Tubby调控miR-195介导低剂量慢性铜暴露致Alzheimer病神经退化的机制研究

Alzheimer’s disease (AD) is a common neurodegenerative disease with severe health impact. Copper may be a key environmental factor contributing to the development of AD. However, the effects of copper exposure (especially low-dose) on AD characteristic pathology-apoptotic neurodegeneration remain elusive. Our preliminary study has demonstrated that trace amount of copper in drinking water (below the copper standard in drinking water set by The U.S. Environmental Protection Agency) caused apoptotic neurodegeneration of hippocampal neurons of AD transgenic mice with concurrent up-regulation of transcriptional factor tubby and miR-195. Inhibition of tubby down-regulated the expression of miR-195, and inhibition of miR-195 expression prevented neurodegeneration in AD caused by low-dose chronic copper exposure. Informatics analysis revealed that there are binding sites between tubby and pre-miR-195 gene, and transient receptor potential canonical type 1 (TRPC1) may be an ideal target gene of miR-195. These findings lead us to raise a hypothesis as follows: low-dose chronic copper exposure may promote the binding of tubby with promoter of pre-miR-195 gene, and therefore up-regulate the expression of miR-195; the latter would down-regulate the expression of its target gene TRPC1, thereby triggering endoplasmic reticulum (ER) stress. ER stress would therefore result in neurodegeneration in AD. We will use the techniques such as recombinant adeno-associated virus (rAAV)-mediated long-term silencing and over-expression of genes and chromatin immunoprecipitation to elucidate the epigenetic regulatory mechanisms by which copper exposure caused neurodegeneration in AD. The accomplishment of this project would be expected to provide new strategies for the prevention and treatment of AD as well as novel molecular targets for the development of AD drugs.

阿尔茨海默病（AD）是危害人类健康的重大疾病。铜暴露是促发AD的关键环境因素。铜暴露（尤其低剂量）对AD特征性病变-凋亡性神经退化的影响未见报道。本课题组前期研究发现：低剂量慢性铜暴露诱导AD神经退化，上调tubby转录因子及miR-195；抑制tubby下调miR-195表达，抑制miR-195显著改善铜致AD神经退化。生物信息学分析发现，tubby与pre-miR-195，miR-195与经典瞬时受体电位通道蛋白1（TRPC1）基因均存在结合位点。因此，我们推测：低剂量铜暴露，通过增加脑内tubby/pre-miR-195基因启动子的结合， 上调miR-195表达，miR-195靶向下调TRPC1表达，激发内质网应激，导致AD神经退化。本项目拟采用基因长效抑制和过表达、染色质免疫共沉淀等手段，阐明低剂量铜暴露促进AD神经退化的表观遗传学调控机制，为AD的防治提供新的策略和药物开发靶点。

阿尔茨海默病（AD）是危害人类健康的重大疾病。铜暴露是促发AD的关键环境因素。铜暴露（尤其低剂量）对AD特征性病变-凋亡性神经退化的影响未见报道。通过国自然面上项目4年的资助，本项目取得以下主要发现：.1、低剂量慢性铜暴露导致早期阶段AD小鼠海马组织铜蓄积；2、低剂量铜暴露导致3xTg-AD小鼠海马显著的氧化损伤（γH2A.X和8-OHdG的水平升高）、显著降低线粒体ATP的含量；3、低剂量慢性铜暴露诱导AD神经退化/海马神经元细胞凋亡，上调tubby转录因子及miR-195抑制tubby下调miR-195表达，抑制miR-195显著改善铜致AD神经退化；4、miR-195与经典瞬时受体电位通道蛋白1（TRPC1）基因是miR-195调控的靶基因，并证实TRPC1 缺失诱导神经细胞凋亡显著增加。敲除TRPC1基因会导致小鼠蛋白质表达显著变化，TRPC1缺失通过干扰包括ER功能，氧化应激和凋亡相关信号在内的多种生物过程而导致神经元丢失或者凋亡；6、此外，我们也发现，低剂量的铜暴露可显著改变野生小鼠和AD转基因小鼠海马蛋白表达谱和磷蛋白表达谱的变化，并揭示了导致AD病理及行为学损伤的关键蛋白。低剂量的铜暴露可引起显著线粒体损伤及线粒体蛋白表达谱的显著改变，结合TRPC1对（线粒体）钙信号通路的调节机制，提示tubby/miR-195/TRPC1通路的异常可能是低剂量铜暴露增加AD发病风险的关键机制。本项目阐明了低剂量铜暴露促进AD神经退化的表观遗传学调控机制，为AD的防治提供新的策略和药物开发靶点。项目按照预期计划完成研究内容，取得以下主要成绩：目前已发表相关SCI论文6篇（通讯作者），获得科技奖项2项，获得发明专利1项。本项目共培养硕士研究生6名，培养博士后1名（出站）。承办国际会议1次（“神经退行性疾病的预防-从基础到临床国际学术会议”（会议网址：http://icpnd.szpma.org.cn/））。

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