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Although the precise mechanisms for neurodegeneration in Parkinson's disease (PD) are unknown, evidence suggests that neuroinflammation is a critical factor in the pathogenic process. Here, we sought to determine whether the voltage-gated proton channel, Hv1 (HVCN1), which is expressed in microglia and regulates NADPH oxidase, is associated with dopaminergic neurodegeneration. We utilized data mining to evaluate the mRNA expression of HVCN1 in the brains of PD patients and controls and uncovered increased expression of the gene encoding Hv1, HVCN1, in the brains of PD patients compared to controls, specifically in male PD patients. In an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 × 16 mg/kg) mouse model of PD, Hvcn1 gene expression was increased 2-fold in the striatum. MPTP administration to wild-type (WT) mice resulted in a ~65% loss of tyrosine hydroxylase positive neurons (TH+) in the substantia nigra (SN), while a ~39% loss was observed in Hv1 knockout (KO) mice. Comparable neuroprotective effects of Hv1 deficiency were found in a repeated-dose LPS model. Neuroprotection was associated with decreased pro-inflammatory cytokine levels and pro-oxidant factors in both neurotoxicant animal models. These in vivo results were confirmed in primary microglial cultures, with LPS treatment increasing Hvcn1 mRNA levels and Hv1 KO microglia failing to exhibit the LPS-mediated inflammatory response. Conditioned media from Hv1 KO microglia treated with LPS resulted in an attenuated loss of cultured dopamine neuron cell viability compared to WT microglia. Taken together, these data suggest that Hv1 is upregulated and mediates microglial pro-inflammatory cytokine production in parkinsonian models and therefore represents a novel target for neuroprotection.
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Preterm birth is a risk factor for growth failure and development of respiratory disease in children and young adults. Their early exposure to oxygen may contribute to lung disease because adult mice exposed to hyperoxia as neonates display reduced lung function, changes in the host response to respiratory viral infections, and develop pulmonary hypertension and heart failure that shortens their lifespan. Here, we provide new evidence that neonatal hyperoxia also impairs growth by inhibiting fat accumulation. Failure to accumulate fat may reflect a systemic defect in adipogenic potential of stem cells because bone marrow-derived mesenchymal cells (BMSCs) isolated from the mice grew slower and were more oxidized compared to controls. They also displayed reduced capacity to accumulate lipid and differentiate into adipocytes. BMSCs from adult mice exposed to neonatal hyperoxia express lower levels of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that drives adipocyte differentiation. The defect in adipogenesis was rescued by expressing PPARγ in these cells. These findings reveal early life exposure to high levels of oxygen may suppresses fat accumulation and impair adipogenic differentiation upstream of PPARγ signaling, thus potentially contributing to growth failure seen in people born preterm.
Assuntos
Hiperóxia , Células-Tronco Mesenquimais , Nascimento Prematuro , Adipogenia , Animais , Medula Óssea , Diferenciação Celular , Células Cultivadas , Feminino , Camundongos , PPAR gama/genética , GravidezRESUMO
Increased pro-inflammatory cytokine levels and proliferation of activated microglia have been found in Parkinson's disease (PD) patients and animal models of PD, suggesting that targeting of the microglial inflammatory response may result in neuroprotection in PD. Microglial proliferation is regulated by many factors, but colony stimulating factor-1 receptor (CSF1R) has emerged as a primary factor. Using data mining techniques on existing microarray data, we found that mRNA expression of the CSF1R ligand, CSF-1, is increased in the brain of PD patients compared to controls. In two different neurotoxic mouse models of PD, acute MPTP and sub-chronic LPS treatment, mRNA and protein levels of CSF1R and CSF-1 were significantly increased. Treatment with the CSF1R inhibitor GW2580 significantly attenuated MPTP-induced CSF1R activation and Iba1-positive cell proliferation, without a reduction of the basal Iba1-positive population in the substantia nigra. GW2580 treatment also significantly decreased mRNA levels of pro-inflammatory factors, without alteration of anti-inflammatory mediators, and significantly attenuated the MPTP-induced loss of dopamine neurons and motor behavioral deficits. Importantly, these effects were observed in the absence of overt microglial depletion, suggesting that targeting CSF1R signaling may be a viable neuroprotective strategy in PD that disrupts pro-inflammatory signaling, but maintains the beneficial effects of microglia.
