PROJECT SUMMARY/ABSTRACT Hematopoietic stem cells (HSCs) give rise to all blood and immune cells throughout life. Aging HSCs exhibit diminished regenerative function, myeloid-biased differentiation, and clonal outgrowth, which contribute to compromised immunity and increased incidence of bone marrow (BM) failure, and hematological malignancies in the aged population. Recent findings demonstrated that HSCs require strict regulation of protein homeostasis (proteostasis) to maintain self-renewal potential, but this declines with age. Translation is the most error prone step in gene expression and is a prominent source of proteostasis dysfunction. Previous studies demonstrated that HSCs depend on lower protein synthesis rates to preserve proteostasis, and increasing protein synthesis diminishes proteome quality and impairs HSC function. This raises the possibility that HSCs depend on enhanced translation fidelity, and that increasing fidelity could mitigate age-related changes in proteostasis and declines in HSC function. Our central hypothesis is that young adult HSCs have higher translation fidelity compared to restricted progenitors and old adult HSCs, and interventions that enhance translation fidelity will improve HSC fitness and longevity. To directly test this hypothesis, I generated a fluorescence-based translation fidelity reporter mouse, and in Aim 1 I will compare translation fidelity in young and old HSCs and progenitors in vivo. To examine the impact of enhancing fidelity, I generated a mouse model with high-fidelity ribosomes (Rps23K60R/K60R). Using this model, I will test if enhancing translation fidelity protects HSCs from the proteostasis disrupting effects associated with increased protein synthesis in a Pten-deficient mouse model in vivo. In Aim 2, I will test if enhancing translation fidelity improves age-associated declines in HSC function by performing comprehensive hematopoietic analyses and competitive transplantation assays. I will also determine if enhancing translation fidelity extends HSC longevity in serial transplantation assays. Finally, I will extend preliminary RNA-sequencing data to confirm that enhancing translation fidelity promotes improved proteostasis maintenance in aging HSCs using a suite of single cell assays to quantify misfolded and unfolded protein. Collectively, these studies will determine if translation fidelity varies across cell types within the blood and is altered upon aging or in the presence of HSC disrupting pathogenic mutations. Research outcomes will reveal if modulating translation fidelity is a potential therapeutic strategy to enhance stem cell fitness in older adults.