Brain-derived neurotrophic factor and its receptor in the human and the sand rat intervertebral disc

Introduction

Brain-derived neurotrophic factor (BDNF) was first identified in the intervertebral disc (IVD) when its molecular upregulation was observed in sections of nucleus pulposus cultured under conditions of increased osmolarity. BDNF is now known to be involved in a number of biologic functions, including regulation of differentiation/survival of sensory neurons, regulation of nociceptive function and central pain modulation, and modulation of inflammatory pain hypersensitivity. In addition, more recent investigations show that BDNF can induce the recruitment of endothelial cells and the formation of vascular structures. The objectives of the present study were to use immunocytochemistry to determine the distribution of BDNF and its receptor (BDNF-tropomyosine receptor kinase B) in the human IVD, and to test for gene expression of BDNF and its receptor in cultured human annulus fibrosus cells.

Methods

We studied immunohistochemical localization of BDNF and its receptor in the human annulus, quantified the percentage of outer annulus and inner annulus cells and nucleus cells positive for BDNF immunolocalization, and studied the gene expression of BDNF and its receptor using microarray analysis.

Results

The percentage (mean ± standard error of the mean) of cells positive for BDNF localization was significantly greater in the outer annulus (32.3 ± 2.7%, n = 22) compared with either the inner annulus (8.1 ± 1.5%, n = 6) or the nucleus (10.4 ± 2.8%, n = 3) (P < 0.0001). BDNF-receptor immunolocalization showed a pattern similar to that of BDNF, but was not quantitatively assessed. BDNF gene expression levels from cultured annulus cells showed a significant positive correlation with increasing levels of IVD degeneration (P = 0.011).

Conclusion

These findings provide data on the presence of BDNF and its receptor in the human IVD at the translational level, and on the expression of BDNF and its receptor by cultured human annulus cells. Our findings point to the need for further studies to define the role of BDNF in the human IVD and to investigate regulatory events within the disc that control the expression of BDNF and its receptor.     

Brain-derived neurotrophic factor and early-life stress: Multifaceted interplay

The brain-derived neurotrophic factor (BDNF) is a key regulator of neural development and plasticity. Long-term changes in the BDNF pathway are associated with childhood adversity and adult depression symptoms. Initially, stress-induced decreases in the BDNF pathway were found in some studies, but subsequent reports indicated the relationship between stress and BDNF to be much more complex, and the concept was significantly revised. In the present mini-review, we focus on the structure and regulation of the Bbnf gene as well as on the stress–BDNF interactions under early-life adverse conditions.     

Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice

The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.     

Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus increases energy expenditure by elevating metabolic rate

Brain-derived neurotrophic factor (BDNF) decreases food intake and body weight, but few central sites of action have been identified. The hypothalamic paraventricular nucleus (PVN) is important in energy metabolism regulation, and expresses both BDNF and its receptor. We tested three hypotheses: 1) PVN BDNF reduces feeding and increases energy expenditure (EE), 2) PVN BDNF-enhanced thermogenesis results from increased spontaneous physical activity (SPA) and resting metabolic rate (RMR), and 3) PVN BDNF thermogenic effects are mediated, in part, by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). BDNF (0.5 microg) was injected into the PVN of Sprague-Dawley rats; and oxygen consumption, carbon dioxide production, food intake, and SPA were measured for 24 h in an indirect calorimeter. SPA was also measured in open-field activity chambers for 48 h after BDNF injection. Animals were killed 6 or 24 h after BDNF injection, and BAT UCP1 gene expression was measured with quantitative real-time PCR. BDNF significantly decreased food intake and body weight gain 24 h after injection. Heat production and RMR were significantly elevated for 7 h immediately after BDNF injection. BDNF had no effect on SPA, but increased UCP1 gene expression in BAT at 6 h, but not 24 h after injection. In conclusion, PVN BDNF reduces body weight by decreasing food intake and increasing EE consequent to increased RMR, which may be due, in part, to BAT UCP1 activity. These data suggest that the PVN is an important site of BDNF action to influence energy balance.     

