Interaction of the Microtubule Cytoskeleton and Perilipin-2 Regulates Hepatic Lipid Droplets - a Potential Therapeutic Target for Fatty Liver Disease

项目总结/摘要 本研究的总体目标是靶向肝细胞脂滴（LDs）， 脂肪肝的治疗方法肝脂肪变性，即肝细胞LD内的脂肪积聚，是一个关键因素， 脂肪性肝炎的前体，可导致肝硬化和肝细胞癌，需要肝脏 移植不幸的是，肝脂肪变性也限制了移植可接受的供体肝脏的数量， 具有严重脂肪变性的供体肝脏具有原发性无功能的高风险。因此， 逆转性脂肪肝对于有脂肪性肝炎和肝硬化风险的患者很重要， 供肝移植的供体库。我们的工作集中在肝细胞LD在此显示三个关键 观察：a）围脂蛋白-2（PLIN 2），一种调节脂肪储存和利用的LD相关蛋白，共 与微管定位并共免疫沉淀; B）用诺考达唑破坏微管， 微管靶向剂（MTA）使PLIN 2与LD解偶联，促进LD融合并产生脱脂（即， 具有最小细胞毒性的脂肪变性肝细胞的LD内容物的胞吐分泌;和c）诺考达唑 促进离体肝组织的脂肪损失，而对组织活力没有影响。根据这些初步数据，我们 提出了中心假设，即肝细胞LD受PLIN 2和 微管细胞骨架，这种相互作用是一个潜在的治疗靶点，以减少脂质负担， 肝细胞在脂肪肝中的作用我们的具体目标将测试三个假设。首先，我们将测试 假设微管直接与LD相关PLIN 2相互作用。第二，我们将检验假设 微管扰动增加脂肪分解并导致脂肪变性肝细胞去脂。最后我们 将检验以下假设，即离体脂肪变性肝组织的治疗和脂肪变性肝的离体灌注 在器官挽救的临床前模型中，MTA将导致肝细胞脱脂和肝功能改善 和康复。这项工作将为LD生物学的新兴领域提供基础信息， 计划证明一种新的细胞机制控制肝细胞LD动力学，并确定 脂肪变性肝脏脱脂的治疗策略。候选人是一位基础科学家和肝脏移植专家， 致力于使用基础科学方法改善肝病患者的生活。她是助理 马约诊所的医学教授，导师团队由Vijay Shah博士和Greg Gores博士组成。她有 保护研究和培训活动的时间，为设备和用品提供专门的机构资金， 一个强有力的职业发展计划，包括具体的活动和基准，使她能够 独立的研究生涯。全面的培训计划将允许候选人完成她的 目前的研究计划，并寻求独立的研究者地位，她将翻译这些和未来的研究 到人体试验和未来对患者的治疗干预。

PROJECT SUMMARY/ABSTRACT The overall objective of this research is to target hepatocyte lipid droplets (LDs) for the development of therapeutic interventions for fatty liver disease. Hepatic steatosis, fat accumulation within hepatocyte LDs, is a key precursor for steatohepatitis which can lead to cirrhosis and hepatocellular cancer, necessitating liver transplantation. Unfortunately, hepatic steatosis also limits the number of donor livers acceptable for transplant as donor livers with severe steatosis have a high risk of primary nonfunction. As such, therapeutic strategies to reverse fatty liver are important for patients at risk for steatohepatitis and cirrhosis as well as to increase the donor pool of viable livers for transplantation. Our work focused on hepatocyte LDs herein shows three key observations: a) perilipin-2 (PLIN2), a LD-associated protein which regulates fat storage and utilization, co- localizes with and co-immunoprecipitates microtubules; b) microtubule disruption with nocodazole, a blunt microtubule targeting agent (MTA), uncouples PLIN2 from the LD, promotes LD fusion and yields delipidation (i.e. exocytotic secretion of LD contents) of steatotic hepatocytes with minimal cytotoxicity; and c) nocodazole promotes fat loss from ex vivo liver tissue with no effect on tissue viability. Based on these preliminary data, we propose the CENTRAL HYPOTHESIS that hepatocyte LDs are regulated by an interaction between PLIN2 and the microtubule cytoskeleton, and that this interaction is a potential therapeutic target to decrease the lipid burden of hepatocytes in fatty liver disease. Our SPECIFIC AIMS will test three hypotheses. FIRST, we will test the hypothesis that microtubules directly interact with LD-associated PLIN2. SECOND, we will test the hypothesis that microtubule perturbation increases lipolysis and leads to delipidation of steatotic hepatocytes. FINALLY, we will test the hypothesis that treatment of ex vivo steatotic liver tissue and ex vivo perfusion of steatotic livers with MTAs will lead to delipidation of hepatocytes and improved liver function in a pre-clinical model of organ salvage and rehabilitation. This work will provide foundational information to the emerging field of LD biology and is projected to demonstrate a novel cellular mechanism controlling hepatocyte LD dynamics and to identify therapeutic strategies to defat steatotic livers. The candidate is a basic scientist and transplant hepatologist, dedicated to improving the lives of patients with liver disease using a basic science approach. She is an Assistant Professor of Medicine at Mayo Clinic with a mentorship team consisting of Drs. Vijay Shah and Greg Gores. She has protected time for research and training activities, dedicated institutional funding for equipment and supplies, and a robust career development plan with specific activities and benchmarks that will position her for an independent research career. The comprehensive training plan will allow for the candidate to complete her current research plan and seek independent investigator status where she will translate these and future studies to human trials and future therapeutic interventions for patients.