The proteasome in aging and neurodegenerative disease

衰老和神经退行性疾病中的蛋白酶体

• 批准号: 10183115
• 负责人: Daniel J Finley
• 金额: $ 43.15万
• 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/10183115
• 关键词: 3T3 Cells; Affect; Age; Aging; Animals; Autophagocytosis; Behavioral; Biochemical; Biological Models; Brain; Brain region; CRISPR/Cas technology; Cells; Data; Disease; Disease Progression; Disease model; Health; Health behavior; Homeostasis; Human; Huntington Disease; Huntington gene; LoxP-flanked allele; Mammals; Mediating; Methods; Molecular Chaperones; Motor; Motor Neurons; Mus; Mutant Strains Mice; Nerve Degeneration; Neurodegenerative Disorders; Nucleosome Core Particle; Output; Pathway interactions; Phenotype; Play; Predisposition; Protein Dynamics; Protein Isoforms; Proteins; Proteome; Proteomics; Protocols documentation; Regulation; Reporting; Resistance; Resolution; Role; Spinal Cord; Stress; System; Tauopathies; Testing; Tissues; Toxic effect; Transgenes; Transgenic Mice; Transgenic Organisms; Ubiquitin; Work; age related; aged; base; cell type; design; in vivo; indexing; interest; multicatalytic endopeptidase complex; mutant; particle; protein degradation; proteostasis; proteotoxicity; success; superoxide dismutase 1; tau Proteins; tissue culture; tool

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.

项目摘要/摘要(项目2) 随着人类和其他哺乳动物年龄的增长，蛋白酶体的活性显著下降。这已经是 在许多组织中观察到，包括大脑和运动神经元。衰老的特征是折衷 蛋白稳定和蛋白酶体活性下降可能在衰老相关的脑功能缺陷中发挥重要作用 蛋白质稳定，蛋白酶体在蛋白质动力学中起着关键作用。通过降解泛素结合物， 蛋白酶体在全球水平上控制蛋白质的稳定性。从历史上看，人们对 蛋白酶体在决定泛素-蛋白酶体途径(UPS)总输出中的潜在作用。 然而，通过最近的工作，现在已经认识到，蛋白酶体相反是一个焦点 UPS的监管。事实上，蛋白酶体的活性水平控制着蛋白质的分解率和压力。 抵抗。值得注意的是，蛋白酶体通常可以在没有毒性的情况下上调。广泛的工作已经完成 还表明，蛋白酶体在许多疾病状态下都受到损害，特别是在与衰老和 神经退行性疾病。然而，更深入和更可靠地了解 蛋白酶体对人类衰老和神经退行性变的作用显然需要使用活体哺乳动物模型 系统。为了直接测试蛋白酶体在老年动物中是否变得有限，三只转基因小鼠 这些线路被设计成允许有条件地提高蛋白酶体水平；其中两条线路已经 已经产生了。每个小鼠品系将有条件地表达不同蛋白酶体的野生型小鼠形式 子单元-RPN6、RPN11或5。之所以选择这些子单元，是因为它们来自现有数据 以限制蛋白酶体组装的速率。提高蛋白酶体水平的多种策略 增加成功的可能性，如果不止一种方法成功，它将增强对 随后的结果。对于每一个转基因，都有一个可抑制dox的构建体整合到rosa26中。 通过CRISPR/Cas9定位。转基因应该以组织特异性和时间控制的方式表达 举止。我们的第一个目标将是验证提高大脑、脊髓中蛋白酶体水平的策略 在™-3T3细胞中，转基因的靶向类似于rosa26。卓越的生物化学和 蛋白质组学方法用于量化蛋白酶体水平的变化。值得注意的是，全球蛋白质组学可以 现在量化蛋白酶体水平升高对这些细胞中数百种底物蛋白控制的影响 设置。我们将继续评估蛋白酶体表达升高对健康和衰老的影响。 这些动物。我们将测试蛋白酶体水平升高是否会影响自噬和蛋白平衡。 整个网络(PN)，并试图通过应用特定的应用程序来识别PN中的年龄相关漏洞 对系统的压力。转基因小鼠工作的中心焦点将是使用疾病模型来 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 肌萎缩侧索硬化症、亨廷顿病和肌萎缩侧索硬化症。