Explore the vast range of titles available.

Small molecules that selectively kill senescent cells (SCs), termed senolytics, have the potential to prevent and treat various age-related diseases and extend healthspan. The use of Bcl-xl inhibitors as senolytics is largely limited by their on-target and dose-limiting platelet toxicity. Here, we report the use of proteolysis-targeting chimera (PROTAC) technology to reduce the platelet toxicity of navitoclax (also known as ABT263), a Bcl-2 and Bcl-xl dual inhibitor, by converting it into PZ15227 (PZ), a Bcl-xl PROTAC, which targets Bcl-xl to the cereblon (CRBN) E3 ligase for degradation. Compared to ABT263, PZ is less toxic to platelets, but equally or slightly more potent against SCs because CRBN is poorly expressed in platelets. PZ effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia. With further improvement, Bcl-xl PROTACs have the potential to become safer and more potent senolytic agents than Bcl-xl inhibitors. Senolytics have the potential to extend healthspan by selectively killing senescent cells (SCs), but senolytics that target Bcl-xl may cause platelet toxicity. Here, the authors generated a Bcl-xl proteolysis-targeting chimera (PROTAC) senolytic, which effectively clears SCs and rejuvenates tissue stem and progenitor cells in naturally aged mice without causing severe thrombocytopenia.

Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E 2 ) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax—two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E 2 . However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases “oxidative phosphorylation” and the expression of mitochondria complex I genes. Additionally, E 2 decreased the activity of complex I and oxygen consumption rate. Similar to E 2 , the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.

Both an increase in osteoclast and a decrease in osteoblast numbers contribute to skeletal aging. Markers of cellular senescence, including expression of the cyclin inhibitor p16, increase with aging in several bone cell populations. The elimination of p16‐expressing cells in old mice, using the INK‐ATTAC transgene, increases bone mass indicating that senescent cells contribute to skeletal aging. However, the identity of the senescent cells and the extent to which ablation of p16‐expressing cells may prevent skeletal aging remain unknown. Using mice expressing the p16‐3MR transgene, we examined whether elimination of p16‐expressing cells between 12 and 24 months of age could preserve bone mass; and whether elimination of these cells from 20 to 26 months of age could restore bone mass. The activation of the p16‐3MR transgene by ganciclovir (GCV) greatly diminished p16 levels in the brain, liver, and osteoclast progenitors from the bone marrow. The age‐related increase in osteoclastogenic potential of myeloid cells was also abrogated by GCV. However, GCV did not alter p16 levels in osteocytes—the most abundant cell type in bone—and had no effect on the skeletal aging of p16‐3MR mice. These findings indicate that the p16‐3MR transgene does not eliminate senescent osteocytes but it does eliminate senescent osteoclast progenitors and senescent cells in other tissues, as described previously. Elimination of senescent osteoclast progenitors, in and of itself, has no effect on the age‐related loss of bone mass. Hence, other senescent cell types, such as osteocytes, must be the seminal culprits.

Marrow adipocytes and osteoblasts differentiate from common mesenchymal progenitors in a mutually exclusive manner, and diversion of these progenitors toward adipocytes in old age has been proposed to account for the decline in osteoblasts and the development of involutional osteoporosis. This idea has been supported by evidence that thiazolidinedione (TZD)‐induced activation of PPARγ, the transcription factor required for adipocyte differentiation, increases marrow fat and causes bone loss. We functionally tested this hypothesis using C57BL/6J mice with conditional deletion of PPARγ from early mesenchymal progenitors targeted by the Prx1‐Cre transgene. Using a longitudinal littermate‐controlled study design, we observed that PPARγ is indispensable for TZD‐induced increase in marrow adipocytes in 6‐month‐old male mice, and age‐associated increase in marrow adipocytes in 22‐month‐old female mice. In contrast, PPARγ is dispensable for the loss of cortical and trabecular bone caused by TZD or old age. Instead, PPARγ restrains age‐dependent development of cortical porosity. These findings do not support the long‐standing hypothesis that increased marrow adipocyte differentiation contributes to bone loss in old age but reveal a novel role of mesenchymal cell PPARγ in the maintenance of cortical integrity. Diversion of mesenchymal progenitors toward adipocytes instead of osteoblasts has been proposed to account for skeletal aging. Using mice with conditional deletion of PPARγ in mesenchymal progenitors, we show that PPARγ decreases cortical bone area but is dispensable for the loss of bone caused by PPARγ stimulators (TZD) or old age. Strikingly, deletion of PPARγ exacerbates the development of cortical porosity but attenuates osteoarthritis with aging.

Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H 2 O 2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H 2 O 2 -inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H 2 O 2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption. Osteoclasts are bone-resorbing cells responsible for the loss of bone mass in diseases such as osteoporosis. Here the authors show that osteoclast proliferation and survival is regulated by FoxO family transcription factors, which control levels of the signalling molecule hydrogen peroxide.

The scavenger receptor class B member 1 (SCARB1), encoded by Scarb1 , is a cell surface receptor for high density lipoproteins, low density lipoproteins (LDL), oxidized LDL (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in multiple cell types, including osteoblasts and macrophages. PC-OxPLs, present on OxLDL and apoptotic cells, adversely affect bone metabolism. Overexpression of E06 IgM – a natural antibody that recognizes PC-OxPLs– increases cancellous and cortical bone at 6 months of age in both sexes and protects against age- and high fat diet- induced bone loss, by increasing bone formation. We have reported that SCARB1 is the most abundant scavenger receptor for OxPLs in osteoblastic cells, and osteoblasts derived from Scarb1 knockout mice ( Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxLDL. Skeletal analysis of Scarb1 KO mice produced contradictory results, with some studies reporting elevated bone mass and others reporting low bone mass. To clarify if Scarb1 mediates the negative effects of PC-OxPLs in bone, we deleted it in osteoblast lineage cells using Osx1-Cre transgenic mice. Bone mineral density (BMD) measurements and micro-CT analysis of cancellous and cortical bone at 6 months of age did not reveal any differences between Scarb1 ΔOSX-l mice and their wild-type (WT), Osx1-Cre, or Scarb1 fl/fl littermate controls. We then investigated whether PC-OxPLs could exert their anti-osteogenic effects via activation of SCARB1 in myeloid cells by deleting Scarb1 in LysM-Cre expressing cells. BMD measurements and micro-CT analysis at 6 months of age did not show any differences between Scarb1 ΔLysM mice and their WT, LysM-Cre, or Scarb1 fl/fl controls. Based on this evidence, we conclude that the adverse skeletal effects of PC-OxPLs in adult mice are not mediated by Scarb1 expressed in osteoblast lineage cells or myeloid cells.

The damaging effects of ionizing radiation (IR) on bone mass are well-documented in mice and humans and are most likely due to increased osteoclast number and function. However, the mechanisms leading to inappropriate increases in osteoclastic bone resorption are only partially understood. Here, we show that exposure to multiple fractions of low-doses (10 fractions of 0.4 Gy total body irradiation [TBI]/week, i.e., fractionated exposure) and/or a single exposure to the same total dose of 4 Gy TBI causes a decrease in trabecular, but not cortical, bone mass in young adult male mice. This damaging effect was associated with highly activated bone resorption. Both osteoclast differentiation and maturation increased in cultures of bone marrow-derived macrophages from mice exposed to either fractionated or singular TBI. IR also increased the expression and enzymatic activity of mitochondrial deacetylase Sirtuin-3 (Sirt3)—an essential protein for osteoclast mitochondrial activity and bone resorption in the development of osteoporosis. Osteoclast progenitors lacking Sirt3 exposed to IR exhibited impaired resorptive activity. Taken together, targeting impairment of osteoclast mitochondrial activity could be a novel therapeutic strategy for IR-induced bone loss, and Sirt3 is likely a major mediator of this effect.

