Khosla, Sundeep

Abstract The aim of this international meta-analysis was to quantify the predictive value of BMI for incident fracture and relationship of this risk with age, sex, follow-up time, and BMD. A total of 1 667 922 men and women from 32 countries (63 cohorts), followed for a total of 16.0 million person-years were studied. 293 325 had FN BMD measured (2.2 million person-years follow-up). An extended Poisson model in each cohort was used to investigate relationships between WHO-defined BMI categories (Underweight: <18.5 kg/m2; Normal: 18.5-24.9 kg/m2; Overweight: 25.0-29.9 kg/m2; Obese I: 30.0-34.9 kg/m2; Obese II: ≥35.0 kg/m2) and risk of incident osteoporotic, major osteoporotic and hip fracture (HF). Inverse-variance weighted β-coefficients were used to merge the cohort-specific results. For the subset with BMD available, in models adjusted for age and follow-up time, the hazard ratio (95% CI) for HF comparing underweight with normal weight was 2.35 (2.10-2.60) in women and for men was 2.45 (1.90-3.17). Hip fracture risk was lower in overweight and obese categories compared to normal weight [obese II vs normal: women 0.66 (0.55-0.80); men 0.91 (0.66-1.26)]. Further adjustment for FN BMD T-score attenuated the increased risk associated with underweight [underweight vs normal: women 1.69 (1.47-1.96); men 1.46 (1.00-2.13)]. In these models, the protective effects of overweight and obesity were attenuated, and in both sexes, the direction of association reversed to higher fracture risk in Obese II category [Obese II vs Normal: women 1.24 (0.97-1.58); men 1.70 (1.06-2.75)]. Results were similar for other fracture outcomes. Underweight is a risk factor for fracture in both men and women regardless of adjustment for BMD. However, while overweight/obesity appeared protective in base models, they became risk factors after additional adjustment for FN BMD, particularly in the Obese II category. This effect in the highest BMI categories was of greater magnitude in men than women. These results will inform the second iteration of FRAX®.

Abstract Germline and osteoblast-directed deletion of G protein-gated inwardly rectifying K+ channel 3 (Girk3) was recently shown to increase bone mass after 18 wk of age in male mice. Here, we show that germline Girk3 deletion also increases trabecular and cortical bone mass and increases the mechanical strength of the femur in female mice after 18 wk of age. Unlike male mice, however, osteoblast-directed Girk3 deletion using 2.3 kb-Col1a1-Cre does not increase bone mass in adult female mice. To discover mechanisms underlying high bone mass in female Girk3−/− mice, bulk RNA-sequencing was performed on 2-d-old calvarial bone, revealing lower expression of proinflammatory cytokines such as IL-1β and IL-6 in Girk3−/− mice. Accordingly, cytokines and chemokines are largely suppressed in the circulation of adult Girk3−/− mice compared to WT littermates. The cytokines GM-CSF, IL-1β, IL-2, and IL-9 are reduced in the serum of both male and female Girk3−/− mice, while eotaxin, IFNγ, MIP-1α, and others are sexually dimorphic. Histomorphometry reveals that osteoclast activity is modestly reduced in Girk3−/− bone, which is supported by in vitro osteoclast resorption assays. However, deletion of Girk3 in myeloid-lineage cells with LysM-Cre is not sufficient to recapitulate high bone mass in either male or female mice. Moreover, female Girk3−/− mice are not protected from ovariectomy-induced bone loss. Finally, single-cell screening using cytometry by time-of-flight in the BM revealed no differences in immune cell abundances due to global Girk3 deletion. Taken together, while Girk3 regulates inflammatory cytokine expression in the bone and serum, deletion of Girk3 in myeloid-lineage cells does not affect bone mass.

ABSTRACT Although animal studies have linked cellular senescence to the pathogenesis and complications of type 2 diabetes mellitus (T2DM), there is a paucity of corroborating data in humans. Thus, we measured a previously validated marker for senescent cell burden in humans, T‐cell expression of p16 mRNA, along with additional biomarkers, to compare the senescence phenotypes of postmenopausal control (lean, N = 37) and T2DM ( N = 27) participants. To control for effects of obesity alone, we included a third group of obese but non‐diabetic women ( N = 29) who were matched for body mass index to the T2DM participants. In addition, given the increase in fracture risk in T2DM despite preserved or even increased bone mineral density, we related these senescence biomarkers in the T2DM participants to skeletal microarchitectural parameters. Relative to the lean participants, T‐cell p16 and p21 Cip1 expression was increased in the T2DM, but not the obese, non‐diabetic participants. Expression of p16 and p21 Cip1 was positively associated with HbA1c and an index of skin advanced glycation end‐products. T2DM was also associated with an increase in a number of SASP factors. Among participants with T2DM, women in the highest tertile for T‐cell expression of p16 had significantly reduced tibial cortical area and thickness as compared to those in the lower two tertiles. Overall, our studies link cellular senescence to metabolic and skeletal alterations in T2DM and point to the need for further studies evaluating the role of cellular senescence in mediating skeletal fragility, as well as potentially other complications in T2DM.

