Project 2: The proteasome in aging and neurodegenerative disease

项目2：衰老和神经退行性疾病中的蛋白酶体

• 批准号: 10411684
• 负责人: RICHARD I MORIMOTO
• 金额: $ 12.68万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10411684
• 关键词: 3T3 Cells; Address; Age; Aging; Alzheimer' s Disease; Animals; Autophagocytosis; Biochemical; Biological Models; Biology; Brain; CRISPR/Cas technology; Data; Disease; Disease model; Health; Human; Huntington Disease; Link; LoxP-flanked allele; Mammals; Methods; Motor Neurons; Mus; Nerve Degeneration; Neurodegenerative Disorders; Output; Pathway interactions; Play; Prevention; Protein Dynamics; Proteins; Proteomics; Regulation; Resistance; Role; Spinal Cord; Stress; System; Tauopathies; Testing; Tissues; Toxic effect; Transgenes; Transgenic Mice; Ubiquitin; Work; age related; aged; design; in vivo; interest; multicatalytic endopeptidase complex; polyglutamine; protein aggregation; protein folding; proteostasis; success; superoxide dismutase 1; tau Proteins

Project Summary / Abstract (Project 2) A marked decline in proteasome activity is observed as humans and other mammals age. This has been observed in many tissues, including the brain and motor neurons. Aging is characterized by compromised proteostasis, and declining proteasome activity may play a significant role in aging-associated deficiencies of proteostasis, given the pivotal role of the proteasome in protein dynamics. By degrading ubiquitin conjugates, the proteasome controls protein stability on a global level. There has historically been little interest in the potential role of the proteasome in determining the overall output of the ubiquitin-proteasome pathway (UPS). Through recent work, however, it is now recognized that the proteasome is on the contrary a focal point of regulation of the UPS. Indeed, the level of proteasome activity controls protein breakdown rates and stress resistance. Remarkably, the proteasome can in general be up-regulated without toxicity. Extensive work has also shown that the proteasome is compromised in many disease states, particularly in aging-associated and neurodegenerative diseases. However, a deeper and more reliable understanding of the relevance of the proteasome to aging and neurodegeneration in humans clearly requires the use of in vivo mammalian model systems. To directly test whether the proteasome becomes limiting in aged animals, three transgenic mouse lines have been designed to allow conditional elevation of proteasome levels; two of these lines have already been generated. Each mouse line will conditionally express the wild-type murine form of a different proteasome subunit–either Rpn6, Rpn11, or 5. These subunits were chosen because they are expected from existing data to be rate-limiting for proteasome assembly. The multiplicity of strategies to elevate proteasome levels increases the likelihood of success, and if more than one method succeeds it will enhance confidence in subsequent results. For each transgene, a floxed and dox-suppressible construct is integrated into the Rosa26 locus via CRISPR/Cas9. The transgenes should be expressed in a tissue-specific and temporally-controlled manner. Our first objective will be to validate the strategy for elevating proteasome levels in the brain, spinal cord, and in Flp-In™-3T3 cells with transgenes similarly targeted to Rosa26. Excellent biochemical and proteomic methods exist for quantifying alterations in proteasome levels. Remarkably, global proteomics can now quantify the impact of elevated proteasome levels on the control of hundreds of substrate proteins in these settings. We will proceed to assess the effects of elevated proteasome expression on the health and aging of these animals. We will test whether elevated proteasome levels influence autophagy and the proteostasis network (PN) as a whole, and seek to identify age-dependent vulnerabilities in the PN by applying specific stresses to the system. A central focus of the work on transgenic mice will be to employ disease models to assess the effect of elevated proteasome levels on the progression of neurodegenerative diseases, particularly tauopathies, Huntington’s disease, and ALS.

项目总结/摘要（项目 2） 随着人类和其他哺乳动物的衰老，蛋白酶体活性显着下降。 在许多组织中观察到，包括大脑和运动神经元，衰老的特点是受损。 蛋白质稳态和蛋白酶体活性下降可能在与衰老相关的蛋白质缺陷中发挥重要作用 鉴于蛋白酶体在蛋白质动力学中的关键作用，通过降解泛素缀合物， 蛋白酶体在全球范围内控制蛋白质的稳定性，历史上人们对它兴趣不大。 蛋白酶体在确定泛素-蛋白酶体途径（UPS）总体输出中的潜在作用。 然而，通过最近的工作，现在人们认识到蛋白酶体恰恰相反，是 事实上，蛋白酶体的活性水平控制着蛋白质的分解率和应激。 值得注意的是，蛋白酶体通常可以在没有毒性的情况下被上调。 还表明，蛋白酶体在许多疾病状态下都会受到损害，特别是在与衰老相关的疾病中 然而，对神经退行性疾病的相关性有更深入、更可靠的了解。 蛋白酶体对人类衰老和神经退行性变的影响显然需要在哺乳动物模型中使用 为了直接测试蛋白酶体是否对老年动物产生限制，使用了三只转基因小鼠。 品系已被设计为允许有条件地提高蛋白酶体水平；其中两条品系已经被设计 每个小鼠品系都会有条件地表达不同蛋白酶体的野生型小鼠形式。 亚基 – Rpn6、Rpn11 或 5 选择这些亚基是因为它们是现有数据的预期值。 提高蛋白酶体水平的策略多种多样。 增加成功的可能性，如果不止一种方法成功，就会增强人们的信心 对于每个转基因，将 floxed 和 dox 抑制构建体整合到 Rosa26 中。 转基因应该以组织特异性和时间控制的方式表达。 我们的首要目标是验证提高大脑、脊髓中蛋白酶体水平的策略。 脐带，以及具有类似针对 Rosa26 的转基因的 Flp-In™-3T3 细胞。 蛋白质组学方法可用于量化蛋白酶体水平的变化，值得注意的是，全局蛋白质组学可以。 现在量化蛋白酶体水平升高对这些中数百种底物蛋白的控制的影响 我们将继续评估蛋白酶体表达升高对健康和衰老的影响。 我们将测试升高的蛋白酶体水平是否影响自噬和蛋白质稳态。 网络（PN）作为一个整体，并通过应用特定的方法来识别 PN 中与年龄相关的漏洞 转基因小鼠工作的一个中心重点是利用疾病模型来研究该系统。 评估蛋白酶体水平升高对神经退行性疾病进展的影响，特别是 tau蛋白病、亨廷顿舞蹈病和 ALS。