Cdk5介导的Drp1磷酸化对线粒体的调控及其在阿尔茨海默病中的作用

Alzheimer's disease (AD) is one of the most common types of chronic neurodegenerative diseases, pathologically characterized by the formation of amyloid-β (Aβ) containing plaques and neurofibrillary tangles. It has been reported that Aβ induces an increase in [Ca2+]i, subsequent hyperactivation of Cdk5 via calpain-dependent cleavage and mitochondrial abnormality. In our previous studies, calcium signalling pathway is found to be invovled in regulation of mitochondrial dynamics via phosphorylation of Drp1(Han XJ, et al, J Cell Biol. 2008).Recently, Cdk5 is also found to be an upstream regulator of mitochondrial fission during neuronal apoptosis. However, the downstream signaling pathway in Cdk5-mediated mitochondrial fission remains elusive. We hypothesize that Cdk5 might regulate mitochondrial dynamics and neuronal function through phosphorylation of Drp1, and this mechanism might be involved in AD. In preliminary experiments, we found that Cdk5 phosphorylated recombinant and endogenous Drp1 at ser579, and Cdk5 inhibitor Roscovitine effectively inhibited Aβ-induced mitochondrial fission and redistribution in hippocampal neurons. To prove our hypothesis, we will further examine the effect of Cdk5-mediated phosphorylation on Drp1 GTPase activity, and investigate the role of Cdk5-mediated Drp1 phosphorylation in the regulation of mitochondrial dynamics and neuronal function in vitro and in vivo. Therefore, this research will provide important new insights into the understanding of Cdk5-mediated mitochondrial dynamics and its role in AD pathogenesis, and it also will be beneficial to the prevention and therapies of AD in the future.

阿尔茨海默病(AD)是常见的慢性神经退行性疾病，特征性病理变化有β-淀粉样斑块(Aβ)、神经原纤维缠结。其中，Aβ可引发神经元胞内钙升高、异常高活性Cdk5和线粒体异常。我们以往的工作表明，钙信号通路可通过线粒体分裂蛋白Drp1来调节线粒体变化（Han XJ,et al, J Cell Biol. 2008）。也有研究报道了Cdk5是神经元线粒体分裂的上游调节因子，但有关下游信号通路尚不清楚。故我们推测Cdk5可能通过对Drp1磷酸化来调控线粒体动态变化及神经元功能，并以此参与AD的发生。预实验已证明：Cdk5能对重组和内源性Drp1磷酸化；且Cdk5抑制剂能有效抑制Aβ引起的线粒体改变。为进一步证实上述假说，本课题还将检测磷酸化对Drp1活性的影响，并在细胞和整体水平上观察Cdk5介导的Drp1磷酸化对线粒体和神经元的影响，旨在阐明Cdk5对线粒体调控的信号机制，为AD防治寻找新的靶点。

阿尔茨海默病是临床上常见的慢性进行性神经退行性疾病之一。其主要的病理学改变有淀粉样斑块和神经原纤维缠结的形成。已有研究表明线粒体异常与神经退行性改变关系密切；淀粉样多肽，如Aβ-42，可通过增高神经元胞内钙浓度异常激活Cdk5活性；Cdk5又是神经元线粒体分裂的上游调节因子，但其下游具体信号通路尚不清楚。本课题中，我们首先利用同位素磷酸化实验在分子水平上检测到Cdk5能对Drp1的579号色氨酸进行磷酸化修饰；并且在神经元中进一步发现Aβ-42刺激能显著上调Drp1-Ser579磷酸化水平，利用Roscovitine或慢病毒敲低Cdk5抑制Cdk5活性后能有效抑制Aβ-42刺激所致Drp1-Ser579磷酸化，这阐明了Aβ-42刺激神经元能通过激活Cdk5介导Drp1-Ser579的磷酸化修饰；我们进一步检测Cdk5介导的Drp1-Ser579磷酸化对神经元线粒体形态的影响，实验结果显示Aβ-42刺激可引起神经元线粒体分裂，Roscovitine、敲低Cdk5或过表达磷酸化缺陷型Drp1-S579A均能有效抑制Aβ-42刺激所致线粒体分裂，而过表达磷酸化模拟型Drp1-S579D即可引发神经元线粒体分裂，这些结果证实了Cdk5介导的Drp1-Ser579磷酸化促进了线粒体分裂活动；我们还发现Roscovitine、敲低Cdk5均能有效抑制Aβ-42刺激所致神经元Caspase依赖性凋亡。本研究阐明了Cdk5介导的Drp1-Ser579磷酸化在Aβ-42促进线粒体分裂和神经元功能损伤中的分子信号机制，具有重要的科学意义；同时，本研究也为AD防治提供了可能的新分子靶点，即有效干扰Cdk5介导的Drp1-Ser579磷酸化为基础的线粒体分裂，故在临床上也具有潜在的应用价值和前景。本课题基本完成研究计划，已发表SCI论文7篇，中文核心期刊论文1篇，并培养了硕士研究生2名。