Biology subjects

Doolittle, M. L.

Publications and source records attributed to Doolittle, M. L..

8 recordsLinked to original sources

bioRxiv2024

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells are key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings establish contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

physiology↗

bioRxiv2023

Distinct secretomes in p16- and p21- positive senescent cells across tissues

Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescence-associated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21Cip1 and p16Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing (scRNA-seq) datasets spanning both murine and human tissues during aging. Our analysis revealed that p21Cip1 and p16Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21Cip1 or p16Ink4a expression. Our study underscores the extraordinary diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent. However, a few SASP factors consistently contribute to a shared "core" SASP. These findings highlight the need for a more nuanced investigation of senescence across a wide array of biological contexts.

cell biology↗

bioRxiv2023

In vitro and in vivo effects of zoledronate on senescence and senescence-associated secretory phenotype markers

In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1{beta}, TNFRSF1A, and TGF{beta}1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

physiology↗

bioRxiv2023

Multiparametric senescent cell phenotyping reveals CD24 osteolineage cells as targets of senolytic therapy in the aged murine skeleton

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we applied mass cytometry by time-of-flight (CyTOF) using carefully validated antibodies to analyze senescent cells at single-cell resolution. We used multiple criteria to identify senescent mesenchymal cells that were growth arrested and resistant to apoptosis (p16+/Ki67-/BCL-2+; "p16KB" cells). These cells were highly enriched for senescence-associated secretory phenotype (SASP) and DNA damage markers and were strongly associated with age. p16KB cell percentages were also increased in CD24+ osteolineage cells, which exhibited an inflammatory SASP in aged mice and were robustly cleared by both genetic and pharmacologic senolytic therapies. Following isolation, CD24+ skeletal cells exhibited growth arrest, SA-{beta}gal positivity, and impaired osteogenesis in vitro. These studies thus provide a new approach using multiplexed protein profiling by CyTOF to define senescent mesenchymal cells in vivo and identify a highly inflammatory, senescent CD24+ osteolineage population cleared by senolytics.

cell biology↗

bioRxiv2022

Identification of a suitable endogenous control miRNA in bone aging and senescence

ObjectiveMicroRNAs (miRNAs) are promising tools as biomarkers and therapeutic agents in various chronic diseases such as osteoporosis, cancers, type I and II diabetes, and cardiovascular diseases. Considering the rising interest in the regulatory role of miRNAs in bone metabolism, aging, and cellular senescence, accurate normalization of qPCR-based miRNA expression data using an optimal endogenous control becomes crucial. MethodsWe used a systematic approach to select candidate endogenous control miRNAs that exhibit high stability with aging from our miRNA sequence data and literature search. Validation of miRNA expression was performed using qPCR and their comprehensive stability was assessed using the RefFinder tool which is based on four statistical algorithms: GeNorm, NormFinder, BestKeeper, and comparative delta CT. The selected endogenous control was then validated for its stability in mice and human bone tissues, and in bone marrow stromal cells (BMSCs) following induction of senescence and senolytic treatment. Finally, the utility of selected endogenous control versus U6 was tested by using each as a normalizer to measure the expression of miR-34a, a miRNA known to increase with age and senescence. ResultsOur results show that Let-7f did not change across the groups with aging, senescence or senolytic treatment, and was the most stable miRNA, whereas U6 was the least stable. Moreover, using Let-7f as a normalizer resulted in significantly increased expression of miR-34a with aging and senescence and decreased expression following senolytic treatment. However, the expression pattern for miR-34a reversed for each of these conditions when U6 was used as a normalizer. ConclusionsWe show that optimal endogenous control miRNAs, such as Let-7f, are essential for accurate normalization of miRNA expression data to increase the reliability of results and prevent misinterpretation. Moreover, we present a systematic strategy that is transferrable and can easily be used to identify endogenous control miRNAs in other biological systems and conditions.

cell biology↗

bioRxiv2021

A New Gene Set Identifies Senescent Cells and Predicts Senescence-Associated Pathways Across Tissues

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.

cell biology↗

bioRxiv2021

Modulation of fracture healing by the transient accumulation of senescent cells

Senescent cells have detrimental effects across tissues with aging but may have beneficial effects on tissue repair, specifically on skin wound healing. However, the potential role of senescent cells in fracture healing has not been defined. Here, we performed an in silico analysis of public mRNAseq data and found that senescence and senescence-associated secretory phenotype (SASP) markers increased during fracture healing. We next directly established that the expression of senescence biomarkers increased markedly during murine fracture healing. We also identified a subset of cells in the fracture callus that displayed hallmarks of senescence, including distension of satellite heterochromatin and telomeric DNA damage. Then, using a genetic mouse model (p16LUC) containing a pl6Ink4a-dnven luciferase reporter, we demonstrated transient in vivo senescent cell accumulation during callus formation. Finally, we intermittently treated young adult mice following fracture with drugs that selectively eliminate senescent cells ("senolytics", Dasatinib plus Quercetin), and showed that this regimen both decreased senescence and SASP markers in the fracture callus and significantly accelerated the time course of fracture healing. Our findings thus demonstrate that senescent cells accumulate transiently in the murine fracture callus and, in contrast to the skin, their clearance does not impair but rather may improve fracture healing.

physiology↗

bioRxiv2019

Genetic Analysis of Osteoblast Activity Identifies Zbtb40 as a Regulator of Osteoblast Activity and Bone Mass

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

genetics↗