectively label the subpopulation of neurons activated during SDS in VTA.Under the management of doxycycline（Dox）diet,hM3D（Gq）-mCherry was selectively tagged in the c-Fos-expressing VTA neurons that were activated by microdefeat stress.6.The mice labelled with hM3D（Gq）-mCherry were manipulated by intraperitoneally injecting with CNO or NS,and two hours later social interaction test or molecular experiments were performed.Results:1.Behavior tests（sucrose preference test,forced swimming test,elevated plus maze test,open field test）associated with depression were performed to assess the responses of Sirt2^+/+mice and Sirt2^-/-mice,but no differences were found.After 10 day repeated social defeat stress,social interaction test,sucrose preference test,forced swimming test,elevated plus maze test,open field test were performed.We found Sirt2 knockout blocks SDS-induced depressive-like behavior.2.SDS-induced depression was established in C57BL/6 mice.Western blotting was used to assessed Sirt2 protein expression in the nuclei suggested to be the most closely associated with depression.No change of total Sirt2 protein levev was observed in these nuclei in SDS mice.However,nuclear Sirt2 protein levels in the amygdala were detected notably increased after SDS with western blotting and immunofluorescence.3.Total proteins of the amygdala were extracted for the assessment of phosphorylated Sirt2 level and activity of CDK5 in depressed mice.It was observed that phosphorylated Sirt2 level increased with elevated p25 and elevated phosphorylated CDK5 in the amygdala of SDS mice.4.A myristoylated membrane-permeabilising peptide（Sirt2-p）was developed to specifically interfere with the phosphorylation of Sirt2 by CDK5 at serine residues 368and 372.The infusion of Sirt2-p prevented SDS-induced behavioral responses in mice.Western blotting was used to assessed subcellular localization of Sirt2 protein.The mice infused with Sirt2-p did not exhibite SDS-induced Sirt2 nuclear translocation in the amygdala.5.The subpopulation of neurons activated during SDS in VTA were selectively label with hM3D（Gq）-mCherry.The expression of hM3D（Gq）-mCherry in the VTA was examined and significantly enhanced virus signals were observed in microdefeat mice when compared with the control groups.6.The mice injected with CNO exhibited the depressive-like behavior compared with the mice injected with NS.Moreover,total proteins in the amygdala were extracted and analysed with western blotting.The CNO injection led to an increase in p25 production and phosphorylated CDK5,while the mice injected with CNO exhibited Sirt2 nuclear translocation in the amygdala.Conclusions:1.Sirt2 knockout blocks SDS-induced depressive-like behavior.2.Phosphorylation of Sirt2 by CDK5 mediates Sirt2 nuclear translocation in the amygdala,contributing to SDS-induced depressive-like behavior.3.Specifically interfering phosphorylation of Sirt2 at serine residues 368 and 372 by CDK5 may be a new target for the treatment of psychiatric disorders,including depression.4.The molecular mechanism underlying VTA-amygdala modulation in depression may be the over activation of CDK5 and the resulting Sirt2 nuclear translocation in the amygdala.更多还原