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

项目总结/摘要： 这项建议是在申请独立之路奖博士特拉维斯李尔在老龄化研究所在 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有着丰富的经验， 炎症、自噬和溶酶体生物学。K99/R 00将是李尔博士的关键一步， 实现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，以寻求新的发现 治疗途径根据这项K99/R 00合同，李尔博士将有时间进行额外的培训， 神经生物学技术和衰老生物学，并为职业发展，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， 衰老研究所的科学环境对李尔博士的训练非常理想。科学技术培训 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)神经变性小鼠模型的产生和表型表征。李尔王博士的领导 培训将侧重于：1）通过网络融入科学界，2）提高他的指导技能， 3)改善花岗岩。成功完成拟议的研究和培训计划将提供 必要的知识和经验，以实现李尔博士的职业目标，成为一个独立的 研究神经元老化和阿尔茨海默病（AD）的研究者。为了实现这一目标，李尔王博士建议： 研究蛋白水解控制溶酶体活化以增强致病性tau加工新机制 AD模型中的蛋白质聚集体。具体来说，李尔博士详细阐述了一个模型，其中一个关键的mTORC 1 抑制蛋白KPTN受E3泛素连接酶PDZRN 3的有效控制，PDZRN 3可泛素化和命运 降解的KPTN。此外，无偏筛选产生了增加KPTN的小分子KPTN激活剂， 蛋白水平，抑制mTORC 1活性，并增加溶酶体数量和活性。令人兴奋的是， KPTN的药理学增强在体外减少了tau蛋白聚集，其具有治疗作用。 对阿尔茨海默病治疗的影响。因此，KPTN扩大的净效应将有助于 通过激活自噬清除毒性蛋白聚集体。这就引出了一个中心假设， KPTN的PDZRN 3控制影响神经元溶酶体活性，并且遗传或药理学上 KPTN的活化可能是减少tau蛋白聚集体的途径。两个目标将审问这个 假设：（1）研究PDZRN 3-KPTN轴对tau聚集的机制和生物学作用， 体外诱导多能干细胞（iPSC）和原代细胞模型，和（2）检查这种机制， 在tau扩散的动物模型中使用遗传和药理学方法的作用。李尔王博士还将继续 一个结构化的培训计划，包括正式的课程工作、会议和新技术的实践培训， 在主要导师Toren Finkel博士和共同导师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.