Assuntos
Anisóis/farmacologia , Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Pirimidinas/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transdução de Sinais/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
Inflammation arising from central and/or peripheral sources contributes to the pathogenesis of multiple neurodegenerative diseases including Parkinson's disease (PD). Emerging data suggest that differential activation of glia could lead to the pathogenesis and progression of PD. Here, we sought to determine the relationship between lipopolysaccharide (LPS) treatment, loss of dopaminergic neurons and differential activation of glia. Using a model of repeated injections with LPS (1mg/kg, i.p. for 4days), we found that LPS induced a 34% loss of dopamine neurons in the substantia nigra 19days after initiation of treatment, but no further cell loss was observed at 36days. LPS induced a strong pro-inflammatory response with increased mRNA expression of pro-inflammatory markers, including tumor necrosis factor-α (4.8-fold), inducible nitric oxide synthase (2.0-fold), interleukin-1 beta (8.9-fold), interleukin-6 (10.7-fold), and robust glial activation were observed at 1day after final dose of LPS. These pro-inflammatory genes were then reduced at 19days after treatment, when there was a rise in the anti-inflammatory genes Ym1 (1.8-fold) and arginase-1 (2.6-fold). Additionally, 36days after the last LPS injection there was a significant increase in interleukin-10 (2.1-fold) expression. The qPCR data results were supported by protein data, including cytokine measurements, western blotting, and immunofluorescence in brain microglia. Taken together, these data demonstrate that progressive neurodegeneration in the substantia nigra following LPS is likely arrested by microglia shifting to an anti-inflammatory phenotype. Thus, strategies to promote resolution of neuroinflammation may be a promising avenue to slow the progressive loss of dopamine neurons in PD.
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Neurônios Dopaminérgicos/imunologia , Lipopolissacarídeos/toxicidade , Microglia/imunologia , Degeneração Neural/imunologia , Animais , Astrócitos/imunologia , Astrócitos/patologia , Morte Celular/fisiologia , Corpo Estriado/imunologia , Corpo Estriado/patologia , Citocinas/metabolismo , Progressão da Doença , Neurônios Dopaminérgicos/patologia , Inflamação/patologia , Inflamação/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/patologia , Degeneração Neural/patologia , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/patologia , Neuroimunomodulação/fisiologia , RNA Mensageiro/metabolismo , Distribuição Aleatória , Fatores de TempoRESUMO
Stress during prenatal development is correlated with detrimental cognitive and behavioral outcomes in offspring. However, the long-term impact of prenatal stress (PS) and disrupted glucocorticoid signaling on bone mass and strength is not understood. In contrast, the detrimental effect of lead (Pb) on skeletal health is well documented. As stress and Pb act on common biological targets via glucocorticoid signaling pathways and co-occur in the environment, this study first sought to assess the combined effect of stress and Pb on bone quality in association with alterations in glucocorticoid signaling. Bone parameters were evaluated using microCT, histomorphometry, and strength determination in 8-month-old male mouse offspring subjected to PS on gestational days 16 and 17, lifetime Pb exposure (100 p.p.m. Pb in drinking water), or to both. Pb reduced trabecular bone mass and, when combined with PS, Pb unmasked an exaggerated decrement in bone mass and tensile strength. Next, to characterize a mechanism of glucocorticoid effect on bone, prednisolone was implanted subcutaneously (controlled-release pellet, 5 mg·kg-1 per day) in 5-month-old mice that decreased osteoblastic activity and increased sclerostin and leptin levels. Furthermore, the synthetic glucocorticoid dexamethasone alters the anabolic Wnt signaling pathway. The Wnt pathway inhibitor sclerostin has several glucocorticoid response elements, and dexamethasone administration to osteoblastic cells induces sclerostin expression. Dexamethasone treatment of isolated bone marrow cells decreased bone nodule formation, whereas removal of sclerostin protected against this decrement in mineralization. Collectively, these findings suggest that bone loss associated with steroid-induced osteoporosis is a consequence of sclerostin-mediated restriction of Wnt signaling, which may mechanistically facilitate glucocorticoid toxicity in bone.
