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.