PARP1通路抑制分子RNF146调控星形胶质细胞凋亡在AD中的作用研究

Beta amyloid peptide (Aβ) deposition in brain tissue is one of the important pathological features in Alzheimer's disease (AD).Traditional view is that Aβ deposition can induce neuronal apoptosis. Recently, researchers have found that Aβ deposition can also induce astrocytes (AS) apoptosis. We screened the E3 ubiquitin ligase RNF146 expressed in the brain primary cultured AS of APP transgenic mice by the expression profile chip. RNF146 can induce poly (adp ribose polymerase 1 (PARP1) ubiquitin degradation, inhibit PARP1 mediated neural death. We found that: (1) Aβ could induce down-regulation of RNF146, increase levels of PARP1 and apoptosis in primary cultured AS; (2) Up-regulation of RNF146 can improve the apoptosis of AS induced by Aβ. However, whether Aβ deposition is the cause of RNF146 down-regulation in primary cultured AS or not? What is the mechanism of RNF146 down-regulation promote apoptosis of AS. Whether related to out of control of ubiquitin degradation of PARP1? In conclusion, we will comprehensively use methods of gene clone, RNAi, virus methods of packaging, electrophysiology, through experiments in vivo and in vitro, systematic research the effect and regulatory mechanism of RNF146 expression and cell apoptosis induced by Aβ deposition in AS. These will provide new targets for prevention and control of the AD.

脑组织β-淀粉样蛋白(Aβ)沉积是阿尔茨海默病(AD)重要的病理特征。传统认为Aβ沉积能够诱导神经元凋亡。最近发现Aβ沉积同样能够诱导星形胶质细胞（AS）凋亡。我们利用表达谱芯片筛选获得了在APP转基因小鼠大脑原代AS中表达下调的E3泛素连接酶RNF146，其能诱导聚腺苷二磷酸核糖聚合酶1（PARP1）泛素化降解，抑制PARP1介导的神经元死亡。我们发现：① Aβ能诱导原代AS中RNF146下调、PARP1水平增加以及细胞凋亡；②上调原代AS中RNF146表达，能改善Aβ诱导的AS凋亡。然而Aβ沉积是否是导致AS中RNF146下调的原因？RNF146下调促进AS凋亡的机制是什么？是否与PARP1的泛素化降解失调控有关？综上所述，我们将综合运用基因克隆、RNAi、病毒包装、电生理等方法，通过体内外实验,系统研究Aβ沉积对AS中RNF146表达和细胞凋亡的影响及调控机制，为AD防治提供新靶点。

阿尔茨海默病（Alzheimer’s disease, AD）是一类以进行性认知障碍和记忆力损伤为主要临床表现的中枢神经系统退行性病变。AD发病机制复杂，目前尚无权威解释。大量研究显示大脑皮质和海马神经细胞外β-淀粉样蛋白（β-amyloid, Aβ）沉积与AD的病理进程关系密切。传统认为Aβ沉积能够诱导神经元凋亡。最近发现Aβ沉积同样能够诱导星形胶质细胞（AS）凋亡。我们通过系统实验研究Aβ沉积对AS中RNF146表达和细胞凋亡的影响及调控机制，为AD防治提供新靶点。（1）建立高纯度原代星形胶质细胞的培养体系。采用“差速贴壁法”、“梯度血清法”和“十字手摇法”，以获取高纯度星形胶质细胞。通过星形胶质细胞特异性表达蛋白即胶质原纤维酸性蛋白（GFAP）行免疫细胞荧光染色和western blot来鉴定细胞纯度。（2）Iduna在Aβ诱导大鼠星型胶质细胞C6损伤中的作用。结果表明，与对照组比较，Aβ组中Aβ能够增加C6细胞培养上清液中MDA含量，降低SOD活性；同时，Aβ可下调C6细胞中Iduna蛋白的表达，上调PARP1的表达，AIF发生核转位。因此，Aβ引起星型胶质细胞损伤可能与神经保护因子Iduna下调从而引起PARP1死亡通路激活有关。（3）通过流式细胞术检测Annexin V/PI 反映C6细胞凋亡情况。Aβ可导致星型胶质细胞凋亡。（4）RNF146在APP转基因小鼠原代星形胶质细胞中的表达水平明显下降。（5）成功构建RNF146过表达慢病毒载体。（6）Aβ暴露能够下调原代星形胶质细胞中RNF146的表达。（7）Aβ暴露能够上调原代星形胶质细胞中PARP1的蛋白水平。（8）小鼠脑室内注射Aβ可导致小鼠学习记忆能力明显下降，采用注射慢病毒上调RNF146可以改善Aβ暴露引起的学习记忆能力下降。小结：Aβ能诱导RNF146下调、PARP1水平增加、AIF核转位以及星型胶质细胞凋亡。Aβ所致星形胶质细胞中RNF146的下调促进PARP1通路激活而使细胞凋亡。在小鼠AD模型中证实RNF146上调可改善Aβ诱导的学习记忆能力的下降。