ScRNA-seq for exploringmitochondrial reactive oxygen species action in bone mesenchymal cells in mice

Mitochondrial reactive oxygen species (mtROS), insufficient NAD+, and cellular senescence all contribute to the decrease in bone mass with aging. ROS can cause senescence and decrease NAD+, but it remains unknown whether these mechanisms mediate the deleterious effects of ROS in vivo. Here, we generated mice lacking the mitochondrial antioxidant enzyme superoxide dismutase 2 (Sod2) in cells targeted by Osx1-Cre to examine the mechanisms via which mtROS impacts cells of the osteoblast lineage. Sod2-Osx1 mice had low trabecular and cortical bone mass. Osteoblastic cell cultures showed that increased mtROS impaired mitochondrial respiration and attenuated NAD+ levels. Supplementation with the NAD+ precursor nicotinamide riboside increased NAD+ levels and improved mitochondrial function in vitro but failed to rescue the low bone mass of Sod2-Osx1 mice. To examine in detail molecular mechanisms mediating the effects of mtROS, we isolated mesenchymal cells from the endosteal surfaces of long bones and performed single-cell (sc) RNA-sequencing. Sod2 deletion decreased the proportion of osteoblasts, while the proportions of the three other major cell clusters, namely pre-osteoblasts, osteo-Cxcl12 abundant reticular (CAR), and adipo-CAR were not overtly impacted. Our analysis suggests that excessive mtROS disrupted parathyroid hormone signaling, iron metabolism, and proteostasis particularly in osteoblasts. No significant effects on cellular senescence markers were detected. These findings provide clues on the interactions, or lack of thereof, between mtROS and other mechanisms of aging, with potential implications for the understanding of the biology of aging in bone cells.