ABSTRACT Hematopoietic stem cells (HSCs) regenerate blood and immune cells throughout life. Unfortunately, HSC function declines with age. Age-related defects in HSCs lead to anemia, impaired immunity, bone marrow failure and cancer. Thus, understanding mechanisms that contribute to HSC aging is critical for developing strategies to enhance regeneration and tissue function in older adults. Protein homeostasis (proteostasis) dysfunction contributes to several age-associated pathologies, but diminished proteostasis has not been examined as a mechanism of HSC aging. We recently discovered that HSCs are particularly dependent on proteostasis to preserve their self-renewal capacity. However, misfolded proteins arise in HSCs and therefore must be eliminated to preserve HSC fitness. Canonically, the proteasome serves as the primary pathway for degradation of misfolded proteins, but we found that proteasome activity is low within HSCs. This raises a fundamental paradox: if HSCs are highly dependent on proteostasis, why do they have such limited proteasome capacity to degrade misfolded proteins? In preliminary studies, we found that mouse and human HSCs preferentially express the co-chaperone Bag3, which can promote delivery of misfolded proteins to aggresomes. Aggresomes are inclusion bodies containing misfolded and aggregated proteins that typically form in response to stress and are substrates for a selective form of autophagy (aggrephagy). We determined that HSCs form aggresomes, even under steady state conditions, and they depend on autophagy to degrade protein aggregates in vivo. Furthermore, we generated data demonstrating that protein aggregates accumulate in aging HSCs and that old adult HSCs activate Hsf1, key proteostasis sensor that helps preserve HSC fitness. Based on these data, our central hypothesis is that HSCs preferentially shuttle misfolded proteins to aggresomes and depend on aggrephagy to maintain proteostasis, fitness and longevity. Furthermore, we propose that accumulation of aggregated proteins contributes to age-related declines in HSC function. In Aim 1, we will test if mouse and human HSCs preferentially form aggresomes. Using conditional Bag3 knockout mice, we will test if disrupting transport of misfolded proteins to aggresomes impairs HSC function, proteostasis and aging. In Aim 2, we will use genetic mouse models to express disease-associated protein aggregates in HSCs to test the effects of protein aggregation on HSC function. We will also determine if aggrephagy regulates HSC fitness, protein synthesis and quiescence. In Aim 3, we will quantify protein aggregates in aging mouse and human HSCs, and test if protein aggregation induces Hsf1 activation. Finally, we will test if enhancing Hsf1 activity rescues age- related declines in HSC function. Research outcomes will uncover how misfolded proteins are eliminated in HSCs and if accumulation of aggregated proteins contributes to HSC aging. These studies will identify strateg...