The autophagy lysosomal pathway: regulation by progranulin and its role in neurodegenerative disease

项目总结 蛋白质错误折叠和破坏正常蛋白质稳态在许多神经退行性变中起关键作用 疾病，包括阿尔茨海默病(AD)和额颞痴呆(FTD)。蛋白质动态平衡失调 是导致衰老的重要因素，也是神经退行性疾病的最重要风险因素。普罗米林 GRN是一种保守的蛋白质，与正常衰老和神经退化有关。单倍体功能不全原因 FTD，无效合子导致神经元蜡样脂褐素沉积症-一种溶酶体储存障碍，以及 GRN基因可能会增加包括阿尔茨海默病在内的常见神经退行性疾病的风险。我们假设 GRN缺乏通过损害自噬/溶酶体途径(ALP)而导致神经退化，a 蛋白质平衡网络(PN)的关键臂，碱性磷酸酶缺陷导致神经退行性疾病 更广泛地说。我们进行了全基因组筛查，以发现GRN水平的修饰物和富含 阿尔卑斯山。我们发现，GRN与初级神经元中的碱性磷酸酶之间存在相互作用关系：GRN水平是 在自噬和GRN缺乏的调控下，溶酶体受损，ALP通量受到抑制。有趣的是，基因 我们屏幕上提高GRN水平的修饰物拯救了GRN引起的溶酶体和自噬缺陷 缺乏症。 我们的项目将研究碱性磷酸酶功能与神经退行性变和 探索碱性磷酸酶作为治疗靶点。在目标1中，我们将研究GRN缺乏对ALP和其他Pn的影响 组件。我们将使用蛋白质组学、脂质组学和RNAseq来定义GRN缺乏如何改变细胞和 溶酶体生理学。此外，我们将使用创新的高通量纵向单细胞分析 机器人显微镜平台，配有碱性磷酸酶和其他PN臂的生物传感器，以确定GRN缺乏 动态地影响他们的功能，并对压力做出反应。在目标2中，我们将调查GRN缺乏如何 而碱性磷酸酶功能障碍导致Tar DNA结合蛋白43(TDP43)积聚，影响细胞代谢 其他与神经退行性疾病有关的蛋白质。在AIM 3中，凯利和芬克贝纳实验室将合作 识别新的小分子来调节自噬途径。在初步研究中，新的自噬 确定了促进几种致病蛋白(例如tau，突触核蛋白， TDP43和突变体Huntingtin)和减轻分化的人神经元的神经退行性变表型 来自ALS和HD患者的IPSCs。新的安全有效的小分子自噬的发现 诱导剂将是有用的研究工具，并可能形成新的治疗方法的基础 神经退行性疾病。 该项目将通过提供ALP的专业知识和新颖的分析方法，与更广泛的计划协同工作， 神经退行性疾病的创新IPSC模型和调节ALP的新工具。与以下项目协作 该计划中的其他项目将帮助该项目将碱性磷酸酶置于更广泛的蛋白质平衡网络的背景下。

PROJECT SUMMARY Protein misfolding and disruption of normal protein homeostasis play critical roles in many neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). Protein dyshomeostasis is an important contributor to aging, the most important risk factor for neurodegenerative disease. Progranulin (GRN) is a conserved protein with links to normal aging and neurodegeneration. Haploinsufficiency causes FTD, nullizygosity causes neuronal ceroid lipofuscinosis—a lysosomal storage disorder, and polymorphisms in the GRN gene may increase the risk of common neurodegenerative diseases, including AD. We hypothesize that GRN deficiency leads to neurodegeneration by impairing the autophagy/lysosomal pathway (ALP), a critical arm of the proteostasis network (PN), and that ALP deficits contribute to neurodegenerative diseases more broadly. We performed a genome-wide screen to discover modifiers of GRN levels and hits enriched in the ALP. We found a reciprocal relationship between GRN and the ALP in primary neurons: GRN levels were regulated by autophagy and GRN deficiency impaired lysosomes and inhibited ALP flux. Interestingly, genetic modifiers from our screen that raised GRN levels rescued lysosomal and autophagy deficits caused by GRN deficiency. Our project will investigate the mechanistic relationship between ALP function and neurodegeneration and explore ALP as a therapeutic target. In Aim 1, we will study how GRN deficiency affects the ALP and other PN components. We will use proteomics, lipidomics, and RNAseq to define how GRN deficiency alters cell and lyososome physiology. In addition, we will use an innovative high-throughput longitudinal single-cell analysis platform, robotic microscopy, with biosensors for ALP and other arms of PN to determine how GRN deficiency affects their function dynamically and in response to stress. In Aim 2, we will investigate how GRN deficiency and ALP dysfunction lead to accumulation of Tar DNA binding protein 43 (TDP43) and affect the metabolism of other proteins linked to neurodegenerative disease. In Aim 3, the Kelly and Finkbeiner labs will collaborate to identify new small molecules to modulate the autophagy pathway. In preliminary studies, novel autophagy inducers were identified that promote the clearance of several disease-causing proteins (e.g., tau, synuclein, TDP43 and mutant huntingtin) and mitigate neurodegeneration phenotypes in human neurons differentiated from iPSCs of patients with ALS and HD. The discovery of new, safe and effective small-molecule autophagy inducers would be useful research tools and may form the basis for new therapeutic approaches to neurodegenerative disease. This project will synergize with the broader program by providing expertise and novel assays of the ALP, innovative iPSC models of neurodegenerative disease and new tools to modulate the ALP. Collaboration with other projects in the program will help this project put ALP in the context of the broader proteostasis network.