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） 蛋白酶体活动的显着下降被认为是人类和其他哺乳动物年龄。这是 在许多组织中观察到，包括大脑和运动神经元。衰老的特征是受损 蛋白质静态和蛋白酶体活性下降可能在与衰老相关的缺陷中起重要作用 蛋白质的蛋白质癌症，蛋白质在蛋白质动力学中的关键作用。通过降解泛素结合物， 蛋白酶体在全球水平上控制蛋白质稳定性。历史上对 蛋白酶体在确定泛素 - 蛋白酶体途径（UPS）的总体输出中的潜在作用。 然而，通过最近的工作，现在已经认识到蛋白酶体是对比度的 UPS的调节。实际上，蛋白酶体活动的水平控制蛋白质分解率和应力 值得注意的是，蛋白酶体通常可以上调而没有毒性。广泛的工作 还表明，蛋白酶体在许多疾病状态下受到损害，特别是在衰老相关和 神经退行性疾病。但是，更深入，更可靠地了解 人类蛋白酶体至衰老和神经变性显然需要使用体内哺乳动物模型 系统。要直接测试蛋白酶体是否在老年动物中限制，三只转基因小鼠 线条已设计为允许有条件的蛋白酶体水平升高。其中两个已经 我们是生成的。每条鼠标线将有条件地表达不同蛋白酶体的野生型鼠形式 亚基 - rpn6，rpn11或5。选择这些亚基是因为它们是从现有数据中预期的 蛋白酶体组件的速率限制。提高蛋白酶体水平的策略多样性 增加成功的可能性，如果多种方法成功，它将增强对 随后的结果。对于每个转换 通过CRISPR/CAS9的基因座。这些翻译应在组织特异性和暂时控制中表达 方式。我们的第一个目标是验证升高大脑蛋白质组水平的策略，脊柱 绳索，在flp-in™-3T3细胞中具有类似针对ROSA26的翻译。优秀的生化和 存在用于量化蛋白酶体水平改变的蛋白质组学方法。值得注意的是，全球蛋白质组学可以 现在量化升高的蛋白质组水平对这些在这些中的数百种底物蛋白的影响 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。我们将测试升高的蛋白酶体水平是否影响自噬和蛋白质抗体 整个网络（PN），试图通过应用特定 对系统的压力。关于转基因小鼠的工作的主要重点是采用疾病模型 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 陶氏病，亨廷顿氏病和ALS。