A decrease in osteoblast number and bone formation are seminal contributors to age‐related osteoporosis. However, the aging‐associated molecular mechanisms that impact osteoblast precursors, osteoblasts, osteocytes, and other bone mesenchymal cell types remain unclear. We performed single‐cell RNA‐sequencing of mesenchymal cells present at the endosteum and periosteum of young and old C57BL/6 mice of both sexes. Osteoblast precursors and osteoblasts from female endosteum exhibited the greatest changes with aging. Transcriptional changes revealed decreased matrix protein production and autophagy, as well as increased senescence, phosphorylation, and hypoxia. Because deficient macroautophagy in osteoblast lineage cells decreases bone formation, we contrasted the transcriptional changes caused by autophagy inactivation in Atg7f/f; Osx1‐Cre mice with those caused by aging and found a causal link between autophagy deficiency and increased senescence in osteoblastic cells. Overall, these findings reveal distinct features of aging in males and females and molecular pathways that might be implicated in the development of intracortical porosity in the female skeleton. The transcriptional changes in periosteal cells indicate mechanisms that might contribute to the decreased response to mechanical loading and delayed fracture healing in the old skeleton. Our data provides a comprehensive resource and serves as a reference for understanding how future genetic and pharmacological interventions impact molecular mechanisms of aging in osteoblasts and other mesenchymal cells in the skeleton. scRNA‐seq analysis reveals age‐related changes in bone mesenchymal cells in male and female mice. Moreover, the comparison of aging and autophagy deficiency in bone mesenchymal cells shows overlap and suggests that age‐related decline in autophagy contributes to skeletal aging.

NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD + deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase ( Nampt ), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cells of the limbs. At birth, Nampt ΔPrx1 exhibit dramatic limb shortening due to death of growth plate chondrocytes. Administration of the NAD precursor nicotinamide riboside during pregnancy prevents the majority of in utero defects. Depletion of NAD post-birth also promotes chondrocyte death, preventing further endochondral ossification and joint development. In contrast, osteoblast formation still occurs in knockout mice, in line with distinctly different microenvironments and reliance on redox reactions between chondrocytes and osteoblasts. These findings define a critical role for cell-autonomous NAD homeostasis during endochondral bone formation. Deficiency in NAD+ has been implicated in skeletal deformities during development in both humans and mice. Here, the authors use mice that lack the critical enzyme of the NAD+ salvage pathway Nampt in mesenchymal lineage cells to show that the NAD salvage pathway is indispensable for endochondral but not intramembranous bone development.

Altered mitochondria activity in osteoblasts and osteoclasts has been implicated in the loss of bone mass associated with aging and estrogen deficiency - the 2 most common causes of osteoporosis. However, the mechanisms that control mitochondrial metabolism in bone cells during health or disease remain unknown. The mitochondrial deacetylase sirtuin-3 (Sirt3) has been earlier implicated in age-related diseases. Here, we show that deletion of Sirt3 had no effect on the skeleton of young mice but attenuated the age-related loss of bone mass in both sexes. This effect was associated with impaired bone resorption. Osteoclast progenitors from aged Sirt3-null mice were able to differentiate into osteoclasts, though the differentiated cells exhibited impaired polykaryon formation and resorptive activity, as well as decreased oxidative phosphorylation and mitophagy. The Sirt3 inhibitor LC-0296 recapitulated the effects of Sirt3 deletion in osteoclast formation and mitochondrial function, and its administration to aging mice increased bone mass. Deletion of Sirt3 also attenuated the increase in bone resorption and loss of bone mass caused by estrogen deficiency. These findings suggest that Sirt3 inhibition and the resulting impairment of osteoclast mitochondrial function could be a novel therapeutic intervention for the 2 most important causes of osteoporosis.