Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

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.

Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice.

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.

Oestradiol and osteoclast differentiation: Effects on p53 and mitochondrial metabolism.

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.

Piezo1 stimulates mitochondrial function via cAMP signaling.

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.

Ionizing Radiation Activates Mitochondrial Function in Osteoclasts and Causes Bone Loss in Young Adult Male Mice.

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.

Blocking the Thrombin Receptor Promotes Repair of Demyelinated Lesions in the Adult Brain.

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knock-out of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knock-out in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knock-out mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.SIGNIFICANCE STATEMENT Replacement of oligodendroglia and myelin regeneration holds tremendous potential to improve function across neurological conditions. Here we demonstrate Protease Activated Receptor 1 (PAR1) is an important regulator of the capacity for myelin regeneration across two experimental murine models of myelin injury. PAR1 is a G-protein-coupled receptor densely expressed in the CNS, however there is limited information regarding its physiological roles in health and disease. Using a combination of PAR1 knock-out mice, oligodendrocyte monocultures and oligodendrocyte-astrocyte cocultures, we demonstrate blocking PAR1 improves myelin production by a mechanism related to effects across glial compartments and linked in part to regulatory actions toward growth factors such as BDNF. These findings set the stage for development of new clinically relevant myelin regeneration strategies.

The thrombin receptor modulates astroglia-neuron trophic coupling and neural repair after spinal cord injury.

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.

The thrombin receptor links brain derived neurotrophic factor to neuron cholesterol production, resiliency and repair after spinal cord injury.

Despite concerted efforts to identify CNS regeneration strategies, an incomplete understanding of how the needed molecular machinery is regulated limits progress. Here we use models of lateral compression and FEJOTA clip contusion-compression spinal cord injury (SCI) to identify the thrombin receptor (Protease Activated Receptor 1 (PAR1)) as an integral facet of this machine with roles in regulating neurite growth through a growth factor- and cholesterol-dependent mechanism. Functional recovery and signs of neural repair, including expression of cholesterol biosynthesis machinery and markers of axonal and synaptic integrity, were all increased after SCI in PAR1 knockout female mice, while PTEN was decreased. Notably, PAR1 differentially regulated HMGCS1, a gene encoding a rate-limiting enzyme in cholesterol production, across the neuronal and astroglial compartments of the intact versus injured spinal cord. Pharmacologic inhibition of cortical neuron PAR1 using vorapaxar in vitro also decreased PTEN and promoted neurite outgrowth in a cholesterol dependent manner, including that driven by suboptimal brain derived neurotrophic factor (BDNF). Pharmacologic inhibition of PAR1 also augmented BDNF-driven HMGCS1 and cholesterol production by murine cortical neurons and by human SH-SY5Y and iPSC-derived neurons. The link between PAR1, cholesterol and BDNF was further highlighted by demonstrating that the deleterious effects of PAR1 over-activation are overcome by supplementing cultures with BDNF, cholesterol or by blocking an inhibitor of adenylate cyclase, Gαi. These findings document PAR1-linked neurotrophic coupling mechanisms that regulate neuronal cholesterol metabolism as an important component of the machinery regulating CNS repair and point to new strategies to enhance neural resiliency after injury.

Simulated Galactic Cosmic Rays Modify Mitochondrial Metabolism in Osteoclasts, Increase Osteoclastogenesis and Cause Trabecular Bone Loss in Mice.

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.

A Western diet impairs CNS energy homeostasis and recovery after spinal cord injury: Link to astrocyte metabolism.

A diet high in fat and sucrose (HFHS), the so-called Western diet promotes metabolic syndrome, a significant co-morbidity for individuals with spinal cord injury (SCI). Here we demonstrate that the spinal cord of mice consuming HFHS expresses reduced insulin-like growth factor 1 (IGF-1) and its receptor and shows impaired tricarboxylic acid cycle function, reductions in PLP and increases in astrogliosis, all prior to SCI. After SCI, Western diet impaired sensorimotor and bladder recovery, increased microgliosis, exacerbated oligodendrocyte loss and reduced axon sprouting. Direct and indirect neural injury mechanisms are suggested since HFHS culture conditions drove parallel injury responses directly and indirectly after culture with conditioned media from HFHS-treated astrocytes. In each case, injury mechanisms included reductions in IGF-1R, SIRT1 and PGC-1α and were prevented by metformin. Results highlight the potential for a Western diet to evoke signs of neural insulin resistance and injury and metformin as a strategy to improve mechanisms of neural neuroprotection and repair.

Antidepressant Effects of Aripiprazole Augmentation for Cilostazol-Treated Mice Exposed to Chronic Mild Stress after Ischemic Stroke.

