Abnormal Late Endosomal Trafficking in Frontotemporal Dementia due to Progranulin Mutation

项目概要/摘要。 候选人：我的目标是成为一名独立的科学家，研究内溶酶体功能障碍， 神经退行性疾病，重点是内体成熟和运输。我的背景是 神经药理学和啮齿动物行为机制，但我最近转向研究异常 前颗粒蛋白不足小鼠的内体运输。在2012年获得博士学位后，这是我的最后一个周期， 获得K99/R 00奖励。我的目标是建立一个实验室来研究内溶酶体功能障碍， 额颞叶痴呆（FTD）的多种遗传模型。在这一领域建立专业知识后，我希望 研究阿尔茨海默病模型，并最终开始探索环境因素（例如， 锻炼、环境丰富等）与神经元内溶酶体功能相互作用以影响行为。 培训：除了我的主要导师Erik Roberson博士，我还组建了一个咨询委员会， 包括内溶酶体功能专家（James Collawn博士）和外泌体分析专家（Andrew West博士）， 还有大卫·斯坦德特博士，他是我们神经病学系的主任，接受过广泛的博士后培训 体验.我将出席国际会议并参加课程（UAB外部和内部）， 技术培训（细胞生物学、内体运输和外泌体分析）、专业发展（实验室 管理，预算，科学写作和任期过程），以及研究的道德行为。 研究：颗粒蛋白前体（GRN）的功能丧失突变是FTD的主要原因， 通过颗粒蛋白前体单倍不足引起FTD。本提案将检验FTD由于 颗粒蛋白前体（GRN）突变（FTD-GRN）是由内溶酶体功能障碍引起的。我们假设 颗粒蛋白前体不足会损害溶酶体活性，从而破坏晚期内体运输， 导致人体内的FTD并扰乱小鼠的行为。颗粒蛋白前体对于正常溶酶体功能至关重要， 完全的颗粒蛋白前体缺乏引起溶酶体贮积症。在初步研究中， 观察到来自FTD-GRN患者的血浆中的外泌体水平升高，以及前颗粒蛋白不足， (Grn+/-）小鼠血浆和原代神经元培养基。在目标1中，我们将测试这是否增强了 外泌体的产生反映了多泡体（MVB）远离溶酶体运输的转变 降解并朝向外泌体分泌。在目标2A中，我们将确定溶酶体功能障碍是否导致这种情况。 增强的外泌体分泌，在目标2B中，我们将确定这种内溶酶体功能障碍是否导致 Grn+/-小鼠的行为缺陷。在目标3中，我们将比较晚期内体/溶酶体蛋白和MVB的水平。 FTD-GRN患者与健康对照和阿尔茨海默病患者的脑形态学对照， 神经变性的非特异性影响。这些研究将给我提供原代神经元培养的经验， 内体追踪分析、shRNA基因敲低和外泌体分离和分析。

Project Summary/Abstract. Candidate: My goal is to become an independent scientist investigating endolysosomal dysfunction in neurodegenerative disease, with a focus on endosomal maturation and trafficking. My background is in neuropharmacology and mechanisms of rodent behavior, but I have recently shifted to investigating abnormal endosomal trafficking in progranulin-insufficient mice. Having received my PhD in 2012, this is my final cycle of eligibility for a K99/R00 award. My goal is to establish a laboratory to investigate endolysosomal dysfunction in multiple genetic models of frontotemporal dementia (FTD). After establishing expertise in this field, I hope to investigate models of Alzheimer’s disease and eventually begin to explore how environmental factors (ex. exercise, environmental enrichment, etc.) interact with neuronal endolysosomal function to influence behavior. Training: In addition to my primary mentor, Dr. Erik Roberson, I have assembled an advisory committee that includes experts on endolysosomal function (Dr. James Collawn) and exosome analysis (Dr. Andrew West), as well as Dr. David Standaert, the chair of our Department of Neurology with extensive postdoctoral training experience. I will present at international meetings and take courses (both external and internal to UAB) for technical training (cell biology, endosomal trafficking, and exosome analysis), professional development (lab management, budgeting, scientific writing, and the tenure process), and ethical conduct of research. Research: Loss-of-function mutations in progranulin (GRN) are a major cause of FTD, and are thought to cause FTD through progranulin haploinsufficiency. This proposal will test the hypothesis that FTD due to progranulin (GRN) mutations (FTD-GRN) is caused by endolysosomal dysfunction. We hypothesize that progranulin insufficiency impairs lysosomal activity, which disrupts late endosomal trafficking and ultimately causes FTD in humans and disrupts behavior in mice. Progranulin is critical for normal lysosomal function as complete progranulin deficiency causes a lysosomal storage disorder. In preliminary studies, we have observed elevated levels of exosome in plasma from FTD-GRN patients, as well as progranulin-insufficient (Grn+/–) mouse plasma and primary neuron culture media. In aim 1, we will test whether this enhanced exosome production reflects a shift in trafficking of multivesicular bodies (MVBs) away from lysosomal degradation and toward exosome secretion. In aim 2A we will determine if lysosomal dysfunction causes this enhanced exosome secretion and in aim 2B we will determine if this endolysosomal dysfunction causes behavior deficits in Grn+/– mice. In aim 3 we will compare levels of late endosomal/lysosomal proteins and MVB morphology in brains of FTD-GRN patients with healthy controls and Alzheimer’s disease patients to control for nonspecific effects of neurodegeneration. These studies will give me experience with primary neuronal culture, endosomal tracking analysis, shRNA gene knockdown, and exosome isolation and analysis.