Organelle dysfunction upon asrij depletion causes aging‐like changes in mouse hematopoietic stem cells

Abstract Aging of the blood system is characterized by increased hematopoietic stem cells (HSCs) and myeloid‐biased differentiation leading to higher propensity for hematological malignancies. Unraveling cell‐intrinsic mechanisms regulating HSC aging could aid reversal or slowing of aging. Asrij/OCIAD1 is an evolutionarily conserved regulator of hematopoiesis and governs mitochondrial, endosomal, and proteasomal function in mammalian stem cells. Asrij deletion in mice causes loss of HSC quiescence, myeloid skewing, reduced p53 and increased DNA damage, features attributed to aged HSCs. Mechanistically, Asrij controls p53 ubiquitination and degradation and AKT/STAT5 activation. Asrij localizes to endosomes and mitochondria. As decline in organelle structure and function are common hallmarks of aging, we asked whether Asrij regulates organelle function in aged HSCs. We find that chronologically aged wild‐type (WT) HSCs had reduced Asrij levels. Expectedly, young asrij KO mice had reduced AcH4K16 levels; however, transcriptome analysis of KO HSCs showed a modest overlap of gene expression with aged WT HSCs. Further, analysis of organelle structure and function in asrij KO mice revealed significant changes, namely damaged mitochondria, elevated ROS; impaired endosomal trafficking seen by increased cleaved Notch1, reduced Rab5; and reduced 26S proteasome activity. Pharmacological correction of mitochondrial and proteasome activity in asrij KO mice restored HSC and myeloid cell frequencies. Furthermore, lysophosphatidic acid‐induced Asrij upregulation in aged WT mice rescued mitochondrial and proteasome activity and restored HSC frequency. Our results highlight a new role for Asrij in preventing HSC aging by regulating organelle homeostasis and will help decipher organelle dynamics in HSC longevity.

activation. Asrij localizes to endosomes and mitochondria. As decline in organelle structure and function are common hallmarks of aging, we asked whether Asrij regulates organelle function in aged HSCs. We find that chronologically aged wild-type (WT) HSCs had reduced Asrij levels. Expectedly, young asrij KO mice had reduced AcH4K16 levels; however, transcriptome analysis of KO HSCs showed a modest overlap of gene expression with aged WT HSCs. Further, analysis of organelle structure and function in asrij KO mice revealed significant changes, namely damaged mitochondria, elevated ROS; impaired endosomal trafficking seen by increased cleaved Notch1, reduced Rab5; and reduced 26S proteasome activity. Pharmacological correction of mitochondrial and proteasome activity in asrij KO mice restored HSC and myeloid cell frequencies. Furthermore, lysophosphatidic acid-induced Asrij upregulation in aged WT mice rescued mitochondrial and proteasome activity and restored HSC frequency. Our results highlight a new role for Asrij in preventing HSC aging by regulating organelle homeostasis and will help decipher organelle dynamics in HSC longevity.

| INTRODUC TION , RE SULTS , AND DISCUSS ION
Bone marrow hematopoietic stem cells (BM HSCs) constantly combat multiple stressors for blood cell homeostasis. This ability reduces with age leading to functional decline characterized by increased HSCs, myeloid skewing, inflammaging, and clonal hematopoiesis (Mejia-Ramirez & Florian, 2020). Multiple cell-intrinsic and cellextrinsic factors regulate the genetic and epigenetic landscape, cell polarity, and autophagy to maintain HSCs (Grigoryan et al., 2018;Ho et al., 2017). The dynamic metabolic requirements of HSCs necessitate strict control of mitochondrial (mt) metabolism, endocytic activity, and proteostasis (Gurumurthy et al., 2010;Warr et al., 2013).
Low mtROS levels and translation along with rapid proteasomemediated protein turnover in HSCs minimize oxidative damage and protein aggregation (Mejia-Ramirez & Florian, 2020;Hidalgo San Jose et al., 2020). Further, endosomal proteins aid asymmetric localization of cellular components, essential for HSC self-renewal (Ting et al., 2012). Thus, integrated organelle function is critical to delay HSC aging. Although altered organelle architecture and function are implicated in aging and age-related diseases (Bouska et al., 2019), very little is known about organelles in HSC aging. Hence, we investigated the role of Asrij, an organelle protein, in HSC aging.
OCIAD1/Asrij controls mtComplex I activity and thereby mtROS, in human pluripotent stem cells (Shetty et al., 2018) and also mitochondrial morphology and dynamics (Ray et al., 2021). Thus, we demonstrate that restoring organelle homeostasis by pharmacological intervention can maintain HSC stemness and lineage choice, thereby reversing phenotypes of premature aging in young asrij KO HSCs. We propose that Asrij is a critical node in organelle control of HSC aging.
In summary, we provide the first report that HSC aging is associated with Asrij-dependent simultaneous dysfunction in mitochondrial, endosomal, and proteasomal machineries. Further, we demonstrate that Asrij links organelle function with genetic and epigenetic programs that promote HSC aging and could serve as a biomarker. How Fellowship to MI and intramural funds from JNCASR.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N S
MSI and SS conceived the project; SS, AS, PD, and KK performed research and collected, analyzed, and interpreted data; SS and MSI prepared figures and wrote the manuscript.

S U PP O RTI N G I N FO R M ATI O N
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