HDAC6在APPswe/PS1ΔE9阿尔茨海默病转基因模型小鼠发病机制中的作用

【作者】 张玲；

【导师】 秦川；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2014， 博士

【摘要】 背景阿尔茨海默病(Alzheimer’s disease, AD)是一种以认知功能进行性减退为主要临床表现的神经退行性病变。其确切的病因尚不清楚,近年来有研究显示组蛋白去乙酰化酶6(histone deacetylase6, HDAC6)在AD病人的海马和皮层中表达增加,但其过表达的作用尚存在争议。方法应用免疫组化的方法检测HDAC6在成年C57BL/6J野生小鼠全脑组织内的表达分布情况；应用免疫荧光双标技术检测HDAC6在各类脑细胞中的表达；应用免疫组织化学和免疫印迹检测HDAC6和其底物乙酰化α-微管蛋白(ace-α-tubulin,K40)在1、3、6、9月龄野生型C57BL/6J小鼠和APPswe/PS1ΔE9(PAP)阿尔茨海默症模型小鼠脑组织中表达的变化。将PAP模型小鼠的Hdac6敲除后获得PAP×Hdac6KO小鼠。PCR鉴定其基因型,免疫印迹和组织化学方法检测HDAC6和ace-α-tubulin在6月龄WT、PAP、Hdac6KO和PAP×Hdac6KO小鼠脑组织的表达情况；应用开放旷场实验检测小鼠的自主活动和焦虑情绪,新物体识别实验和Morris水迷宫实验检测小鼠学习记忆能力的改变；硫磺素-S荧光染色观察老年斑的改变；ELISA检测小鼠脑内可溶性和不可溶性Aβ40/42的含量；免疫印迹检测小鼠脑内过度磷酸化tau蛋白(P-tau)和GSK3β磷酸化的水平；透射电镜分析微管超微结构的改变。另外,本实验选取了两种具有临床应用前景的HDAC6特异性抑制剂tubastatinA和ACY-1215,观察它们对6月龄PAP小鼠认知功能的影响并进行初步的机制研究。将6月龄PAP小鼠随机分为模型组、溶剂对照组(vehicle, DMSO/生理盐水=1：10)、阳性对照组(SAHA,50mg/kg)、tubastatin A组(25mg/kg)和ACY-1215组(25mg/kg)。并以同窝阴性的C57BL/6J小鼠作为正常对照组(WT),每组16只,雌雄各半,腹腔注射连续给药20天。每4天称一次小鼠体重。注射结束后进行旷场实验、新物体识别实验和Morris水迷宫实验来观察tubastatin A和ACY-1215对AD模型小鼠自主活动和认知功能的影响；检测脑重和肝肾功能各项指标；应用开放旷场实验检测小鼠的自主活动和焦虑情绪,新物体识别实验和Morris水迷宫实验检测小鼠学习记忆能力的改变；检测脑重和肝肾功能各项指标；免疫印迹和免疫组织化学检测小鼠皮层和海马内HDAC6;和α-tubulin的乙酰化水平；6E10免疫染色和硫磺素-S染色检测脑内老年斑沉积；ELISA检测小鼠脑内可溶性和非可溶性Aβ40和Aβ42含量；免疫印迹检测脑内APP降解相关酶和产物,自噬相关蛋白的表达,P-tau的水平,脑信号转导通路分子P-GSK3β、P-Akt及P-mTOR的变化；透射电镜检测脑组织超微结构的变化。结果HDAC6在成年C57BL/6J小鼠脑组织内分布较为广泛,但具有相对区域特异性：在海马、皮层和杏仁核等与认知相关脑区的表达最为丰富；有细胞特异性：主要分布在各类神经元的细胞浆中,而在胶质细胞中无表达。HDAC6在WT和PAP小鼠海马和皮层的表达随年龄增加而明显增多。与同龄WT小鼠相比,AD小鼠脑中HDAC6的表达从3月龄开始明显增多,且随年龄增加HDAC6过表达的趋势更明显,ace-a-tubulin相应减少。与PAP小鼠相比,Hdac6敲除后并不影响小鼠的自主活动和焦虑情绪,但能明显改善其认知功能,PAP×Hdac6KO小鼠脑内老年斑沉积未有明显变化,但ELISA检测结果显示其脑内可溶性Aβ42的含量明显减少,tau蛋白的磷酸化水平显著降低,P-GSK3p的表达明显上调,Hdac6敲除可明显改善PAP小鼠脑中微管的超微结构。Tubastatin A和ACY-1215能明显抑制HDAC6勺活性,改善PAP小鼠的认知功能,减少老年斑的沉积和可溶性Aβ40/42的含量,抑制APP的β/γ-切割和GSK3(3的活性,降低tau蛋白的磷酸化水平,促进自噬清除过程,并改善微管的超微结构,而对小鼠的自主活动和焦虑水平及体重、脑重和肝肾功能无明显影响。结论HDAC6的正常表达可能对维持认知相关脑区神经元的正常功能具有重要作用。HDAC6过表达在AD中很可能通过影响Ap/P-tau的生成和自噬清除发挥了神经损伤作用。HDAC6是很可能是AD的治疗靶点,HDAC6特异性抑制剂tubastatinA和ACY-1215或许是治疗AD的潜在药物。更多还原

