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: 10600015
• 负责人: Ling Hao
• 金额: $ 38.83万
• 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/10600015
• 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; Half-Life; Human; Impairment; Induced pluripotent stem cell derived neurons; Knock-out; Label; Lysosomes; Mass Spectrum Analysis; Measures; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Mutation; Neurites; Neurodegenerative Disorders; Neurons; Organelles; PGRN gene; Pathologic; Pathway interactions; Patients; Peptide Hydrolases; Peroxidases; Persons; Phenotype; Proteasome Inhibitor; Proteins; Proteomics; Reporting; Research; Site; Techniques; Technology; Therapeutic; ascorbate; curative treatments; extracellular; improved; innovation; insight; lysosomal proteins; macromolecule; neuronal growth; pharmacologic; 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 分子机制仍不清楚。GRN基因突变，导致缺乏 颗粒蛋白前体（PGRN）是一种溶酶体糖蛋白，可引起额颞叶痴呆（FTD）。PGRN丢失 破坏溶酶体活性，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 缺乏高通量的方法来鉴定和定量参与高度动态的蛋白质， 有时是短暂的溶酶体活性。长期目标是确定药物途径， FTD的治疗靶点。本提案的总体目标是：1）发展质谱分析法 （MS）为基础的蛋白质组学策略，以表征时空溶酶体的相互作用，组成， 在人类诱导多能干细胞（iPSC）衍生的神经元中的功能退化，以及2）评估如何 PGRN缺乏损害人神经元中的动态溶酶体活性和表型。中央 假设是颗粒蛋白前体损失损害了人中溶酶体相互作用和降解功能 神经元，可以通过基于MS的蛋白质组学策略和细胞生物学的组合来确定 接近。这些目标将通过以下三个具体目标实现： 确定颗粒蛋白前体的丢失是否损害动力学的时空溶酶体邻近标记方法 溶酶体的相互作用和组成。目的2：开发动态SILAC蛋白质组学 一种确定颗粒蛋白前体的丢失是否改变了依赖自噬的蛋白质的周转率的方法， 神经元的退化目的3：确定细胞外添加颗粒蛋白前体是否可以拯救蛋白质组 人神经元中颗粒蛋白前体缺乏引起的变化和FTD表型。拟议研究 在技术和生物学方面都具有很高的创新性。它独特地集成了创新的基于MS的 蛋白质组学技术与先进的人类iPSC-神经元平台和细胞生物学方法， 表征FTD的动态溶酶体活性和分子机制。如果细胞外 颗粒蛋白前体确实挽救了蛋白质组学变化和FTD表型， 是一种非常有前景的治疗方法，可用于开发FTD的疾病缓解治疗。