PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) is the most common neurodegenerative disorder, and is characterized by progressive neurological decline associated with accumulation of misfolded amyloid peptides and neurofibrillary tangles. Identifying mechanisms that drive both sporadic and genetic forms of AD is arguably one of the most important avenues of research in modern medicine. Several longstanding mechanisms have been proposed to explain the etiology and pathway of neuron impairment in AD. Principle among them is the longstanding and prevailing amyloid cascade hypothesis. The central feature of this hypothesis, which applies to both sporadic and inherited AD, is the presence of toxic, misfolded beta-amyloid peptides that are generated by cleavage of the amyloid precursor protein (APP). These short peptides form protein aggregates that disrupt a number of intracellular processes. A major question in the field has been identifying mechanisms by which Ab peptides impact cell health. Numerous studies ranging from yeast to humans have identified several potential targets and effects of Ab aggregation, including alterations in membrane trafficking via the ESCRT/MVB endocytic pathway, perturbed iron homeostasis, mitochondrial demise, and increased reactive oxygen species (ROS) generation. However, it remains unclear how these numerous defects are connected to each other to promote disease pathology. Excitingly, recent work from our lab based on the parent R01 award has focused on understanding how endolysosomal failure in aging and disease states impacts cell health. These studies have uncovered a cascade of intracellular connections that provide a previously unappreciated link between endolysosomal impairment, iron and amino acid homeostasis and mitochondrial health. Specifically, we discovered that the major way age- related lysosome impairment impacts cells is by causing perturbations in amino acid homeostasis. These alterations, which stem from excess aa pools in cytoplasm, lead to impairment of the bio-available iron pool, ROS production, and mitochondrial dysfunction. Based on these observations, as well as recent work connecting Ab to endolysosomal impairment, we propose to test a new line of AD-related investigation as a supplement to our current R01. The goal of this supplement application is to test the hypothesis that Ab peptides cause mitochondrial decline and iron dyshomeostasis in AD through an amino acid toxicity mechanism that occurs downstream of Ab-mediated impairment of the endolysosomal system. Excitingly, this proposed mechanism brings together numerous observations related to Ab pathology into a single unifying model. The experiments proposed herein are designed to rigorously test this model in a humanized yeast system, and will utilize common assays and readouts developed in our parent R01. Overall, the results of these studies will provide key preliminary data for a subsequent future R01 application, and also identify...