Ikeno, Yuji

Abstract Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16 Ink4a or p21 Cip1 , but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21 ‐ ATTAC mouse with the p21 Cip1 promoter driving a “suicide” transgene encoding an inducible caspase‐8 which, upon induction, selectively kills p21 Cip1 ‐expressing senescent cells. Next, we used the p21 ‐ ATTAC mouse and the established p16 ‐ INK ‐ ATTAC mouse to directly compare the contributions of p21 Cip1 versus p16 Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence—specifically, radiation‐induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation‐induced p21 Cip1 ‐ or p16 Ink4a ‐driven transcripts following senescent cell clearance in both models. However, only clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells prevented both radiation‐induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21 Cip1 +, but not p16 Ink4a +, senescent cells also reduced several of the radiation‐induced pro‐inflammatory senescence‐associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation‐induced osteoporosis is driven predominantly by p21 Cip1 ‐ rather than p16 Ink4a ‐mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence‐associated conditions, including aging across tissues.

Abstract Although cellular senescence is increasingly recognized as driving multiple age-related co-morbidities through the senescence-associated secretory phenotype (SASP), in vivo senescent cell identification, particularly in bulk or single cell RNA-sequencing (scRNA-seq) data remains challenging. Here, we generated a novel gene set (SenMayo) and first validated its enrichment in bone biopsies from two aged human cohorts. SenMayo also identified senescent cells in aged murine brain tissue, demonstrating applicability across tissues and species. For direct validation, we demonstrated significant reductions in SenMayo in bone following genetic clearance of senescent cells in mice, with similar findings in adipose tissue from humans in a pilot study of pharmacological senescent cell clearance. In direct comparisons, SenMayo outperformed all six existing senescence/SASP gene sets in identifying senescent cells across tissues and in demonstrating responses to senescent cell clearance. We next used SenMayo to identify senescent hematopoietic or mesenchymal cells at the single cell level from publicly available human and murine bone marrow/bone scRNA-seq data and identified monocytic and osteolineage cells, respectively, as showing the highest levels of senescence/SASP genes. Using pseudotime and cellular communication patterns, we found senescent hematopoietic and mesenchymal cells communicated with other cells through common pathways, including the Macrophage Migration Inhibitory Factor (MIF) pathway, which has been implicated not only in inflammation but also in immune evasion, an important property of senescent cells. Thus, SenMayo identifies senescent cells across tissues and species with high fidelity. Moreover, using this senescence panel, we were able to characterize senescent cells at the single cell level and identify key intercellular signaling pathways associated with these cells, which may be particularly useful for evolving efforts to map senescent cells (e.g., SenNet). In addition, SenMayo represents a potentially clinically applicable panel for monitoring senescent cell burden with aging and other conditions as well as in studies of senolytic drugs.