PRMT6–SIRT5–HADHA signaling safeguards osteoblasts from R-loop-induced DNA damage during age-related osteoporosis

Age-related osteoporosis is characterized by impaired bone formation and progressive deterioration of trabecular microarchitecture. Declining osteoblast abundance and function, together with the accumulation of cellular senescence, are recognized as key contributors to skeletal aging. During aging, increased genomic instability and persistent DNA damage responses further impair osteogenic capacity. Aberrant accumulation of R-loops (RNA–DNA hybrids) can induce transcription–replication conflicts and DNA damage; however, the regulatory mechanisms maintaining R-loop homeostasis during osteoblast aging remain largely unknown. SIRT5, a key mitochondrial desuccinylase that regulates metabolic adaptation through protein lysine desuccinylation, has been implicated in cellular stress responses, but whether SIRT5 controls R-loop homeostasis and bone aging through succinylation remodeling remains unclear. Public mouse bone marrow single-cell RNA-seq datasets from GEO were integrated to delineate age-dependent changes in bone marrow cellular composition and the osteoblast lineage. Naturally aged mice as well as Sirt5 −/− and Prmt6 −/− models were analyzed, and skeletal phenotypes were assessed by micro-computed tomography and histological examination. In vitro, primary osteoblasts were subjected to replicative senescence and H₂O₂-induced oxidative stress. RNA-seq, lysine succinylation proteomics, S9.6-based R-loop CUT&Tag, Western blotting, and immunofluorescence were employed to characterize molecular alterations and phenotypic outcomes. Causality was further tested using AAV5-mediated Sirt5 overexpression in vivo and in vitro as rescue approaches. Single-cell analysis revealed a reduction in the proportion of osteoblasts during aging accompanied by decreased Sirt5 expression. Compared with wild-type controls, Sirt5 −/− mice exhibited more pronounced trabecular bone loss and impaired osteogenesis, with reduced Runx2, ALPL, and OCN and elevated p16/p21, decreased ALP activity, and increased SA-β-gal-positive cells. Transcriptomic profiling indicated activation of stress-related programs and suppression of osteogenic and metabolic pathways. Mechanistically, Sirt5 deficiency caused global succinylation accumulation and reshaped the succinylation landscape of metabolic proteins, leading to elevated multi-site succinylation of HADHA and reduced HADHA protein abundance. Concurrently, R-loops were broadly increased around transcription start site (TSS) regions, accompanied by elevated S9.6 and γH2AX signals and enhanced DNA damage accumulation. Prmt6 deficiency downregulated Sirt5 and phenocopied Sirt5 loss, including trabecular deterioration and increased R-loop signals. AAV5-mediated Sirt5 overexpression restored HADHA expression, attenuated R-loop accumulation and senescence phenotypes induced by oxidative stress or Prmt6 loss, and alleviated trabecular bone loss. Together, these findings identify the PRMT6–SIRT5 axis as a critical regulator of osteoblast aging. Disruption of this pathway promotes succinylation remodeling, compromises R-loop homeostasis, and enhances DNA damage accumulation, thereby accelerating osteoblast senescence and age-related bone loss. Targeting the PRMT6–SIRT5 signaling pathway may represent a potential therapeutic strategy for preventing osteoporosis associated with aging.