3130 – CELL-AUTONOMOUS STRESS SIGNALING IN AGED HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells (HSC) ensure lifelong blood production, and decline in function with age. Understanding the underlying mechanisms of HSC aging will allow for interventions that delay blood aging. We previously demonstrated that aging HSCs accumulate features of replication stress, with DNA damage histone marks (gH2AX) persisting at nucleolar ribosomal DNA loci (Flach et al, Nature 2014). Here, we aim to uncover the impact of nucleolar gH2AX in old HSCs. As HSCs depend on stringent proteostasis regulation for functionality (Signer et al, Nature 2014), we determined differences in protein translation between HSCs from young and old mice. We found a defect in translation specifically in old HSCs with nucleolar gH2AX, indicating that these marks impair old HSC function. We integrated the role of nucleolar gH2AX in repressing old HSC activation using aged Fucci cell cycle reporter mice and single-cell RNA-sequencing. We next investigated the nucleolus more deeply. Imaging of nucleolar markers revealed a disruption of nucleolar architecture in old HSCs, with nucleoplasmic dispersion of FBL and NPM1, and formation of stereotypical “necklace” structures, consistent with activation of the p53-dependent nucleolar stress pathway (Boulon et al, Mol Cell 2010). We are now testing the ability of nucleolar stress to drive HSC aging using genetic models of necessity (Mdm2C305F mutant mice with a disrupted nucleolar stress pathway) and sufficiency (TIF-IAflox/flox:Mx1-Cre mice with enhanced nucleolar stress). Our results indicate that nucleolar stress drives a damaging positive feedback loop in old HSCs by inducing replication stress via p53, which in turn causes accumulation of nucleolar gH2AX. These findings uncover new mechanisms of HSC aging, providing opportunities for therapeutic targeting that may have implications for other somatic stem cells. Hematopoietic stem cells (HSC) ensure lifelong blood production, and decline in function with age. Understanding the underlying mechanisms of HSC aging will allow for interventions that delay blood aging. We previously demonstrated that aging HSCs accumulate features of replication stress, with DNA damage histone marks (gH2AX) persisting at nucleolar ribosomal DNA loci (Flach et al, Nature 2014). Here, we aim to uncover the impact of nucleolar gH2AX in old HSCs. As HSCs depend on stringent proteostasis regulation for functionality (Signer et al, Nature 2014), we determined differences in protein translation between HSCs from young and old mice. We found a defect in translation specifically in old HSCs with nucleolar gH2AX, indicating that these marks impair old HSC function. We integrated the role of nucleolar gH2AX in repressing old HSC activation using aged Fucci cell cycle reporter mice and single-cell RNA-sequencing. We next investigated the nucleolus more deeply. Imaging of nucleolar markers revealed a disruption of nucleolar architecture in old HSCs, with nucleoplasmic dispersion of FBL and NPM1, and formation of stereotypical “necklace” structures, consistent with activation of the p53-dependent nucleolar stress pathway (Boulon et al, Mol Cell 2010). We are now testing the ability of nucleolar stress to drive HSC aging using genetic models of necessity (Mdm2C305F mutant mice with a disrupted nucleolar stress pathway) and sufficiency (TIF-IAflox/flox:Mx1-Cre mice with enhanced nucleolar stress). Our results indicate that nucleolar stress drives a damaging positive feedback loop in old HSCs by inducing replication stress via p53, which in turn causes accumulation of nucleolar gH2AX. These findings uncover new mechanisms of HSC aging, providing opportunities for therapeutic targeting that may have implications for other somatic stem cells.