The aim of this study was to determine the effects and underlying mechanism of aripiprazole (APZ) augmentation for cilostazol (CLS)-treated post-ischemic stroke mice that were exposed to chronic mild stress (CMS). Compared to treatment with either APZ or CLS alone, the combined treatment resulted in a greater reduction in depressive behaviors, including anhedonia, despair-like behaviors, and memory impairments. This treatment also significantly reduced atrophic changes in the striatum, cortex, and midbrain of CMS-treated ischemic mice, and inhibited neuronal cell apoptosis, particularly in the striatum and the dentate gyrus of the hippocampus. Greater proliferation of neuronal progenitor cells was also observed in the ipsilateral striatum of the mice receiving combined treatment compared to mice receiving either drug alone. Phosphorylation of the cyclic adenosine monophosphate response element binding protein (CREB) was increased in the striatum, hippocampus, and midbrain of mice receiving combined treatment compared to treatment with either drug alone, particularly in the neurons of the striatum and hippocampus, and dopaminergic neurons of the midbrain. Our results suggest that APZ may augment the antidepressant effects of CLS via co-regulation of the CREB signaling pathway, resulting in the synergistic enhancement of their neuroprotective effects.

Sirtuin1 (Sirt1) promotes cortical bone formation by preventing ß-catenin sequestration by FoxO transcription factors in osteoblast progenitors.

A decline of the levels and activity of Sirtuin1 (Sirt1), a NAD(+) class III histone deacetylase, with age contributes to the development of several diseases including type 2 diabetes, neurodegeneration, inflammation, and cancer. The anti-aging effects of Sirt1 evidently result from the deacetylation of many transcription factors and co-factors including members of the Forkhead box O (FoxO) family and ß-catenin. Wnt/ß-catenin is indispensable for osteoblast generation. FoxOs, on the other hand, sequester ß-catenin and inhibit osteoprogenitor proliferation. Here, we have deleted Sirt1 in osteoprogenitors expressing Osterix1 (Osx1)-Cre and their descendants. Sirt1(ΔOsx1) mice had lower cortical thickness in femora and vertebrae because of reduced bone formation at the endocortical surface. In line with this, osteoprogenitor cell cultures from the Sirt1(ΔOsx1) mice exhibited lower alkaline phosphatase activity and mineralization, as well as decreased proliferation and increased apoptosis. These changes were associated with decreased Wnt/ß-catenin signaling and expression of cyclin D1 and resulted from increased binding of FoxOs to ß-catenin. These findings demonstrate that Sirt1-induced deacetylation of FoxOs unleashes Wnt signaling. A decline in Sirt1 activity in osteoblast progenitors with aging may, therefore, contribute to the age-related loss of bone mass. Together with evidence that Sirt1 activators increase bone mass in aged mice, our results also suggest that Sirt1 could be a therapeutic target for osteoporosis.

Protease activated receptor-1 antagonist ameliorates the clinical symptoms of experimental autoimmune encephalomyelitis via inhibiting breakdown of blood-brain barrier.

To evaluate the question of whether protease activated receptor-1 (PAR-1) antagonist is a potential therapeutic target in multiple sclerosis, we treated experimental autoimmune encephalomyelitis (EAE) mice with two PAR-1 antagonists, KC-A0590 and SCH-530348. Treatment with both antagonists resulted in a significant decrease in the clinical characteristics of EAE mice by suppressing demyelination and infiltration of inflammatory cells in the spinal cord and brain, as well as a significantly reducing the increased thrombin and tumor necrosis factor-α. Profound leakage of dextran was observed in the brain of EAE mice. However, treatment with PAR-1 antagonists resulted in the stabilization of vascular endothelial cells and reduced blood-brain barrier breakdown with suppression of inflammatory response. Treatment with PAR-1 antagonists also resulted in down-regulated expression of matrix metalloproteinase-9 and preserved expression of occludin and zonula occludens (ZO)-1 in the brain and their significant expression was confirmed in neurons, astrocytes, and vascular endothelial cells. Finally, endothelial cells and primary cultured astrocytes were treated with PAR-1 antagonists; both antagonists suppressed thrombin-induced breakdown of ZO-1 in endothelial cells and secretion of matrix metalloproteinase-9 in astrocytes. Collectively, our results suggest that PAR-1 antagonist is effective in attenuation of the clinical symptoms of EAE mice by stabilizing the blood-brain barrier and may have therapeutic potential for treatment of multiple sclerosis.

A single recurrent mutation in the 5'-UTR of IFITM5 causes osteogenesis imperfecta type V.

Osteogenesis imperfecta (OI) is a heterogenous group of genetic disorders of bone fragility. OI type V is an autosomal-dominant disease characterized by calcification of the forearm interosseous membrane, radial head dislocation, a subphyseal metaphyseal radiodense line, and hyperplastic callus formation; the causative mutation involved in this disease has not been discovered yet. Using linkage analysis in a four-generation family and whole-exome sequencing, we identified a heterozygous mutation of c.-14C>T in the 5'-untranslated region of a gene encoding interferon-induced transmembrane protein 5 (IFITM5). It completely cosegregated with the disease in three families and occurred de novo in five simplex individuals. Transfection of wild-type and mutant IFITM5 constructs revealed that the mutation added five amino acids (Met-Ala-Leu-Glu-Pro) to the N terminus of IFITM5. Given that IFITM5 expression and protein localization is restricted to the skeletal tissue and IFITM5 involvement in bone formation, we conclude that this recurrent mutation would have a specific effect on IFITM5 function and thus cause OI type V.

Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia.

BACKGROUND: Four traditional Korean medicinal herbs which act in retarding the aging process, Polygonum multiflorum Thunb., Rehmannia glutinosa (Gaertn) Libosch., Polygala tenuifolia Willd., and Acorus gramineus Soland., were prepared by systematic investigation of Dongeuibogam (Treasured Mirror of Eastern Medicine), published in the early 17th century in Korea. This study was performed to evaluate beneficial effects of four herbal mixture extract (PMC-12) on hippocampal neuron and spatial memory. METHODS: High performance liquid chromatography (HPLC) analysis was performed for standardization of PMC-12. Cell viability, lactate dehydrogenase, flow cytometry, reactive oxygen species (ROS), and Western blot assays were performed in HT22 hippocampal cells and immunohistochemistry and behavioral tests were performed in a mouse model of focal cerebral ischemia in order to observe alterations of hippocampal cell survival and subsequent memory function. RESULTS: In the HPLC analysis, PMC-12 was standardized to contain 3.09% 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, 0.35% 3',6-disinapoyl sucrose, and 0.79% catalpol. In HT22 cells, pretreatment with PMC-12 resulted in significantly reduced glutamate-induced apoptotic cell death. Pretreatment with PMC-12 also resulted in suppression of ROS accumulation in connection with cellular Ca(2+) level after exposure to glutamate. Expression levels of phosphorylated p38 mitogen-activated protein kinases (MAPK) and dephosphorylated phosphatidylinositol-3 kinase (PI3K) by glutamate exposure were recovered by pretreatment with either PMC-12 or anti-oxidant N-acetyl-L-cysteine (NAC). Expression levels of mature brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (CREB) were significantly enhanced by treatment with either PMC-12 or NAC. Combination treatment with PMC-12, NAC, and intracellular Ca(2+) inhibitor BAPTA showed similar expression levels. In a mouse model of focal cerebral ischemia, we observed higher expression of mature BDNF and phosphorylation of CREB in the hippocampus and further confirmed improved spatial memory by treatment with PMC-12. CONCLUSIONS: Our results suggest that PMC-12 mainly exerted protective effects on hippocampal neurons through suppression of Ca(2+)-related ROS accumulation and regulation of signaling pathways of p38 MAPK and PI3K associated with mature BDNF expression and CREB phosphorylation and subsequently enhanced spatial memory.

Electroacupuncture preconditioning reduces cerebral ischemic injury via BDNF and SDF-1α in mice.

BACKGROUND: This study was designed to determine if electroacupuncture (EA) preconditioning improves tissue outcome and functional outcome following experimentally induced cerebral ischemia in mice. In addition, we investigated whether the expression of brain-derived neurotrophic factor (BDNF) and stromal cell derived factor-1α (SDF-1α) and infarct volume were related with improvement in neurological and motor function by interventions in this study. METHODS: After treatment with EA at the acupoints 'Baihui (GV20)' and 'Dazhui (GV14)' for 20 min, BDNF was assessed in the cortical tissues based on Western blot and the SDF-1α and vascular endothelial growth factor (VEGF) levels in the plasma determined by ELISA. To assess the protective effects of EA against ischemic injury, the mice received once a day 20 min EA preconditioning for three days prior to the ischemic event. Focal cerebral ischemia was then induced by photothrombotic cortical ischemia. Infarct volumes, neurobehavioral deficit and motor deficit were evaluated 24 h after focal cerebral ischemia. RESULTS: The expression of BDNF protein increased significantly from 6 h, reaching a plateau at 12 h after the end of EA treatment in the cerebral cortex. Furthermore, SDF-1α, not VEGF, increased singnificantly from 12 h to 48 h after EA stimulation in the plasma. Moreover, EA preconditioning reduced the infarct volume by 43.5% when compared to control mice at 24 h after photothrombotic cortical ischemia. Consistent with a smaller infarct size, EA preconditioning showed prominent improvement of neurological function and motor function such as vestibule-motor function, sensori-motor function and asymmetric forelimb use. The expression of BDNF colocalized within neurons and SDF-1α colocalized within the cerebral vascular endothelium was observed throughout the ischemic cortex by EA. CONCLUSIONS: Pretreatment with EA increased the production of BDNF and SDF-1α, which elicited protective effects against focal cerebral ischemia. These results suggest a novel mechanism of EA pretreatment-induced tolerance against cerebral ischemic injury.

Advances in molecular therapies for targeting pathophysiology in spinal cord injury.

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.

The role of reactive oxygen species in bone cell physiology and pathophysiology.

Hydrogen peroxide (H2O2), superoxide anion radical (O2-•), 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.

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens.

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.

RETRACTED: Deletion of the scavenger receptor Scarb1 in myeloid cells does not affect bone mass

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.