ABSTRACT Lysosomes, the essential organelle for cellular waste and pathogen clearance through all autophagic and endocytic pathways, are highly vulnerable in aging neurons and strongly implicated in neurodegenerative disease pathogenesis, particularly Alzheimer’s Disease (AD). We showed that presenilin 1 (PS1) is required for vATPase activity necessary to acidify lysosomes and initiate digestion: loss of PS1 function in familial AD impedes substrate degradation, leading to neuronal dysfunction and AD-related pathology. Recently, we reported that the direct molecular trigger of the endosomal dysfunction linked to cholinergic neurodegeneration in AD is the APP -site cleaved C-terminal fragment (CTF) (studied in Projects 1, 3, and 4). Our newest data indicate a significant impact of CTF on lysosome function, including impaired acidification. Our working hypothesis is that other risk factors, including ones for late-onset AD (ApoE4, cholesterol, brain aging), impair lysosomal function by CTF-dependent and -independent mechanisms. Much of this lysosomal dysfunction stems from the compromise of vATPase, occuring via multiple mechanisms shown to disrupt its function and thereby raise lysosomal pH and promote neurodegeneration. In 3 specific aims, we address both the mechanisms underlying pathogenic lysosomal dysfunction in AD and strategies to remediate this dysfunction. In Aim 1, we will define the molecular basis for lysosomal dysfunction in a late-onset AD model of wild-type APP overexpression and a Down syndrome model with known CTF-driven endosome phenotypes in comparison to models in which lysosome compromise is induced by known AD risk factors (ApoE4 genotype and dietary fat/cholesterol). Models in which CTF are also experimentally modulated will be used to ascertain the nature and extent of the βCTF contributions, believed to be major, in compromising lysosomal pH regulation, substrate hydrolysis, and signaling functions. A novel role of chaperone-mediated autophagy (CMA) in CTF metabolism will be assessed in relation to known aging-related declines in the efficiency of CMA and lysosomes. In Aim 2, we will define in neurons and isolated lysosomes multiple suspected mechanisms underlying the loss of vATPase function seen in AD models. In Aim 3, we will characterize a novel lysosomal pH modulation mechanism via the ClC7 chloride channel and the basis for its ability to reverse acidification deficits and fully rescue lysosomal and autophagy deficits in PS1 deficient and FAD patient cells. The detailed mechanism by which certain beta-adrenergic agonists induce this rescue, the applicability of this remediation to non-PS AD models, and the evaluation of more optimal brain-penetrant compounds that promote lysosomal acidification are additional goals of Aim 3. Novel genetic models with altered endosomal-lysosomal function or that report on neuronal lysosomal network function in vivo are introduced in our project and serve to enhance expectations...