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OBJECTIVES: The mitochondrial deacetylase sirtuin-3 (SIRT3) is necessary for the increased bone resorption and enhanced function of mitochondria in osteoclasts that occur with advancing age; how SIRT3 drives bone resorption remains elusive. METHODS: To determine the role of SIRT3 in osteoclast mitochondria, we used mice with conditional loss of Sirt3 in osteoclast lineage and mice with germline deletion of either Sirt3 or its known target Pink1. RESULTS: SIRT3 stimulates mitochondrial quality in osteoclasts in a PINK1-independent manner, promoting mitochondrial activity and osteoclast maturation and function, thereby contributing to bone loss in female but not male mice. Quantitative analyses of global proteomes and acetylomes revealed that deletion of Sirt3 dramatically increased acetylation of osteoclast mitochondrial proteins, particularly ATPase inhibitory factor 1 (ATPIF1), an essential protein for mitophagy. Inhibition of mitophagy via mdivi-1 recapitulated the effect of deletion of Sirt3 or Atpif1 in osteoclast formation and mitochondrial function. CONCLUSIONS: Decreasing mitophagic flux in osteoclasts may be a promising pharmacotherapeutic approach to treat osteoporosis in older adults.
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Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
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Células-Tronco Mesenquimais , Osteoblastos , Osteócitos , Periósteo , Animais , Camundongos , Condrócitos/metabolismo , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteócitos/metabolismo , Osteócitos/citologia , Periósteo/citologia , Periósteo/metabolismo , Análise de Célula Única , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Oestrogen deficiency increases bone resorption, contributing to osteoporosis development. Yet, the mechanisms mediating the effects of oestrogen on osteoclasts remain unclear. This study aimed to elucidate the early metabolic alteration induced by RANKL, the essential cytokine in osteoclastogenesis and 17-beta-oestradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and primary bone marrow-derived macrophages as biological models. RESULTS: This research demonstrated that, in osteoclast precursors, RANKL stimulates complex I activity, oxidative phosphorylation (OXPHOS) and mitochondria-derived ATP production as early as 3 h of exposure. This effect on mitochondrial bioenergetics is associated with an increased capacity to oxidize TCA cycle substrates, fatty acids and amino acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3 h. Further, the pro-apoptotic effects of E2 during osteoclast differentiation were associated with an accumulation of p392S-p53 in mitochondria. CONCLUSIONS: These findings elucidate the early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.
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Diferenciação Celular , Estradiol , Mitocôndrias , Osteoclastos , Proteína Supressora de Tumor p53 , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Estradiol/farmacologia , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Ligante RANK/metabolismo , Células RAW 264.7 , Proteína Supressora de Tumor p53/metabolismoRESUMO
Single-cell RNA sequencing has led to numerous novel designations for mesenchymal cell types associated with bone. Consequently, there are now multiple designations for what appear to be the same cell type. In addition, existing datasets contain relatively small numbers of mature osteoblasts and osteocytes and there has been no comparison of periosteal bone cells to those at the endosteum and trabecular bone. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. To do this, we created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in any cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of skeletal stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
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Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.
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Citrulinação , Displasia Cleidocraniana , Animais , Camundongos , Osteogênese , Cromatografia Líquida , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Espectrometria de Massas em Tandem , Camundongos KnockoutRESUMO
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, NamptΔPrx1 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.
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Metabolismo Energético , NAD , Humanos , Camundongos , Animais , NAD/metabolismo , Oxirredução , Homeostase , Camundongos Knockout , Citocinas/metabolismoRESUMO
Introduction: Estrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17ß-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors. Methods: Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity. Results: We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. Discussion: These findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.
