Lagnado, Anthony B.

Abstract Senescent cells drive age-related tissue dysfunction partially via the induction of a chronic senescence-associated secretory phenotype (SASP). Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated. Mitochondria also control apoptosis, a cell fate generally perceived as distinct from cellular senescence, which is apoptosis resistant. Here, we show that mitochondrial outer membrane permeability (MOMP) occurring in a small subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), depends on the formation of BAX and BAK macropores and results in release of mitochondrial DNA (mtDNA) into the cytosol, which in turn activates the cGAS/STING pathway, a major regulator of the SASP. Importantly, we found that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan parameters in aged mice. Our results propose the novel concept that apoptosis and senescence are regulated by similar mitochondrial-dependent mechanisms, and that sub-lethal mitochondrial apoptotic stress is a major driver of the SASP. We also provide proof-of-concept that inhibition of miMOMP may be a new therapeutic avenue to improve healthspan.

ABSTRACT Oxidative stress-induced reactive oxygen species, DNA damage, apoptosis, and cellular senescence have been associated with reduced osteoprogenitors in a reciprocal fashion to bone marrow adipocyte tissue (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. Here we show by RNA-Sequencing data that BMAT-related genes were the most upregulated gene subset in radiated bones of C57BL/6 mice. Using focal radiation as a model to understand age-associated changes in bone, we performed a longitudinal assessment of cellular senescence and BMAT. Using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA in situ hybridization of p21 transcripts and histological assessment of telomere dysfunction as a marker of senescence, we observed an increase in senescent cell burden of bone cells from day 1 postradiation, 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 postradiation. Similarly, elevation in BMAT-related genes was observed in bones of aged mice. The senolytic cocktail of Dasatinib (D) plus Quercetin (Q) (ie, D + Q), which clears senescent cells, reduced BMAT in aged and radiated bones. MicroRNAs (miRNAs or miRs) linked with senescence marker p21 were downregulated in radiated and aged bones, whereas 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 postradiation. 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. © 2022 American Society for Bone and Mineral Research (ASBMR). Abstract Schematic representing a senescent bone environment during aging and radiation. The schematic represents changes in an oxidatively stressed, senescent microenvironment in which mesenchymal progenitors are preferentially forming adipocytes, regulated by a common process shared between aging bone and radiation-induced bone damage, and with identical expression patterns of bone marrow adipocyte tissue (BMAT)-related genes and microRNAs (miRNAs or miRs). Increase in senescent markers p21 and p16Ink4a induce a cellular state in which production of the senescence-associated secretory phenotype (SASP) increases and influences the bone marrow environment. Secreted adipokines can promote mesenchymal stem cell (MSC) fate, switching to an adipocyte lineage. Several adipokines are also released in circulation, causing systemic effects. These changes in senescence markers and genes that regulate BMAT are in turn regulated by their corresponding miRNAs. These changes can be blocked or reversed by the clearance of senescent cells using senolytic drugs. The figure was created with BioRender.com.

The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a onetime pharmacological intervention (streptozotocin, STZ) initiated in adulthood combined with high-fat diet–induced (HFD-induced) obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic β cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE CML in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique proinflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.

ABSTRACT Clinical radiotherapy treats life-threatening cancers, but the radiation often affects neighboring normal tissues including bone. Acute effects of ionizing radiation include oxidative stress, DNA damage, and cellular apoptosis. We show in this study that a large proportion of bone marrow cells, osteoblasts, and matrix-embedded osteocytes recover from these insults only to attain a senescent profile. Bone analyses of senescence-associated genes, senescence-associated beta-galactosidase (SA-β-gal) activity, and presence of telomere dysfunction-induced foci (TIF) at 1, 7, 14, 21, and 42 days post–focal radiation treatment (FRT) in C57BL/6 male mice confirmed the development of senescent cells and the senescence-associated secretory phenotype (SASP). Accumulation of senescent cells and SASP markers were correlated with a significant reduction in bone architecture at 42 days post-FRT. To test if senolytic drugs, which clear senescent cells, alleviate FRT-related bone damage, we administered the senolytic agents, dasatinib (D), quercetin (Q), fisetin (F), and a cocktail of D and Q (D+Q). We found moderate alleviation of radiation-induced bone damage with D and Q as stand-alone compounds, but no such improvement was seen with F. However, the senolytic cocktail of D+Q reduced senescent cell burden as assessed by TIF+ osteoblasts and osteocytes, markers of senescence (p16 Ink4a and p21), and key SASP factors, resulting in significant recovery in the bone architecture of radiated femurs. In summary, this study provides proof of concept that senescent cells play a role in radiotherapy-associated bone damage, and that reduction in senescent cell burden by senolytic agents is a potential therapeutic option for alleviating radiotherapy-related bone deterioration. © 2020 American Society for Bone and Mineral Research.

AbstractIncreased activation of the major pro‐inflammatory NF‐κB pathway leads to numerous age‐related diseases, including chronic liver disease (CLD). Rapamycin, an inhibitor of mTOR, extends lifespan and healthspan, potentially via suppression of inflammaging, a process which is partially dependent on NF‐κB signalling. However, it is unknown if rapamycin has beneficial effects in the context of compromised NF‐κB signalling, such as that which occurs in several age‐related chronic diseases. In this study, we investigated whether rapamycin could ameliorate age‐associated phenotypes in a mouse model of genetically enhanced NF‐κB activity (nfκb1−/−) characterized by low‐grade chronic inflammation, accelerated aging and CLD. We found that, despite showing no beneficial effects in lifespan and inflammaging, rapamycin reduced frailty and improved long‐term memory, neuromuscular coordination and tissue architecture. Importantly, markers of cellular senescence, a known driver of age‐related pathology, were alleviated in rapamycin‐fed animals. Our results indicate that, in conditions of genetically enhanced NF‐κB, rapamycin delays aging phenotypes and improves healthspan uncoupled from its role as a suppressor of inflammation.