Biology subjects

Monroe, D.

Publications and source records attributed to Monroe, D..

10 recordsLinked to original sources

bioRxiv2026

MicroRNA Networks Driving Skeletal Aging and WNT Pathway Modulation

Cellular senescence is a key mechanism of skeletal aging in both physiological and accelerated conditions, such as radiotherapy. This study aimed to identify common differentially regulated microRNAs (miRs) across these contexts. We performed miR sequencing on three models: femurs from young (5-month-old) versus old (24-month-old) mice; focally radiated versus non-radiated femurs; and osteocytes from young versus old mice. Osteocytes were included in the comparison, as they have the longest lifespan in the mineralized bone matrix and they form 90-95% of all mesenchymal bone cell types. Among the three groups, miR-135a-5p and miR-671-5p were the common (i.e., shared) miRs that were downregulated, and miR-183-5p, a miR that regulates the WNT pathway, was the only shared upregulated miR, while miR-155-5p, a miR that regulates the Senescence-Associated Secretory Phenotype (SASP), was elevated in two conditions. The WNT-pathway has been positively associated with bone health and Sclerostin, a WNT-pathway inhibitor produced and secreted by osteocytes, has been implicated in accelerated skeletal deterioration following radiation. Thus, we used a neutralizing antibody to Sclerostin (Scl-Ab), to assess genes related to the WNT pathway and senescence, which are regulated by miR-183-5p and miR-155-5p, respectively. We further performed miR sequencing in radiated bones from mice treated with Scl-Ab and identified miR-133a-3p, a key miR that inhibits bone metabolism and function, which is upregulated in accelerated skeletal aging (i.e., focal radiation) downregulated by Scl-Ab. Overall, our study identifies potential regulatory gene pathways that modulate skeletal aging in the presence and absence of a WNT activator, Scl-Ab.

genomics↗

bioRxiv2025

Comprehensive healthspan assessments and influence of sex as a biological variable in aging rats

Rats share a significant amount of genetic and physiological similarity with humans. Many biological processes and pathways are conserved between rats and humans, making rats a suitable model for studying various aspects of human health and disease. Using rats as an aging model offers a more ethical alternative to using larger, longer-lived animals like primates. Rats are easier to handle in laboratory settings, as compared to non-human primates, both of which have physiological functions like humans. To date, there are very few studies which have comprehensively studied age-related changes in rat physiology. Here we present a longitudinal assessment of several aspects of Brown-Norway rat physiology and histopathology using molecular and functional assessments at 6-, 17- and 27 months of age. Our studies thus provide age-related healthspan parameters, which can be used as reference for genetic or pharmacological rat models of aging.

animal behavior and cognition↗

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

Roles of cellular senescence in driving bone marrow adiposity in radiation-and aging-associated bone loss

Osteoporosis is associated with an increase in marrow adipocytes, collectively termed bone marrow adipose tissue (BMAT). An increase in BMAT is linked with decline of mesenchymal progenitors that give rise to osteoblasts which are responsible for bone accrual. Oxidative stress-induced reactive oxygen species, DNA damage, apoptosis and cellular senescence have been associated with reduced osteoprogenitors in a reciprocal fashion to BMAT; however, a direct (causal) link between cellular senescence and BMAT is still elusive. Accumulation of senescent cells occur in naturally aged and in focally radiated bone tissue, but despite amelioration of age- and radiation-associated bone loss after senescent cell clearance, molecular events that precede BMAT accrual are largely unknown. Using a mouse model here we show by RNA-Sequencing data that BMAT-related genes were the most upregulated gene subset in radiated bones. Using focal radiation as a model to understand age-associated changes in bone, we performed a longitudinal assessment of cellular senescence and BMAT. Using qRT-PCR, RNA in situ hybridization and histological assessment of telomere dysfunction as a marker of senescence, we observed increased p21 transcripts in bone lining cells, osteocytes and bone marrow cells, and elevated dysfunctional telomeres in osteocytes starting from day 1 post-radiation, without the presence of BMAT. BMAT was significantly elevated in radiated bones at day 7, confirming the qRT-PCR data in which most BMAT-related genes were elevated by day 7, and the trend continued until day 42 post-radiation. Similarly, elevation in BMAT-related genes was observed in aged bones. The senolytic cocktail of Dasatinib (D) plus Quercetin (Q) - D+Q, which clears senescent cells, reduced BMAT in aged and radiated bones. MicroRNAs (miRs) linked with senescence marker p21 were downregulated in radiated- and aged-bones, while miR-27a, a miR that is associated with increased BMAT, was elevated both in radiated- and aged-bones. D+Q downregulated miR-27a in radiated bones at 42 days post-radiation. Overall, our study provides evidence that BMAT occurrence in oxidatively stressed bone environments, such as radiation and aging, is induced following a common pathway and is dependent on the presence of senescent cells.

molecular 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↗