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The heavy metal lead (Pb) has a deleterious effect on skeletal health. Because bone mass is maintained through a balance of bone formation and resorption, it is important to understand the effect of Pb levels on osteoblastic and osteoclastic activity. Pb exposure is associated with low bone mass in animal models and human populations; however, the correlation between Pb dosing and corresponding bone mass has been poorly explored. Thus, mice were exposed to increasing Pb and at higher levels (500 ppm), there was unexpectedly an increase in femur-tibial bone mass by 3 months of age. This is contrary to several studies alluded to earlier. Increased bone volume (BV) was accompanied by a significant increase in cortical thickness of the femur and trabecular bone that extended beyond the epiphyseal area into the marrow cavity. Subsequent evaluations revealed an increase in osteoclast numbers with high Pb exposure, but a deficiency in osteoclastic activity. These findings were substantiated by observed increases in levels of the resorption-altering hormones calcitonin and estrogen. In addition we found that pro-osteoclastic nuclear factor-kappa beta (NF-κB) pathway activity was dose dependently elevated with Pb, both in vivo and in vitro. However, the ability of osteoclasts to resorb bone was depressed in the presence of Pb in media and within test bone wafers. These findings indicate that exposure to high Pb levels disrupts early life bone accrual that may involve a disruption of osteoclast activity. This study accentuates the dose dependent variation in Pb exposure and consequent effects on skeletal health.
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Densidade Óssea/efeitos dos fármacos , Chumbo/toxicidade , Osteoclastos/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Envelhecimento , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Resistência à Tração/efeitos dos fármacosRESUMO
Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/ß-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the ß-catenin pathway using a pharmacological inhibitor of GSK-3ß ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFß canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/ß-catenin pathway.
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Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Chumbo/toxicidade , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/anatomia & histologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Linhagem Celular , Células Cultivadas , Exposição Ambiental/efeitos adversos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Lead (Pb) exposure and obesity are co-occurring risk factors for decreased bone mass in the young, particularly in low socioeconomic communities. OBJECTIVES: The goal of this study was to determine whether the comorbidities of Pb exposure and high-fat diet-induced obesity amplify skeletal deficits independently associated with each of these risk factors, and to explore associated mechanisms of the observed deficiencies. METHODS: Five-week-old male C57BL/6J mice were placed on low-fat (10% kcal, LFD) or high-fat (60% kcal, HFD) diets for 12 weeks. Mice were exposed to lifetime Pb (50 ppm) through drinking water. RESULTS: HFD was associated with increased body mass and glucose intolerance. Both HFD and Pb increased fasting glucose and serum leptin levels. Pb and HFD each reduced trabecular bone quality and together had a further detrimental effect on these bone parameters. Mechanical bone properties of strength were depressed in Pb-exposed bones, but HFD had no significant effect. Both Pb and HFD altered progenitor cell differentiation, promoting osteoclastogenesis and increasing adipogenesis while suppressing osteoblastogenesis. In support of this lineage shift being mediated through altered Wnt signaling, Pb and non-esterified fatty acids in MC3T3 cells increased in vitro PPAR-γ activity and inhibited ß-catenin activity. Combining Pb and non-esterified fatty acids enhanced these effects. CONCLUSIONS: Pb and HFD produced selective deficits in bone accrual that were associated with alterations in progenitor cell activity that may involve reduced Wnt signaling. This study emphasizes the need to assess toxicants together with other risk factors relevant to human health and disease.
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Desenvolvimento Ósseo/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Poluentes Ambientais/toxicidade , Chumbo/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Obesity is a severe health problem in children, afflicting several organ systems including bone. However, the role of obesity on bone homeostasis and bone cell function in children has not been studied in detail. Here we used young mice fed a high-fat diet (HFD) to model childhood obesity and investigate the effect of HFD on the phenotype of cells within the bone marrow environment. Five-week-old male mice were fed a HFD for 3, 6, and 12 weeks. Decreased bone volume was detected after 3 weeks of HFD treatment. After 6 and 12 weeks, HFD-exposed mice had less bone mass and increased osteoclast numbers. Bone marrow cells, but not spleen cells, from HFD-fed mice had increased osteoclast precursor frequency, elevated osteoclast formation, and bone resorption activity, as well as increased expression of osteoclastogenic regulators including RANKL, TNF, and PPAR-gamma. Bone formation rate and osteoblast and adipocyte numbers were also increased in HFD-fed mice. Isolated bone marrow cells also had a corresponding elevation in the expression of positive regulators of osteoblast and adipocyte differentiation. Our findings indicate that in juvenile mice, HFD-induced bone loss is mainly due to increased osteoclast bone resorption by affecting the bone marrow microenvironment. Thus, targeting osteoclast formation may present a new therapeutic approach for bone complications in obese children.
