Project Summary/Abstract This proposal presents a five-year research career development program on the study of lysosomal pH and function in aging and disease to expand our understanding of the mechanisms by which aging contributes to neurodegenerative diseases, like Alzheimer’s disease (AD). The candidate, Dr. Courtney Lane-Donovan, is currently a Clinical Fellow in Neurology at the University of California, San Francisco, in the division of Memory and Aging. The outlined proposal builds on Dr. Lane-Donovan’s previous research experience in mouse models of AD to gain new domains of expertise represented by her mentor team of primary mentor Dr. Aimee Kao and co-mentor Dr. Anna Molofsky of the departments of neurology and psychiatry, respectively, at UCSF. The proposed experiments, didactic work, and training in academic skills will provide Dr. Lane-Donovan with a unique skillset that will enable her transition to independence as a physician scientist leader in the field of aging and neurodegenerative diseases. As our nation ages, the burden of the aging-related neurodegenerative diseases has increased substantially. How aging promotes protein aggregation in certain brain regions – and more importantly, how to reverse the effect – remains unknown. Protein aggregates can accumulate when protein degradation by proteases in the lysosome is impaired, and several genetic risk factors for AD encode proteins involved in endolysosomal function and autophagy. Lysosomal proteases function optimally at an acidic pH, and data from invertebrate models suggest that age and stress cause lysosomes to lose their acidity, resulting in impaired function; however, the relevance of these findings to human aging and disease is unclear. Regional variation in lysosomal protease activity may contribute to the selective vulnerability of certain brain regions to the accumulation of protein aggregates; however, regional lysosomal function is not fully characterized and thereby poorly understood. Together, this suggests a tantalizing hypothesis - regional variability of lysosomal protease expression leaves certain neurons more vulnerable to the lysosomal dysfunction caused by lysosomal alkalinization with age. To test this hypothesis, Aim 1 utilizes the novel lysosomal pH reporter, FIRE-pHLy (Fluorescent Indicator Reporting pH of the Lysosome) to delineate the effect of aging and amyloid beta accumulation on lysosomal pH. Aim 2 will determine regional changes in lysosomal protease activity and expression in the aging brain by combining lysosome isolations, immunohistochemistry, and spatial transcriptomics. The data generated by this study can be used to propose new models of lysosomal dysfunction in aging and disease and identify new therapeutic targets for neuroprotection in neurodegenerative disease.