The role of CDK6 in Alzheimer's Disease

摘要 最近，细胞衰老成为导致阿尔茨海默氏症的一种基本衰老机制 疾病(AD)。在众多治疗衰老相关疾病的临床试验中，没有一项能够证明 对患者有益的影响。此外，缺乏临床前的衰老进程模型是阻碍 治疗方面的发展。通过使用我们的CDK6小鼠模型和生化分析，我们发现小鼠 缺乏CDK6蛋白(KO)或其激活区(K43M)复制了许多人类衰老的特征。相比之下， 具有构成激酶活性(R31C)的小鼠在KO/K43M小鼠中观察到相反的表型。我们有 在KO/K43M脑的海马区和内嗅皮层(EC)区也发现了衰老细胞，但 而WT/R31C在7个月龄时的脑组织中则没有。尽管R31C突变使细胞具有结构性 蛋白激酶的活性，它不会增强肿瘤的敏感性，这使得我们提出的研究更具可行性。 结合CDK6的其他特异性作用 制作的电影 神经干细胞(NSC)6和 抗氧化剂14， 诱导血管生成19，并介导参与抑制Aβ5的Notch1信号通路 生产，所有描述的证据使我们假设，CDK6激酶活性是需要抑制 AD的发展，从而增强CDK6激酶的活性或靶向其效应器可能是一种新的 阿尔茨海默病治疗中的治疗性干预。我们研究的长期目标是验证CDK6作为一种 老年性阿尔茨海默病有吸引力的靶点，目的是识别新的治疗靶点基因。 CDK6在AD中的表达。这项提案的短期目标是集中于中心目标，以定义 衰老(目标1)和确定CDK6影响衰老的分子机制(S) 定义衰老细胞(目标2)。在可靠的初步数据的指导下，中心假设将在两个阶段进行检验 具体目标：(Aim1)通过Flow确定导致海马和EC衰老的细胞 细胞计数、免疫组织化学、RT-PCR和Western blotting。(目标2a)：确定 CDK6在神经干细胞自我更新和分化中的作用 SOX-2，然后进行自我更新和分化鉴定，如所述8，9。 标记物将通过流式细胞仪分析、Western blotting和免疫组织化学确定；(目标2b)： 通过CDK6在细胞上的特异性再表达来确定CDK6对衰老的细胞自主作用 明确衰老细胞，观察CDK6的重新表达能否逆转KO脑内观察到的衰老； (目标2c)：确定小鼠CDK6激酶活性丧失是否会引发氧化应激和炎症反应 已知的引发神经炎症的级联反应。总的来说，拟议的研究将产生足够的数据来 支持设计和启动为下一阶段设计的实验方法。

Summary Cellular senescence has recently emerged as a fundamental aging mechanism contributing to Alzheimer’s Disease (AD). None of the numerous clinical trials to treat aging related diseases has been able to demonstrate beneficial impact on patients. Moreover, the lack of a preclinical model of aging progression is a barrier to therapeutic development. By using our Cdk6 mouse models and biochemical analyses, we have found that mice lacking the CDK6 protein (KO) or its kinase domain (K43M) replicated many features of human aging. In contrast, mice with constitutive kinase activity (R31C) had opposite phenotypes observed in KO/K43M mice. We have also discovered senescent cells in the hippocampus and entorhinal cortex (EC) regions of KO/K43M brains but not in those of WT/R31C brains at the age of 7 months. Although R31C mutation confers cells with constitutive kinase activity, it does not enhance tumor susceptibility, which makes our proposed study more feasible. Combined with other specific effects of CDK6 on production of the Neural Stem Cells (NSCs)6 and antioxidants14, induction of angiogenesis19, and mediating Notch1 signaling pathway which is involved in inhibition of Aβ5 production, all the evidence described leads us to hypothesize that CDK6 kinase activity is required for inhibiting the development of AD, and therefore enhancing CDK6 kinase activity or targeting its effectors could be a novel therapeutic intervention in the treatment of AD. The long-term goal of our studies is to validate CDK6 as an attractive target for aging related AD, with the aim of identifying the novel therapeutic target genes regulated by CDK6 in AD. The short-term goal of this proposal is to focus on central aims to define the cells attribute to senescence (Aim 1) and to determine the molecular mechanism(s) whereby CDK6 affects senescence in the defined senescent cells (Aim 2). Guided by solid preliminary data, the central hypothesis will be tested in two specific Aims: (Aim1) Defining the cells which attributed to the senescence in hippocampus and EC by flow cytometry, immunohistochemistry, RT-PCR, and Western Blotting. (Aim 2a): Determining the requirement of CDK6 for self-renewal and differentiation of the NSCs by isolating undifferentiated NSCs positive for Nestin and Sox-2 and then by performing self-renewal and differentiation assays as described8,9. Quantification of expressed markers will be determined by Flow cytometry analysis, Western Blotting, and immunohistochemistry; (Aim 2b): Determining the cell autonomous effect of CDK6 on senescence by re-expression of CDK6 specifically on the defined senescent cells to observe if re-expression of CDK6 can reverse the senescence observed in KO brain; (Aim 2c): Determining if loss of CDK6 kinase activity in mice may trigger oxidative stress and the inflammatory cascades known to induce neuroinflammation. Overall, the proposed studies will generate adequate data to support the design and initiation of experimental approaches designed for the next phase.