TDP43 Degradation by the Lysosomal Proteases in Amyotrophic Lateral Sclerosis

项目总结/摘要 肌萎缩侧索硬化症（ALS）是一种进行性的、一致致命的神经退行性疾病， 失去上下运动神经元。尽管进行了多项先进的临床试验， 可以稳定或逆转ALS运动缺陷的治疗。我追求K 08指导临床的目标 科学家研究生涯发展奖是为了获得知识和实践训练，使 这是我们对ALS中神经元丢失机制理解的重大进展。我的职业生涯，我 寻求开发新的治疗方法，以防止运动神经元死亡和保护功能。 2006年，Neumann及其同事发现TAR DNA结合蛋白43（TDP 43）在细胞内积累， 在患病的神经元内异常，并代表ALS的统一的终末期神经病理学标志。 有了这一观察结果，该领域随后集中在自噬的细胞过程上-- 细胞使用溶酶体来清除蛋白质聚集体并维持体内平衡--这是一个关键的途径， 在ALS发病机制中的作用然而，溶酶体是非常复杂的， 在神经元内。溶酶体内有许多复杂的水解酶，它们协助适应性应激 反应;它们广泛地促进细胞存活。虽然已经表明TDP 43可以被引导到 过去的努力已经停止了他们在溶酶体膜上的研究，假设 假设此后通过一系列未指定的酶有效地发生了TDP 43降解。 我的初步数据表明，只有一个溶酶体酶的子集（组织蛋白酶B，D，E，G，L，K，S， 和V）能够以pH依赖性方式降解TDP 43，并且导致ALS的TDP 43突变具有 可能会破坏这些酶的功能。因此，我假设组织蛋白酶B、D、E、G、L、K， S和V负责TDP 43降解，致病性TDP 43突变能够赋予 对这些组织蛋白酶的耐药性，以及受损的组织蛋白酶活性（无论是由于TDP 43突变还是年龄- 相关的pH变化）有助于随时间推移的TDP 43积累、TDP 43聚集和神经变性。 这个建议建立在我在神经生物学和神经学方面的坚实基础上，我已经培养了作为 我在本科、医学院、住院医师、研究员和早期教师期间都是一名基础研究科学家 年我目前的研究导师在以下领域有着有影响力的发现记录： 神经变性我还在加州大学旧金山分校组建了一个非常有成就的顾问团队来指导我 通过下一阶段的培训，我将成为一名独立调查员。我的训练计划是 专门为我提供指导，高级实验技能培训， 管理研究小组所需的经验。完成研究并获得技能， 本建议书中概述的指导将使我做好准备，以获得R 01或同等资金，开始我的 作为一名独立调查员。

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive, uniformly fatal neurodegenerative disorder caused by the loss of upper and lower motor neurons. Despite multiple advanced clinical trials, there are currently no therapies that can stabilize or reverse the motor deficits of ALS. My goal in pursuing a K08 Mentored Clinical Scientist Research Career Development Award is to acquire the knowledge and practical training to make major advances in our understanding of the mechanisms underlying neuronal loss in ALS. With my career, I seek to develop novel therapies that will prevent motor neuron death and preserve function. In 2006 Neumann and colleagues discovered that TAR DNA-binding protein 43 (TDP43) accumulated abnormally within diseased neurons and represented a unifying, end-stage neuropathologic hallmark of ALS. With this observation, the field then converged on the cellular process of autophagy — the method by which cells use lysosomes to purge proteins aggregates and maintain homeostasis — as a critical pathway involved in ALS’ pathogenesis. Lysosomes, however, are tremendously intricate and not well understood compartments within neurons. Lysosomes house many complex, hydrolytic enzymes; they assist in adaptive stress responses; and they promote cell survival broadly. While it has been shown that TDP43 can be directed to the lysosome for clearance, past efforts have stopped their investigations at the lysosomal membrane, presuming that presuming that TDP43 degradation happens efficiently by an array of unspecified enzymes thereafter. My preliminary data demonstrates that only a subset of lysosomal enzymes (cathepsins B, D, E, G, L, K, S, and V) can degrade TDP43 in a pH-dependent manner and that ALS-causing TDP43 mutations have the potential to disrupt the functions of these enzymes. Therefore, I hypothesize that cathepsins B, D, E, G, L, K, S, and V are responsible for TDP43 degradation, pathogenic TDP43 mutations are capable of conferring resistance to these cathepsins, and that impaired cathepsin activity (whether due to TDP43 mutations or age- related changes in pH) contributes to TDP43 buildup, TDP43 aggregation, and neurodegeneration over time. This proposal builds upon my solid foundation in neurobiology and neurology that I have cultivated working as a basic research scientist during my undergraduate, medical school, residency, fellowship, and early faculty years. My current research mentor has an established record of impactful discoveries in the field of neurodegeneration. I have also assembled a team of highly accomplished advisors at UCSF to guide me through this next phase of training on my path to becoming an independent investigator. My training plan is specifically designed to provide me with the mentorship, training in advanced experimental skills, and experience required to run a research group. Completing the research and obtaining the skill sets and mentorship outlined in this proposal will prepare me well to obtain R01 or equivalent funding to begin my career as an independent investigator.