Lysosomes are membrane-bound degradative compartments that break down macromolecules from endocytic, phagocytic and autophagic pathways, and serve the role of key metabolic and signaling hubs. Accumulating evidence suggests that in Alzheimer's disease and other neurodegenerative disorders lysosomes fail to correctly perform their functions. However, due to the paucity of tools to study organelles in vivo, so far there has been no systematic assessment of lysosomal alterations during progression of Alzheimer's disease, and the exact molecular nature of the proposed impairments is not known. Our understanding of the involvement of the lysosome in the disease is further limited because lysosomes are rare, constituting <3% of the cell. Here, we seek to combine powerful, state-of-the-art approaches including recently developed rapid lysosomal isolations (LysoIPs) and unbiased proteomic and metabolomic analyses to determine if and how lysosomes change in vivo in murine models of Alzheimer's disease. We propose to focus on lysosomes isolated from neurons and microglia which we expect to be critical to the pathology of the disease. Complex reciprocal interactions between neurons and microglia are essential for regulation of the most important aspects of brain function, and we hypothesize that alterations of the endolysosomal systems in these two cell types compromise the integrity of the central nervous system. Here, in Aim I we propose to define lysosomal alterations in neurons and microglia over a time course of Alzheimer's disease progression generating a dynamic atlas of lysosomal proteins and metabolites in these cells. This aim will generate novel mouse models and robust protocols enabling rapid lysosomal isolations from neurons and microglia. In Aim 2 we will validate bioinformatically filtered candidates from Aim 1, paving the way for future mechanistic dissections. The proposed research utilizes innovative technologies and concepts to address fundamental molecular aspects of pathobiology of Alzheimer's disease, building a comprehensive atlas of in vivo lysosomal changes in neurons and microglia. We believe that this work will shed light on novel aspects of lysosomal biology in the brain and has the potential to transform our understanding of the mechanistic basis of Alzheimer's disease, informing future developments in the treatment of this devastating disorder.