Central depletion of brain-derived neurotrophic factor in mice results in high bone mass and metabolic phenotype.

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal differentiation/survival, the regulation of food intake, and the pathobiology of obesity and type 2 diabetes mellitus. BDNF and its receptor are expressed in osteoblasts and chondrocyte. BDNF in vitro has a positive effect on bone; whether central BDNF affects bone mass in vivo is not known. We therefore examined bone mass and energy use in brain-targeted BDNF conditional knockout mice (Bdnf(2lox/2lox)/93). The deletion of BDNF in the brain led to a metabolic phenotype characterized by hyperphagia, obesity, and increased abdominal white adipose tissue. Central BDNF deletion produces a marked skeletal phenotype characterized by increased femur length, elevated whole bone mineral density, and bone mineral content. The skeletal changes are developmentally regulated and appear concurrently with the metabolic phenotype, suggesting that the metabolic and skeletal actions of BDNF are linked. The increased bone development is evident in both the cortical and trabecular regions. Compared with control, Bdnf(2lox/2lox)/93 mice show greater trabecular bone volume (+50% for distal femur, P < 0.001; +35% for vertebral body, P < 0.001) and midfemoral cortical thickness (+11 to 17%, P < 0.05), measured at 3 and 6 months of age. The skeletal and metabolic phenotypes were gender dependent, with female being more affected than male mice. However, uncoupling protein-1 expression in brown fat, a marker of sympathetic tone, was not different between genotypes. We show that deletion of central BDNF expression in mice results in increased bone mass and white adipose tissue, with no significant changes in sympathetic signaling or peripheral serotonin, associated with hyperphagia, obesity, and leptin resistance.

Mecp2 deficiency decreases bone formation and reduces bone volume in a rodent model of Rett syndrome.

Rett syndrome (RTT), a neurological disorder characterized by neurological impairment and a high frequency of osteopenia which often manifests early in childhood, most often is caused by inactivating mutations in the X-linked gene encoding a regulator of epigenetic gene expression, methyl CpG binding protein, MeCP2. Clinical data show that, along with neurological defects, females with RTT frequently have marked decreases in bone mineral density (BMD) beyond that expected from disuse atrophy. To investigate the relationship between loss of Mecp2 and reduced BMD, we used a Mecp2 null mouse model, Mecp2 (-/yBIRD), for our histological and biochemical studies. Mecp2 (-/yBIRD) mice have significantly shorter femurs and an overall reduced skeletal size compared to wild-type mice by post-natal day 60 (P60). Histological and histomorphometric studies identified growth plate abnormalities as well as decreased cortical and trabecular bone in P21 and especially in P60 Mecp2 (-/yBIRD) mice. Dynamic histomorphometry revealed decreased mineral apposition rates (MAR) in Mecp2 null femoral trabecular bone as well as in calvarial bone samples. While changes in MAR of cortical bone were not significant, loss of Mecp2 significantly reduced cortical, trabecular and calvarial bone volume compared with age-matched wild-type animals. These differences indicate that Mecp2 deficiency leads to osteoblast dysfunction, which translates into reduced osteoid deposition accounting for the reduced bone volume phenotype. While individual variations were observed in OPG and Rankl concentrations, molar ratios of OPG:Rankl at P21 and P60 were comparable between wild-type and Mecp2 (-/yBIRD) mice and showed a consistent excess of OPG. In tibial sections, TRAP staining demonstrated equivalent osteoclast number per bone surface measurements between wild-type and null animals. Our work with a Mecp2 null mouse model suggests epigenetic regulation of bone in the Mecp2 (-/yBIRD) mice which is associated with decreased osteoblast activity rather than increased osteoclastic bone loss.

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts.

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

Extracellular glucose influences osteoblast differentiation and c-Jun expression.

Insulin dependent diabetes mellitus, marked by high blood glucose levels and no insulin secretion, is associated with decreased bone mass and increased fracture rates. Analysis of bone histology suggests that osteoblast phenotype and function are influenced by diabetes. To determine if elevated extracellular glucose levels could directly influence osteoblast phenotype we treated mouse osteoblasts, MC3T3-E1 cells, with 22 mM glucose and analyzed osteoblast gene expression. Collagen I mRNA levels significantly increased while osteocalcin mRNA levels decreased 24 h after the addition of glucose. Expression of other genes, actin, osteopontin, and histone H4, was unaffected. Effects on collagen I expression were seen as early as 1 h after treatment. c-Jun, an AP-1 transcription factor involved in the regulation of osteoblast gene expression and growth, was also modulated by glucose. Specifically, an increase in c-jun expression was found at 1 h and maintained for 24 h following glucose treatment. Treatment of osteoblasts with an equal concentration of mannitol completely mimicked glucose treatment effects on collagen I and c-jun expression, demonstrating that osmotic stress rather than glucose metabolism is responsible for the effects on osteoblast gene expression and phenotype. Additional studies using staurosporine and Ro-31-8220 demonstrate that protein kinase C is required for the glucose up regulation of collagen I and c-jun. Taken together, our results demonstrate that osteoblasts respond to increasing extracellular glucose concentration through an osmotic response pathway that is dependent upon protein kinase C activity and results in upregulation of c-jun and modulation of collagen I and osteocalcin expression.