ABSTRACT Hematopoietic stem cells (HSCs) regenerate blood and immune cells throughout life. However, aging HSCs exhibit reduced self-renewal activity, diminished regenerative capacity, and myeloid-biased differentiation that contribute to an increased incidence of immune dysfunction, bone marrow failure, anemia, clonal hematopoiesis, and cancer in older adults. Translational control has emerged as fundamentally and preferentially important for stem cells. Young adult HSCs have lower protein synthesis rates than other hematopoietic cells and increases in protein synthesis impair HSC function by increasing the biogenesis of misfolded proteins and disrupting protein homeostasis (proteostasis). In preliminary studies, we found that old HSCs preserve low protein synthesis rates, but increase the biogenesis of misfolded proteins, raising the possibility that translation fidelity declines in HSCs during aging and contributes to age-related HSC dysfunction by disrupting proteostasis. The goal of this proposal is to test if young adult HSCs have elevated translation fidelity as compared to restricted progenitors and old HSCs, and to determine if enhancing translation fidelity increases HSC fitness and longevity. To our knowledge, translation fidelity has not yet been studied in a cell-type- or age-specific manner in vivo, and interventions that specifically boost translation fidelity in mammals have not been reported. To overcome these limitations, we developed a single cell assay to quantify relative translation fidelity, and developed a new genetic mouse model with high-fidelity ribosomes (Rps23K60R) that enhances translation fidelity and reduces proteostasis stress in aging HSCs. In Aim 1, we will use translation fidelity reporter mice to quantify relative translation fidelity in HSCs and progenitors in vivo, and will determine if genetically or environmentally increasing protein synthesis reduces fidelity in HSCs. Furthermore, we will test if enhancing translation fidelity in Rps23K60R mice protects HSCs from the proteostasis disrupting effects associated with increased protein synthesis. In Aim 2, we will test if translation fidelity declines in HSCs during aging, and if enhancing fidelity delays/mitigates age-related declines in HSC function. We will also determine if enhancing translation fidelity delays/prevents stem cell exhaustion in serial transplantation assays and extends organismal healthspan and lifespan. Finally, in Aim 3, we will determine the effects of enhancing translation fidelity on proteostasis using a suite of single cell assays to investigate proteome quality and quantitative proteomics to investigate proteome content throughout life. We will also test if enhancing fidelity alleviates the need to activate proteotoxic stress response pathways to preserve HSC fitness during aging. These studies will elucidate if cell-type-specific differences in translation fidelity are employed to confer stem cell longevity and will reveal if modul...