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NAD , Osteogênese , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Diferenciação Celular/fisiologia , Estrogênios/farmacologia , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , NAD/metabolismo , Osteoclastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
INTRODUCTION: Spinal cord injury (SCI) affects 25,000-50,000 people around the world each year and there is no cure for SCI patients currently. The primary injury damages spinal cord tissues and secondary injury mechanisms, including ischemia, apoptosis, inflammation, and astrogliosis, further exacerbate the lesions to the spinal cord. Recently, researchers have designed various therapeutic approaches for SCI by targeting its major cellular or molecular pathophysiology. AREAS COVERED: Some strategies have shown promise in repairing injured spinal cord for functional recoveries, such as administering neuroprotective reagents, targeting specific genes to promote robust axon regeneration of disconnected spinal fiber tracts, targeting epigenetic factors to enhance cell survival and neural repair, and facilitating neuronal relay pathways and neuroplasticity for restoration of function after SCI. This review focuses on the major advances in preclinical molecular therapies for SCI reported in recent years. EXPERT OPINION: Recent progress in developing novel and effective repairing strategies for SCI is encouraging, but many challenges remain for future design of effective treatments, including developing highly effective neuroprotectants for early interventions, stimulating robust neuronal regeneration with functional synaptic reconnections among disconnected neurons, maximizing the recovery of lost neural functions with combination strategies, and translating the most promising therapies into human use.
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Axônios , Traumatismos da Medula Espinal , Humanos , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , NeurôniosRESUMO
The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a glycosylated cell surface receptor for high density lipoproteins (HDL), oxidized low density lipoproteins (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in macrophages and has been shown to have both pro- and anti-atherogenic properties. It has been reported that global deletion of Scarb1 in mice leads to either high or low bone mass and that PC-OxPLs decrease osteoblastogenesis and increase osteoclastogenesis. PC-OxPLs decrease bone mass in 6-month-old mice and are critical pathogenetic factors in the bone loss caused by high fat diet or aging. We have investigated here whether Scarb1 expression in myeloid cells affects bone mass and whether PC-OxPLs exert their anti-osteogenic effects via activation of Scarb1 in macrophages. To this end, we generated mice with deletion of Scarb1 in LysM-Cre expressing cells and found that lack of Scarb1 did not affect bone mass in vivo. These results indicate that Scarb1 expression in cells of the myeloid/osteoclast lineage does not contribute to bone homeostasis. Based on this evidence, and earlier studies of ours showing that Scarb1 expression in osteoblasts does not affect bone mass, we conclude that Scarb1 is not an important mediator of the adverse effects on PC-OxPLs in osteoclasts or osteoblasts in 6-month-old mice.
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Densidade Óssea , Osso e Ossos , Animais , Camundongos , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Osso e Ossos/metabolismo , Osteoclastos/metabolismo , OsteogêneseRESUMO
Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.
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Proteínas Adaptadoras de Transdução de Sinal , Reabsorção Óssea , Osteoclastos , Animais , Feminino , Masculino , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Camundongos Knockout , Osteoclastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Hydrogen peroxide (H2O2), superoxide anion radical (O2-â¢), and other forms of reactive oxygen species (ROS) are produced by the vast majority of mammalian cells and can contribute both to cellular homeostasis and dysfunction. The NADPH oxidases (NOX) enzymes and the mitochondria electron transport chain (ETC) produce most of the cellular ROS. Multiple antioxidant systems prevent the accumulation of excessive amounts of ROS which cause damage to all cellular macromolecules. Many studies have examined the contribution of ROS to different bone cell types and to skeletal physiology and pathophysiology. Here, we discuss the role of H2O2 and O2-⢠and their major enzymatic sources in osteoclasts and osteoblasts, the fundamentally different ways via which these cell types utilize mitochondrial derived H2O2 for differentiation and function, and the molecular mechanisms that impact and are altered by ROS in these cells. Particular emphasis is placed on evidence obtained from mouse models describing the contribution of different sources of ROS or antioxidant enzymes to bone resorption and formation. Findings from studies using pharmacological or genetically modified mouse models indicate that an increase in H2O2 and perhaps other ROS contribute to the loss of bone mass with aging and estrogen deficiency, the two most important causes of osteoporosis and increased fracture risk in humans.