【Abstract】 Background Alzheimer’s disease (AD), as the most common form of dementia among older individuals, is clinically characterized by progressive loss of cognitive function. Despite much progress, the pathogenesis of AD remains to be elucidated. Histone deacetylase6(HDAC6) was reported to increase in hippocampus and cortex in both patients and animal models of AD. However, whether HDAC6overexpression is an underlying cause of AD or a condition resulting from AD is controversial.Methods The regional distribution of HDAC6in the adult C57BL/6J mouse brain was analyzed by immunochemical staining. Double immunofluorescent staining was used to detect the co-location of HDAC6with markers of multiple brain cell types. The temporal expression patterns and expression changes of HDAC6and acetylation of a-tubulin (ace-a-tubulin) in the brain of wild type (WT) and APPswe/PS1ΔAE9(PAP) mice were analyzed by immunohistochemistry and western blotting.Next, we detected the effects of Hdac6knockout on the cognitive deficits of PAP mice. The genotype of mice was detected by PCR. The expression of HDAC6and ace-a-tubulin were analyzed by immunohistochemistry and western blotting. The locomotor activity was examined by open field test and cognitive abilities were detected using novel object recognition test and Morris water maze. Senile plaques were observed by Thioflavin-S staining. The levels of Aβ40/42were quantified by ELISA. The expression of hyperphosphorylated tau protein (P-tau) and P-GSK3β were examined by western blotting. The microtubule ultrastructures were observed by transmission electronic microscopy.Then we examined the effects of two selective HDAC6inhibitors-tubastatin A and ACY-1215on the cognition of PAP mice. PAP mice were randomly divided into model group, vehicle (DMSO/saline=1:10), SAHA (50mg/kg), tubastatin A (25mg/kg) and ACY-1215(25mg/kg) groups. The wild-type littermates were chosen as normal control group (WT). The mice (n=16/group, male/female=1:1) were treated for20consecutive days by daily intraperitoneal injection. After administration, a battery of behavioral tests including open field, novel object recognition and Morris water maze were performed. All animals were sacrificed and the brain weight and brain coefficient were tested. Amyloid depositions were quantified using immunohistochemistry and thioflavine-S staining. The soluble and insoluble Aβ40/42peptides were determined by ELISA. Western blotting and immunohistochemistry staining were used to detect levels of HDAC6and acetylated a-tubulin. The expression of proteins and intermediate products involved in processing of APP, autophagy-associated proteins, P-tau, P-GSK3(3, P-Akt and P-mTOR were detected by western blotting. The ultrastructures of brain tissues were observed by transmission electronic microscopy.Results Our data showed that HDAC6, primarily located in cytoplasm of various kinds of neurons, is ubiquitously expressed throughout the mouse brain. Interestingly, the highest HDAC6signals were observed in regions relevant to cognition, such as cortex, hippocampus and amygdala. The HDAC6expression level was significantly increased in the brain of PAP transgenic mice compared with WT mice. It was increased with age in both WTand PAP transgenic mice accompanied by the decrease of ace-a-tubulin.Hdac6knockout improved learning and memory abilities of PAP transgenic mice without obvious effect on the anxiety level of mice. Compared with PAP transgenic mice, the soluble Aβ42peptide of PAP×Hdac6KO mice was significantly decreased, though the deposition of senile plaques was not altered. The deletion of Hdac6in PAP mice down-regulated the hyperphosphorylation of tau protein and inhibited the activity of GSK3β In addition, ultrastructural analysis showed obviously microtubule morphological abnormality of PAP mice was rescued by loss of Hdac6.Both tubastatin A and ACY-1215alleviated the cognitive decline without obvious effect on the locomotor activity of PAP mice through inhibiting the activity of HDAC6rather than its expression level. They reduced soluble and insoluble Aβ levels and the deposition of plaques, and decreased the levels of BACE1, PS1, sAPPβ and β-CTF. They also upregulated the expression of autophagy-associated proteins and reduced the expression of P-tau, P-GSK3β and P-mTOR. Moreover, they decreased the accumulation of autophagy vesicles and rescued the damaged microtubule in PAP mice. In addition, there were no significant adverse effects of tubastatin A and ACY-1215on brain weight, brain coefficient, liver and kidney function of mice.Conclusion The normal expression of HDAC6may play an important role in the function of neurons in cognition-related regions. HDAC6overexpression is probably associated with AD etiopathogenesis and plays a potential detrimental role in AD. And HDAC6is likely to be a therapeutic target for AD. Our preclinical results offer prospective approaches for using tubastatin A/ACY-1215as potential therapeutic strategy for AD.更多还原