Characterization of senescent cell populations in skeletal aging

There is now extensive evidence that cellular senescence drives age-related bone loss and multiple other aging co-morbidities. Characteristics of senescent cells include increased expression of p16Ink4a and p21Cip1 as well as resistance to apoptosis. In previous work, we have systematically identified senescent cells in the bone microenvironment and demonstrated a causal role, specifically for p16+ senescent cells, in mediating age- related bone loss in mice. It is becoming clear, however, that p16+ cells in the bone microenvironment are a heterogeneous population, and a better understanding of this heterogeneity is critical to developing new approaches to target these cells and prevent age-related bone loss, along with ameliorating other aging co- morbidities. To facilitate this, we have recently developed and validated multiparametric single-cell protein analysis by cytometry by time of flight (CyTOF) to perform a deep characterization of p16+ cells in mice. These studies identified a specific sub-population of p16+ cells that are growth-arrested (Ki67-) and express high levels of senescent-associated secretory phenotype (SASP) markers as well as the anti-apoptosis protein, BCL-2 (p16+/Ki67-/BCL-2+, “p16KB” cells), thus meeting all of the criteria for defining a senescent cell. Interestingly, we also identified non-senescent p16+/Ki67+ cells that express a SASP unique from that of the p16KB cells. These proliferating, inflammatory p16+ cells have also been identified by others in the lung using a highly sensitive p16Ink4a reporter, yet the role of this population in aging remains unclear. Taken together, these findings lead to the hypothesis that p16KB cells are truly senescent, while p16+/Ki67+ cells are inflammatory cycling cells, and perhaps “pre-senescent.” However, the role of each population in driving age-related disease remains unclear and Aim 1 proposes to characterize these populations in detail. In additional studies, we have developed and validated a novel mouse model, p16-LOX-ATTAC, capable of temporal- and cell-specific senescent cell clearance. Using these mice, we found that in contrast to global clearance of senescent cells using the (p16)INK- ATTAC model, clearance specifically of senescent osteocytes only partially replicated the beneficial skeletal effects of global senescent cell clearance, indicating an important role for other cells in the bone microenvironment in contributing to skeletal aging. Consistent with this, our CyTOF analyses have identified a novel p16+, highly inflammatory (SASP+) CD24high osteolineage population (negative for stem cell markers, e.g., Sca-1, CD200) in aged mice that, along with the late osteoblast/osteocytic cells, is robustly cleared by senolytic interventions. These studies indicate that although osteocyte senescence clearly contributes to age-related bone loss, p16+, CD24high osteolineage cells are also potential candidate cells that mediate skeletal aging and thus need to be further characterized, as proposed in Aim 2. Thus, the overall goal of our application is to leverage our novel tools and mouse models in order to better define the mechanisms driving cellular senescence in the bone microenvironment and to characterize the key senescent cell populations that contribute to skeletal aging.