The role of CDK6 in Alzheimer's Disease

CDK6 在阿尔茨海默病中的作用

• 批准号: 10740243
• 负责人: MIAOFEN G HU
• 金额: $ 48.95万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740243
• 关键词: Abeta synthesis; Affect; Age; Aging; Alzheimer' s Disease; Antibodies; Antioxidants; Benchmarking; Biochemical; Biological Assay; Brain; Cell Aging; Cell Separation; Cells; Clinical Trials; Data; Development; Disease; Enzymes; Flour; Flow Cytometry; Fluorescein; Generations; Genes; Goals; Hippocampus; Human; Hyperactivity; Immunofluorescence Immunologic; Immunohistochemistry; Inflammatory; Intervention; Label; Magnetism; Mediating; Metabolism; Molecular; Mus; Mutant Strains Mice; Mutation; Neurons; Oxidative Stress; Patients; Phase; Phenotype; Phosphotransferases; Physiological; Play; Pre-Clinical Model; Predisposition; Production; Proliferating; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Solid; Sorting; Stains; Testing; Therapeutic; Undifferentiated; Western Blotting; angiogenesis; anti aging; antibody conjugate; beta-galactoside; cyclin-dependent kinase 6; design; entorhinal cortex; immunocytochemistry; insight; mouse model; nerve stem cell; nestin protein; neuroinflammation; new therapeutic target; novel; novel therapeutic intervention; pharmacologic; prevent; self-renewal; senescence; stem cell self renewal; therapeutic development; tumor

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.

概括 细胞衰老最近已成为导致阿尔茨海默病的基本衰老机制 疾病（AD）。众多治疗衰老相关疾病的临床试验都未能证明这一点。 此外，缺乏衰老进展的临床前模型也是阻碍这一研究的一个障碍。 通过使用我们的 Cdk6 小鼠模型和生化分析，我们发现小鼠 相比之下，缺乏CDK6蛋白（KO）或其激酶结构域（K43M）则复制了人类衰老的许多特征。 具有组成型激酶活性（R31C）的小鼠具有与 KO/K43M 小鼠相反的表型。 还在 KO/K43M 大脑的海马体和内嗅皮层 (EC) 区域发现了衰老细胞，但 7 个月大时，WT/R31C 大脑中没有这种现象，尽管 R31C 突变赋予细胞组成性。 激酶活性，它不会增强肿瘤的易感性，这使得我们提出的研究更加可行。 与 CDK6 的其他特定作用相结合 的生产 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 生产中，所描述的所有证据使我们发现 CDK6 激酶活性是抑制 AD 的发展，因此增强 CDK6 激酶活性或靶向其效应子可能是一种新的方法 AD 治疗的治疗干预 我们研究的长期目标是验证 CDK6 作为一种治疗干预措施。 衰老相关 AD 的有吸引力的靶标，目的是确定受调节的新治疗靶基因 AD 中的 CDK6 该提案的短期目标是集中于定义细胞属性的中心目标。 衰老（目标 1）并确定 CDK6 影响衰老的分子机制 在可靠的初步数据的指导下，中心假设将在两个方面进行检验。 具体目的：（目的1）通过流式定义海马和EC中导致衰老的细胞 细胞计数、免疫组织化学、RT-PCR 和蛋白质印迹（目标 2a）：确定以下要求。 CDK6 通过分离巢蛋白阳性的未分化 NSC 来实现 NSC 的自我更新和分化 Sox-2，然后通过进行自我更新和分化测定，如所描述的 8,9 进行定量。 标记物将通过流式细胞术分析、蛋白质印迹和免疫组织化学测定（目标 2b）： 通过在细胞上特异性重新表达 CDK6 来确定 CDK6 对衰老的细胞自主作用 确定衰老细胞，观察 CDK6 的重新表达是否可以逆转 KO 脑中观察到的衰老现象； （目标 2c）：确定小鼠 CDK6 激酶活性的丧失是否可能引发氧化应激和炎症 总体而言，拟议的研究将产生足够的数据来诱发神经炎症。 支持下一阶段实验方法的设计和启动。