Development of Mass Spectrometry Strategies to Decipher Dynamic Lysosomal Dysfunctions in Frontotemporal Dementia

开发质谱策略来破译额颞叶痴呆的动态溶酶体功能障碍

• 批准号: 10392451
• 负责人: Ling Hao
• 金额: $ 38.88万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10392451
• 关键词: Age; Autophagocytosis; Biological; Cells; Cellular biology; Climacteric; Data; Defect; Dementia; Development; Disease; Engineering; Etiology; Exhibits; Frontotemporal Dementia; Functional disorder; GRN gene; Genetic; Glycoproteins; Goals; Growth; Half-Life; Human; Impairment; Induced pluripotent stem cell derived neurons; Knock-out; Label; Lead; Lysosomes; Mass Spectrum Analysis; Measures; Membrane; Methodology; Methods; Modeling; Molecular; Mutation; Neurites; Neurodegenerative Disorders; Neurons; Organelles; PGRN gene; Pathologic; Pathway interactions; Patients; Peptide Hydrolases; Peroxidases; Persons; Pharmacology; Phenotype; Proteasome Inhibitor; Proteins; Proteomics; Reporting; Research; Site; Techniques; Therapeutic; ascorbate; base; curative treatments; extracellular; improved; induced pluripotent stem cell; innovation; insight; lysosomal proteins; macromolecule; protein TDP-43; protein degradation; spatiotemporal; therapeutic target

Lysosomal dysfunctions have been extensively associated with neurodegenerative diseases, but the molecular mechanisms remain unclear. Mutations in the GRN gene, which lead to the deficiency of progranulin (PGRN), a lysosomal glycoprotein, cause Frontotemporal dementia (FTD). Loss of PGRN impairs lysosomal activity, but the precise function of PGRN in lysosomes is unknown. This is, in part, due to a lack of high-throughput methodologies to identify and quantify proteins involved in the highly dynamic and sometimes transient lysosomal activities. The long-term goal is to identify druggable pathways and therapeutic targets for FTD. The overall objectives of this proposal are to 1) develop mass spectrometry (MS)-based proteomic strategies to characterize spatiotemporal lysosomal interaction, composition, and degradative function in human Induced pluripotent stem cell (iPSC)-derived neurons and 2) evaluate how PGRN deficiency impairs dynamic lysosomal activities and phenotypes in human neurons. The central hypothesis is that loss of progranulin impairs lysosomal interaction and degradative function in human neurons, which can be determined by a combination of MS-based proteomic strategies and cell biology approaches. The objectives will be achieved by the following three specific aims: Aim 1: Develop spatiotemporal lysosomal proximity labeling methods to determine if loss of progranulin impairs dynamic lysosomal interaction and composition in human neurons. Aim 2: Develop a dynamic SILAC proteomic method to determine if loss of progranulin alters the turnover rates of proteins that rely on autophagy for degradation in neurons. Aim 3: Determine if extracellular addition of progranulin can rescue the proteomic changes and FTD phenotype caused by progranulin deficiency in human neurons. The proposed research is highly innovative both in technological and biological aspects. It uniquely integrates innovative MS-based proteomic techniques with the advanced human iPSC-neuron platform and cell biology approaches to characterize dynamic lysosomal activities and molecular mechanisms underlying FTD. If extracellular progranulin does rescue the proteomic changes and FTD phenotype, then increasing progranulin levels can be a very promising therapeutic method to develop disease modifying treatments for FTD.

溶酶体功能障碍与神经退行性疾病广泛相关，但 分子机制尚不清楚。GRN基因突变，导致GRN缺乏 前颗粒蛋白(PGRN)是一种溶酶体糖蛋白，可引起额颞叶痴呆(FTD)。PGRN的丢失 损害溶酶体的活性，但PGRN在溶酶体中的确切功能尚不清楚。这在一定程度上是由于 由于缺乏高通量方法来鉴定和量化参与高度动态的蛋白质 有时还有一过性的溶酶体活动。长期目标是确定可用药的途径和 FTD的治疗靶点。这项提议的总体目标是：1)发展质谱学 基于(MS)的蛋白质组学策略来表征时空溶酶体的相互作用、组成和 人诱导多能干细胞(IPSC)来源神经元的退化功能和2)评估 PGRN缺乏会损害人神经元的动态溶酶体活性和表型。中环 假设原颗粒的丢失会损害人类溶酶体的相互作用和降解功能 神经元，可以通过基于MS的蛋白质组策略和细胞生物学的组合来确定 接近了。这些目标将通过以下三个具体目标来实现：目标1：发展 时空溶酶体邻近标记方法确定原颗粒丢失是否损害动力 人类神经元中溶酶体的相互作用和组成。目的2：建立动态SILAC蛋白质组 确定原颗粒丢失是否改变依赖于自噬的蛋白质的周转率的方法 神经元的退化。目的3：确定细胞外添加原颗粒是否可以挽救蛋白质组 原颗粒缺乏引起的人类神经元的变化和FTD表型。拟议的研究 在技术和生物方面都具有很高的创新性。它独一无二地集成了基于MS的创新 采用先进的人iPSC-神经元平台的蛋白质组学技术和细胞生物学方法 描述FTD的动态溶酶体活性和分子机制。如果在细胞外 原颗粒能挽救蛋白质组变化和FTD表型，然后提高原颗粒水平 可能是一种非常有前途的治疗方法，用于开发FTD的疾病修正治疗方法。