TDP43 Degradation by the Lysosomal Proteases in Amyotrophic Lateral Sclerosis

项目摘要/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，由 上下运动神经元的缺失。尽管有多项先进的临床试验，但目前还没有 可以稳定或逆转肌萎缩侧索硬化症运动障碍的治疗方法。我追求K08指导临床的目标 科学家研究职业发展奖是为了获得知识和实践训练而做出的 我们对肌萎缩侧索硬化症神经元丢失机制的了解取得了重大进展。在我的职业生涯中，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和他的同事发现TAR DNA结合蛋白43(TDP43)积累 在病变神经元内出现异常，代表ALS统一的终末期神经病理特征。 有了这种观察，磁场就会聚在自噬的细胞过程上--通过这种方法 细胞使用溶酶体清除蛋白质聚集体并维持体内平衡--这是参与其中的关键途径 在ALS的发病机制中起重要作用。然而，溶酶体是极其复杂的，并且没有被很好地理解。 在神经元内部。溶酶体内含有许多复杂的水解酶；它们有助于适应压力。 反应；它们广泛地促进细胞存活。虽然已经表明TDP43可以定向到 为了清除溶酶体，过去的努力已经停止了他们对溶酶体膜的调查，假设 假设TDP43随后通过一系列未指明的酶有效地降解。 我的初步数据表明，只有溶酶体酶(组织蛋白酶B、D、E、G、L、K、S、 和V)可以pH依赖的方式降解TDP43，并且导致ALS的TDP43突变具有 有可能破坏这些酶的功能。因此，我假设组织蛋白酶B，D，E，G，L，K， S和V负责TDP43的降解，致病的TDP43突变能够赋予 对这些组织蛋白酶的抵抗，以及组织蛋白酶活性受损(无论是由于TDP43突变还是年龄- PH的相关变化)有助于TDP43的积聚、TDP43的聚集和随时间推移的神经退化。 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，这是我在工作中培养出来的 我在本科、医学院、实习、研究员和早期教职期间是一名基础研究科学家 好几年了。我目前的研究导师在以下领域有着卓有成效的发现： 神经退行性变。我还在加州大学旧金山分校组建了一个由非常有成就的顾问组成的团队来指导我 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，以及 管理一个研究小组所需的经验。完成研究并获得技能集和 这份建议书中概述的导师计划将使我做好充分准备，以便获得R01或同等资金，开始我的 独立调查员的职业生涯。

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.