Abstract Bisphosphonates (BPs) have been the major class of medicines used to treat disorders of excessive bone loss for over five decades. Recently it has been recognized that BPs may also have additional significant beneficial extra-skeletal effects. These include a reduction of all-cause mortality and of conditions commonly linked to ageing, such as cancer and cardiovascular disease. Here we show that bisphosphonates co-localize with lysosomal and endosomal organelles in non-skeletal cells and stimulate cell growth at low doses. In vivo spatial transcriptomic analysis revealed differentially expressed senescence markers in multiple organs of aged BP-treated mice, and a shift in cellular composition toward those of young counterparts. Similarly, a 5000-plex plasma proteome analysis from osteopenic patients before and after BP-treatment showed significant alterations in ∼400 proteins including GTPase regulators and markers of senescence, autophagy, apoptosis, and inflammatory responses. Furthermore, treatment with BPs protected against the onset of senescence in vitro . Proteome-wide target deconvolution using 2D thermal profiling revealed novel BP-binding targets (PHB2, ASAH1), and combined with RNA- and ATAC-seq of BP-treated cells and patient data, suggests downstream regulation of the MEF2A transcription factor within the heart. Collectively, these results indicate how BPs may beneficially modify the human plasma proteome, and directly impact multiple non-skeletal cell types through previously unidentified proteins, thereby influencing a range of pathways related to senescence and ageing.

ABSTRACT There is an increasing need for biomarkers of senescent cell burden to facilitate the selection of participants for clinical trials. p16 Ink4a is encoded by the CDKN2A locus, which produces five variant transcripts in humans, two of which encode homologous p16 proteins: p16 Inka4a , encoded by p16_variant 1 , and p16ɣ, encoded by p16_variant 5. While distinct quantitative polymerase chain reaction primers can be designed for p16_variant 5, primers for p16_variant 1 also measure p16_variant 5 ( p16_variant 1 + 5 ). In a recent clinical trial evaluating the effects of the senolytic combination, dasatinib + quercetin (D + Q), on bone metabolism in postmenopausal women, we found that women in the highest tertile for T‐cell expression of p16_variant 5 had the most robust skeletal responses to D + Q. Importantly, the assessment of p16_variant 5 was more predictive of these responses than p16_variant 1 + 5 . Here, we demonstrate that in vitro, p16_variant 1 + 5 increased rapidly (Week 1) following the induction of DNA damage, whereas p16_variant 5 increased later (Week 4), suggesting that p16_variant 5 becomes detectable only when the abundance of senescent cells reaches some threshold. Further analysis identified a SASP panel in plasma that performed as well in identifying postmenopausal women with a positive skeletal response to D + Q. Collectively, our findings provide further support for the T‐cell p16_variant 5 assay as a biomarker for selecting participants in clinical trials of senolytic interventions. In addition, our data indicate that correlated plasma SASP markers could be used in lieu of the more technically challenging T‐cell p16 assay. Trial Registration: ClinicalTrials.gov identifier: NCT04313634.

Abstract Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

AbstractCellular senescence is an established cause of cell and tissue aging. Senescent cells have been shown to increase in multiple organs during aging, including the skin. Here we hypothesized that senescent cells residing in the skin can spread senescence to distant organs, thereby accelerating systemic aging processes. To explore this hypothesis, we initially observed an increase in several markers of senescence in the skin of aging mice. Subsequently, we conducted experiments wherein senescent fibroblasts were transplanted into the dermis of young mice and assessed various age‐associated parameters. Our findings reveal that the presence of senescent cells in the dermal layer of young mice leads to increased senescence in both proximal and distal host tissues, alongside increased frailty, and impaired musculoskeletal function. Additionally, there was a significant decline in cognitive function, concomitant with increased expression of senescence‐associated markers within the hippocampus brain area. These results support the concept that the accumulation of senescent cells in the skin can exert remote effects on other organs including the brain, potentially explaining links between skin and brain disorders and diseases and, contributing to physical and cognitive decline associated with aging.

Abstract Single‐cell RNA sequencing and spatial transcriptomics enable unprecedented insight into cellular and molecular pathways implicated in human skin aging and regeneration. Senescent cells are individual cells that are irreversibly cell cycle arrested and can accumulate across the human lifespan due to cell‐intrinsic and ‐extrinsic stressors. With an atlas of single‐cell RNA‐sequencing and spatial transcriptomics, epidermal and dermal senescence and its effects were investigated, with a focus on melanocytes and fibroblasts. Photoaging due to ultraviolet light exposure was associated with higher burdens of senescent cells, a sign of biological aging, compared to chronological aging. A skin‐specific cellular senescence gene set, termed SenSkin™, was curated and confirmed to be elevated in the context of photoaging, chronological aging, and non‐replicating CDKN1A + (p21) cells. In the epidermis, senescent melanocytes were associated with elevated melanin synthesis, suggesting haphazard pigmentation, while in the dermis, senescent reticular dermal fibroblasts were associated with decreased collagen and elastic fiber synthesis. Spatial analysis revealed the tendency for senescent cells to cluster, particularly in photoaged skin. This work proposes a strategy for characterizing age‐related skin dysfunction through the lens of cellular senescence and suggests a role for senescent epidermal cells (i.e., melanocytes) and senescent dermal cells (i.e., reticular dermal fibroblasts) in age‐related skin sequelae.

Abstract Extracellular vesicles (EVs) are key mediators of cell–cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the “Snorkel-tag,” which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed “CAGS-Snorkel,” which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.