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Medula Óssea/patologia , Reabsorção Óssea/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Osteoclastos/citologia , Adipócitos/citologia , Animais , Biomarcadores/sangue , Glicemia/análise , Densidade Óssea , Medula Óssea/metabolismo , Osso e Ossos/patologia , Diferenciação Celular , Separação Celular , Citometria de Fluxo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Osteoblastos/citologia , Osteoclastos/metabolismo , PPAR gama/metabolismo , Ligante RANK/metabolismo , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Microtomografia por Raio-XRESUMO
There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased ß-catenin levels. Reduction of ß-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. ß-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in ß-catenin signaling, suggesting the Wnt/ß-catenin as a pivotal pathway in the influence of Pb on fracture repair.
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Consolidação da Fratura/efeitos dos fármacos , Chumbo/química , Células-Tronco Mesenquimais/citologia , Fraturas da Tíbia/fisiopatologia , beta Catenina/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicoproteínas/metabolismo , Indóis/química , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteogênese , Oximas/química , Transdução de Sinais , Fraturas da Tíbia/metabolismo , Fatores de Tempo , Proteínas Wnt/metabolismo , Cicatrização , Microtomografia por Raio-X , beta Catenina/metabolismoRESUMO
Twenty-one risk factors affecting laborers in manual materials handling tasks were analyzed to determine what, if any, statistically significant relationships existed between the factors and the emergence of occupational back injury. The statistically significant risk factors (p ≤ .05) in the univariate analysis were determined to be weight lifted per hour (work intensity), trunk twists per hour, weight lifted per day, frequency of lift, trunk motions per hour, and trunk flexions per hour, with odds ratios (ORs) of 1.28-2.88. In addition, self-reported discomfort in the neck, middle back, knees, and lower back was associated with the outcome of back injury (p ≤ .05, OR 1.75-2.66). In the multivariate analysis, the statistically significant risk factors (p ≤ .05) were weight lifted per hour (work intensity), average weight of lift, and number of trunk twists per hour, with ORs of 1.74-4.98.
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Lesões nas Costas/etiologia , Remoção/efeitos adversos , Traumatismos Ocupacionais/etiologia , Carga de Trabalho , Feminino , Humanos , Masculino , Movimento , Postura , Fatores de RiscoRESUMO
BACKGROUND: Exposure to lead (Pb) from environmental and industrial sources remains an overlooked serious public health risk. Elucidating the effect of Pb on bone cell function is therefore critical for understanding its risk associated with diseases of low bone mass. OBJECTIVES: We tested the hypothesis that Pb negatively affects bone mass. We also assessed the underlying mechanisms of Pb on bone signaling pathways. METHODS: We used a model of low-level Pb exposure in a rodent beginning before conception and continuing over 18 months. We characterized the effect of Pb on bone quality using dual-energy X-ray absorptiometry (DXA), micro-computed tomography, Raman spectroscopy, and histology. We assessed the effect of Pb on bone and adipocyte formation by mineral deposition, lipid droplet formation, and Western blot and RNA analysis. RESULTS: Pb-exposed animals had decreased bone mass that resulted in bones that were more susceptible to fracture. Pb decreased osteoblastic cell number leading to a depression of bone formation. Accompanying this, Pb exposure elevated sclerostin protein levels in the skeleton, and correspondingly reduced levels of ß-catenin and Runx2 in stromal precursor cells. Pb also increased skeletal expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). These results indicate a shift in mesenchymal differentiation wherein Pb promoted enhanced adipogenesis and decreased osteoblastogenesis. Substantial differences in bone marrow composition were observed, highlighted by an increase in adipocytes. CONCLUSIONS: The disruption Pb has on bone mass and bone homeostasis is principally explained by inhibition of the Wnt/ß-catenin pathway, which may provide a molecular basis for novel therapeutic strategies to combat Pb-induced bone pathologies.
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Densidade Óssea/efeitos dos fármacos , Chumbo/toxicidade , Osteoporose/induzido quimicamente , Osteoporose/metabolismo , Proteínas Wnt/metabolismo , Animais , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genéticaRESUMO
Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-ß signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-ß signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF-ß signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase 3 and 8 were dose-dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-ß reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix.