PROJECT SUMMARY/ABSTRACT Aging is accompanied by the gradual deterioration within each individual cellular compartments/organelles as well as the collapse of the interconnections among them, leading to the establishment of different hallmarks of aging. However, these hallmarks of aging, including loss of proteostasis, mitochondrial dysfunction, and cellular senescence, are traditionally studied separately and leave the connections among these hallmarks largely un- known. With a growing understanding of these inter-organelle interactions within young cells, it has become of interest to explore how losses in compartmental crosstalk contribute to the age-associated degeneration of or- ganelle function and the establishment/connection of hallmarks of aging. We recently discovered an unexpected crosstalk between mitochondria and the lysosome (vacuole in yeast) that explains age-associated vacuole/lysosome de-acidification, a process conserved from yeast to human cells and which contributes to the loss of proteostasis during aging and in many age-related diseases. We found that mitochondria contribute to the acidification of lysosome/vacuole via membrane contact sites. Moreover, our pre- liminary studies suggest that the collapse of mitochondria-vacuole contact during the replicative aging of yeast causes age-dependent de-acidification of vacuole. Similarly, we observed a close correlation between mitochon- dria and lysosome acidification in cultured human cells and in C. elegans, suggesting evolutionary conservation of this process. To address how aging affects the mitochondria-lysosome/vacuole contact and whether this mi- tochondria-to-lysosome axis plays a critical role in lysosome acidification in cellular senescence and animals, we here propose to investigate the mitochondria-lysosome/vacuole contact and lysosome/vacuole acidification dur- ing aging and in different longevity paradigms. Specifically, we will combine the expertise of our different model systems to (1) dissect the mechanism(s) underlying the age-related loss of mitochondria-vacuole connection in yeast; (2) determine mitochondrial contribution to lysosome acidification in human cells; and (3) dissect the roles of mitochondria-lysosome connection in lysosome acidification in C. elegans. Our studies will investigate a novel, possibly evolutionarily conserved inter-organelle communication connect- ing mitochondrial dysfunction and lysosome/vacuole de-acidification across different organisms and longevity paradigms. Although traditional thinking has held that age-associated lysosome/vacuole dysfunction is respon- sible for inducing mitochondrial dysfunction in a uni-directional manner, our study will determine whether mito- chondrial dysfunction may conversely impact on lysosome/vacuole dysfunction during aging revealing a here- to-fore unappreciated two-way crosstalk between these tightly connected organelles which may together con- tribute to several important hallmarks of aging. As such, the long-term implications of this study could be the identification of novel conserved therapeutic targets for age-related diseases with lysosome defects.

PROJECT NARRATIVE Aging is accompanied by the gradual deterioration within each individual cellular compartments/organelles as well as the collapse of the interconnections among them, leading to the establishment of different hallmarks of aging. We recently discovered an unexpected crosstalk between mitochondria and the lysosome (vacuole in yeast) that explains age-associated vacuole/lysosome de-acidification and provides a novel interconnection between age-associated mitochondrial dysfunction and lysosome defects that conserved from yeast to human cells and cause many age-related diseases. In this grant, we will integrate the complementary expertise, techniques, and model organisms from multiple labs, including yeast, human cells, and C. elegans, to investigate how aging affects the mitochondria-lysosome/vacuole contact and validate whether this mitochondria-to- lysosome axis plays a critical role in lysosome acidification in cellular senescence and animals during aging and in longevity paradigms in turn leading to new therapeutic targets for many age-related diseases with lysosome defects.