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Mechanical signals stimulate mitochondrial function but the molecular mechanisms are not clear. Here, we show that the mechanically sensitive ion channel Piezo1 plays a critical role in mitochondrial adaptation to mechanical stimulation. The activation of Piezo1 induced mitochondrial calcium uptake and oxidative phosphorylation (OXPHOS). In contrast, loss of Piezo1 reduced the mitochondrial oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production in calvarial cells and these changes were associated with increased expression of the phosphodiesterases Pde4a and lower cyclic AMP (cAMP) levels. In addition, Piezo1 increased cAMP production and the activation of a cAMP-responsive transcriptional reporter. Consistent with this, cAMP was sufficient to increase mitochondrial OCR and the inhibition of phosphodiesterases augmented the increase in OCR induced by Piezo1. Moreover, the inhibition of cAMP production or activity of protein kinase A, a kinase activated by cAMP, prevented the increase in OCR induced by Piezo1. These results demonstrate that cAMP signaling contributes to the increase in mitochondrial OXPHOS induced by activation of Piezo1.
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AMP Cíclico , Mitocôndrias , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mitocôndrias/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de SinaisRESUMO
The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal cells, but functional evidence for the mediators of these effects has only recently begun to emerge. We report that the matrix metalloproteinase 13 (MMP13) is the highest up-regulated gene in mesenchymal cells from mice lacking the estrogen receptor alpha (ERα). In sham-operated female mice with conditional Mmp13 deletion in Prrx1 expressing cells (Mmp13ΔPrrx1), the femur and tibia length was lower as compared to control littermates (Mmp13f./f). Additionally, in the sham-operated female Mmp13ΔPrrx1 mice cortical thickness and trabecular bone volume in the femur and tibia were higher and osteoclast number at the endocortical surfaces was lower, whereas bone formation rate was unaffected. Notably, the decrease of cortical thickness caused by ovariectomy (OVX) in the femur and tibia of Mmp13f./f mice was attenuated in the Mmp13ΔPrrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell-derived MMP13 may regulate osteoclast number and/or activity, bone resorption, and bone mass. And increased production of mesenchymal cell-derived factors may be important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone.
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Densidade Óssea , Doenças Ósseas Metabólicas , Metaloproteinase 13 da Matriz/metabolismo , Animais , Osso e Ossos/diagnóstico por imagem , Osso Cortical , Estrogênios , Feminino , Proteínas de Homeodomínio , Humanos , Metaloproteinase 13 da Matriz/genética , Camundongos , Ovariectomia/efeitos adversosRESUMO
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.
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Reabsorção Óssea/patologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Osteoclastos/metabolismo , Osteoclastos/efeitos da radiação , Radiação Ionizante , Animais , Osso Esponjoso/patologia , Osso Esponjoso/efeitos da radiação , Respiração Celular/efeitos da radiação , Fracionamento da Dose de Radiação , Masculino , Camundongos Endogâmicos C57BL , Sirtuína 3/metabolismoRESUMO
Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth's magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.
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Osso Esponjoso/efeitos da radiação , Radiação Cósmica/efeitos adversos , Mitocôndrias/efeitos da radiação , Osteoclastos/efeitos da radiação , Osteogênese/efeitos da radiação , Animais , Masculino , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismoRESUMO
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.
