The proteasome in aging and neurodegenerative disease

项目概要/摘要（项目2） 随着人类和其他哺乳动物年龄的增长，蛋白酶体活性显著下降。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 蛋白质代谢停滞和蛋白酶体活性下降可能在衰老相关的 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， 蛋白酶体在整体水平上控制蛋白质的稳定性。从历史上看， 蛋白酶体在决定泛素-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，现在认识到蛋白酶体恰恰相反是蛋白酶体的焦点。 UPS的监管。事实上，蛋白酶体的活性水平控制蛋白质的分解率和压力 阻力值得注意的是，蛋白酶体通常可以被上调而没有毒性。了大量的工作 还表明蛋白酶体在许多疾病状态中受损，特别是在与衰老相关的疾病中， 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影响显然需要使用体内哺乳动物模型 系统.为了直接测试蛋白酶体是否在老年动物中变得受限， 已经设计了允许蛋白酶体水平有条件升高的细胞系;其中两个细胞系已经 被生成。每个小鼠系将有条件地表达不同蛋白酶体的野生型鼠形式 亚单位-Rpn 6、Rpn 11或Rpn 5。之所以选择这些子单元，是因为它们是从现有数据中期望得到的 限制蛋白酶体组装的速率。提高蛋白酶体水平的多种策略 增加了成功的可能性，如果一种以上的方法成功，它将增强信心， 随后的结果。对于每种转基因，将floxed和dox可抑制的构建体整合到Rosa 26中。 基因座通过CRISPR/Cas9。转基因应在组织特异性和时间控制下表达 方式我们的第一个目标将是验证提高脑、脊髓和神经系统中蛋白酶体水平的策略。 Cord中，以及在具有类似地靶向Rosa 26的转基因的Flp-In™-3T3细胞中。优秀的生化和 存在定量蛋白酶体水平变化的蛋白质组学方法。值得注意的是，全球蛋白质组学可以 现在，他们量化了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的升高是否会影响自噬和蛋白稳态， 网络（PN）作为一个整体，并寻求通过应用特定的 对系统的压力。转基因小鼠研究的一个中心焦点是采用疾病模型， 评估蛋白酶体水平升高对神经退行性疾病进展的影响， tau蛋白病、亨廷顿病和ALS。

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.