Modulating Alzheimer’s Disease by mTORC1 inhibition to augment lysosomal activity

项目摘要/摘要： 这项提案是为老年研究所的特拉维斯·李尔博士申请独立之路奖的，地址是 匹兹堡大学。李尔博士在衰老过程中泛素化的分子生物学方面拥有丰富的经验， 炎症、自噬和溶酶体生物学。这款K99/R00将是李尔博士作为他的 达到研究独立的职业目标。李尔博士未来实验室的重点将是研究 神经退行性变中蛋白质降解和溶酶体活性的机制 治疗途径。在这个K99/R00奖项下，李尔博士将保护额外的培训时间 神经生物学技术和衰老生物学，以及职业发展，以促进成功过渡到 研究独立性。这个由衰老、溶酶体和神经生物学专家组成的导师团队结合了 老龄研究所的科学环境将是李尔博士训练的理想环境。科学技能培训 将需要1)体外原代组织培养技能，2)IPSC发育、分化和基因编辑的研究， 3)小鼠神经退行性变模型的建立及表型特征。李尔博士的领导力 培训的重点是1)通过网络融入科学界，2)提高他的指导技能， 3)提高授权度。成功完成拟议的研究和培训计划将提供 向李尔博士的事业目标--成为独立人士--迈进所需的知识和经验 研究神经元老化和阿尔茨海默病(AD)的研究员。为了实现这一目标，李尔博士建议 蛋白水解性调控溶酶体激活增强病原性牛磺酸加工的新机制研究 阿尔茨海默病模型中的蛋白质聚集。具体地说，李尔博士详细阐述了一个模型，在该模型中，一个关键的mTORC1 抑制蛋白Kptn受E3泛素连接酶PDZRN3的有效控制，该酶泛素化和命运 Kptn用于降解。此外，无偏筛选产生了小分子Kptn激活剂，它增加了Kptn 蛋白水平，抑制mTORC1活性，增加溶酶体数量和活性。令人兴奋的是， Kptn的药理增强作用可降低tau蛋白的体外聚集，具有治疗作用 对阿尔茨海默病治疗的启示。因此，Kptn扩大的净影响将有助于 通过激活自噬来清除有毒蛋白质聚集体。这导致了一个中心假设，即 PDZRN3调控Kptn影响神经元溶酶体活性以及遗传或药理学 Kptn的激活可能是减少tau蛋白聚集的途径之一。两个目标将审问这一点 假设：(1)研究PDZRN3-Kptn轴对Tau聚集的机制和生物学效应。 使用可诱导多能干细胞(IPSC)和原代细胞模型进行体外培养，以及(2)研究这一机制和 遗传学和药理学方法在tau扩散动物模型中的作用。李尔博士还将继续 包括正式课程工作、会议和新技术实践培训在内的结构化培训计划； 在主要导师Toren Finkel博士、共同导师Stacey Rizzo博士和Bill Chen博士的指导下。

PROJECT SUMMARY/ABSTRACT: This proposal is in application for a Pathway to Independence Award for Dr. Travis Lear at the Aging Institute at the University of Pittsburgh. Dr. Lear has extensive experience in the molecular biology of ubiquitination in aging, inflammation, autophagy, and lysosome biology. This K99/R00 would be a crucial step for Dr. Lear as part of his career goal of reaching research independence. The focus of Dr. Lear’s future lab will be to study the mechanisms of protein degradation and lysosomal activity in neurodegeneration in pursuit of uncovering new therapeutic avenues. Under this K99/R00 award, Dr. Lear would have protected time for additional training in neurobiology techniques and aging biology, and for career development to facilitate a successful transition to research independence. This mentorship team of experts in aging, lysosomal, and neurobiology combined with the scientific environment at the Aging Institute will be ideal for Dr. Lear’s training. The scientific expertise training will entail 1) in vitro primary tissue culture skill, 2) study of iPSC development, differentiation, and gene-editing, 3) generation and phenotypic characterization of mouse models of neurodegeneration. Dr. Lear’s leadership training will focus on 1) integration to scientific community through networking, 2) enhancing his mentoring skills, 3) improving grantsmanship. Successful completion of the proposed research and training plan will provide the knowledge and experience necessary to progress toward Dr. Lear’s career goal of becoming an independent investigator studying neuronal aging and Alzheimer’s Disease (AD). To accomplish this, Dr. Lear proposes to study a new mechanism of proteolytic control of lysosomal activation to augment processing of pathogenic tau protein aggregates in models of AD. Specifically, Dr. Lear has elaborated a model in which a key mTORC1 inhibitor protein, KPTN, is potently controlled by the E3 ubiquitin ligase PDZRN3, which ubiquitinates and fates KPTN for degradation. Also, unbiased screening yielded a small molecule KPTN activator which increases KPTN protein level, inhibits mTORC1 activity, and increases lysosomal number and activity. Excitingly, pharmacological augmentation of KPTN reduces tau protein aggregation in vitro, which has therapeutic implications for the treatment of Alzheimer’s Disease. The net effect of KPTN augmentation would therefore aid in clearance of toxic protein aggregates by activation of autophagy. This leads to the central hypothesis that PDZRN3 control of KPTN affects neuronal lysosomal activity and that genetic or pharmacological activation of KPTN may be an avenue to reduce tau protein aggregates. Two aims will interrogate this hypothesis: (1) To examine the mechanism and biologic effect of the PDZRN3-KPTN axis on tau-aggregation in vitro with inducible pluripotent stem cell (iPSC) and primary cell models, and (2) to examine this mechanism and effect using genetic and pharmacological approaches in animal models of tau spreading. Dr. Lear will also pursue a structured training plan including formal course work, conferences, and hands-on training of new techniques, under the guidance of primary mentor Dr. Toren Finkel, and co-mentors Dr. Stacey Rizzo and Dr. Bill Chen.