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Envelhecimento/fisiologia , Mitocôndrias/metabolismo , Osteoporose/metabolismo , Sirtuína 3 , Animais , Estrogênios/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismoRESUMO
Excessive activation of the thrombin receptor, protease activated receptor 1 (PAR1) is implicated in diverse neuropathologies from neurodegenerative conditions to neurotrauma. PAR1 knockout mice show improved outcomes after experimental spinal cord injury (SCI), however information regarding the underpinning cellular and molecular mechanisms is lacking. Here we demonstrate that genetic blockade of PAR1 in female mice results in improvements in sensorimotor co-ordination after thoracic spinal cord lateral compression injury. We document improved neuron preservation with increases in Synapsin-1 presynaptic proteins and GAP43, a growth cone marker, after a 30 days recovery period. These improvements were coupled to signs of enhanced myelin resiliency and repair, including increases in the number of mature oligodendrocytes, their progenitors and the abundance of myelin basic protein. These significant increases in substrates for neural recovery were accompanied by reduced astrocyte (Serp1) and microglial/monocyte (CD68 and iNOS) pro-inflammatory markers, with coordinate increases in astrocyte (S100A10 and Emp1) and microglial (Arg1) markers reflective of pro-repair activities. Complementary astrocyte-neuron co-culture bioassays suggest astrocytes with PAR1 loss-of-function promote both neuron survival and neurite outgrowth. Additionally, the pro-neurite outgrowth effects of switching off astrocyte PAR1 were blocked by inhibiting TrkB, the high affinity receptor for brain derived neurotrophic factor. Altogether, these studies demonstrate unique modulatory roles for PAR1 in regulating glial-neuron interactions, including the capacity for neurotrophic factor signaling, and underscore its position at neurobiological intersections critical for the response of the CNS to injury and the capacity for regenerative repair and restoration of function.
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Receptor PAR-1 , Traumatismos da Medula Espinal , Animais , Astrócitos/metabolismo , Feminino , Camundongos , Neurônios/metabolismo , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismoRESUMO
Age-related osteoporosis is caused by a deficit in osteoblasts, the cells that secrete bone matrix. The number of osteoblast progenitors also declines with age associated with increased markers of cell senescence. The forkhead box O (FoxO) transcription factors attenuate Wnt/ß-catenin signaling and the proliferation of osteoprogenitors, thereby decreasing bone formation. The NAD+-dependent Sirtuin1 (Sirt1) deacetylates FoxOs and ß-catenin in osteoblast progenitors and, thereby, increases bone mass. However, it remains unknown whether the Sirt1/FoxO/ß-catenin pathway is dysregulated with age in osteoblast progenitors. We found decreased levels of NAD+ in osteoblast progenitor cultures from old mice, associated with increased acetylation of FoxO1 and markers of cell senescence. The NAD+ precursor nicotinamide riboside (NR) abrogated FoxO1 and ß-catenin acetylation and several marker of cellular senescence, and increased the osteoblastogenic capacity of cells from old mice. Consistent with these effects, NR administration to C57BL/6 mice counteracted the loss of bone mass with aging. Attenuation of NAD+ levels in osteoprogenitor cultures from young mice inhibited osteoblastogenesis in a FoxO-dependent manner. In addition, mice with decreased NAD+ in cells of the osteoblast lineage lost bone mass at a young age. Together, these findings suggest that the decrease in bone formation with old age is due, at least in part, to a decrease in NAD+ and dysregulated Sirt1/FoxO/ß-catenin pathway in osteoblast progenitors. NAD+ repletion, therefore, represents a rational therapeutic approach to skeletal involution.
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Aging is characterized by systemic declines in tissue and organ functions. Interventions that slow these declines represent promising therapeutics to protect against age-related disease and improve the quality of life. In this study, several interventions associated with lifespan extension in invertebrates or improvement of age-related disease were tested in mouse models to determine if they were effective in slowing tissue aging in a broad spectrum of functional assays. Benzoxazole, which extends the lifespan of Caenorhabditis elegans, slowed age-related femoral bone loss in mice. Rates of change were established for clinically significant parameters in untreated mice, including kyphosis, blood glucose, body composition, activity, metabolic measures, and detailed parameters of skeletal aging in bone. These findings have implications for the study of preclinical physiological aging and therapies targeting aging. Finally, an online application was created that includes the calculated rates of change and that enables power and variance to be calculated for many clinically important metrics of aging with an emphasis on bone. This resource will help in future study designs employing novel interventions in aging mice. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.