Brain-derived Neurotrophic Factor Regulates Energy Expenditure Through the Central Nervous System in Obese Diabetic Mice

It has been previously demonstrated that brain-derived neurotrophic factor (BDNF) regulates glucose metabolism and energy expenditure in rodent diabetic models such as C57BL/KsJ-lepr^db/lepr^db (db/db) mice. Central administration of BDNF has been found to reduce blood glucose in db/db mice, suggesting that BDNF acts through the central nervous system. In the present study we have expanded these investigations to explore the effect of central administration of BDNF on energy metabolism. Intracerebroventricular administration of BDNF lowered blood glucose and increased pancreatic insulin content of db/db mice compared with vehicle-treated pellet pair-fed db/db mice. While body temperatures of the pellet pair-fed db/db mice given vehicle were reduced because of restricted food supply in this pair-feeding condition, BDNF treatment remarkably alleviated the reduction of body temperature suggesting the enhancement of thermogenesis. BDNF enhanced norepinephrine turnover and increased uncoupling protein-1 mRNA expression in the interscapular brown adipose tissue. Our evidence indicates that BDNF activates the sympathetic nervous system via the central nervous system and regulates energy expenditure in obese diabetic animals.     

Brain-derived neurotrophic factor: Its role in energy balance and cancer cachexia

Brain-derived neurotrophic factor (BDNF) plays an important role in the development of the central and peripheral nervous system during embryogenesis. In the mature central nervous system, BDNF is required for the maintenance and enhancement of synaptic transmissions and the survival of neurons. Particularly, it is involved in the modulation of neurocircuits that control energy balance through food intake, energy expenditure, and locomotion. Regulation of BDNF in the central nervous system is complex and environmental factors affect its expression in murine models which may reflect to phenotype dramatically. Furthermore, BDNF and its high-affinity receptor tropomyosin receptor kinase B (TrkB), as well as pan-neurotrophin receptor (p75^NTR) is expressed in peripheral tissues in adulthood and their signaling is associated with regulation of energy balance. BDNF/TrkB signaling is exploited by cancer cells as well and BDNF expression is increased in tumors. Intriguingly, previously demonstrated roles of BDNF in regulation of food intake, adipose tissue and muscle overlap with derangements observed in cancer cachexia. However, data about the involvement of BDNF in cachectic cancer patients and murine models are scarce and inconclusive. In the future, knock-in and/or knock-out experiments with murine cancer models could be helpful to explore potential new roles for BDNF in the development of cancer cachexia.     

Explore the Features of Brain-Derived Neurotrophic Factor in Mood Disorders

Objectives

Brain-derived neurotrophic factor (BDNF) plays important roles in neuronal survival and differentiation; however, the effects of BDNF on mood disorders remain unclear. We investigated BDNF from the perspective of various aspects of systems biology, including its molecular evolution, genomic studies, protein functions, and pathway analysis.

Methods

We conducted analyses examining sequences, multiple alignments, phylogenetic trees and positive selection across 12 species and several human populations. We summarized the results of previous genomic and functional studies of pro-BDNF and mature-BDNF (m-BDNF) found in a literature review. We identified proteins that interact with BDNF and performed pathway-based analysis using large genome-wide association (GWA) datasets obtained for mood disorders.

Results

BDNF is encoded by a highly conserved gene. The chordate BDNF genes exhibit an average of 75% identity with the human gene, while vertebrate orthologues are 85.9%-100% identical to human BDNF. No signs of recent positive selection were found. Associations between BDNF and mood disorders were not significant in most of the genomic studies (e.g., linkage, association, gene expression, GWA), while relationships between serum/plasma BDNF level and mood disorders were consistently reported. Pro-BDNF is important in the response to stress; the literature review suggests the necessity of studying both pro- and m-BDNF with regard to mood disorders. In addition to conventional pathway analysis, we further considered proteins that interact with BDNF (I-Genes) and identified several biological pathways involved with BDNF or I-Genes to be significantly associated with mood disorders.

Conclusions

Systematically examining the features and biological pathways of BDNF may provide opportunities to deepen our understanding of the mechanisms underlying mood disorders.     

Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: Role of DNA methylation and histone acetylation

Thyroid hormones have long been known to play important roles in the development and functions of the central nervous system, however, the precise molecular mechanisms that regulate thyroid hormone-responsive gene expression are not well understood. The present study investigated the role of DNA methylaion and histone acetylation in the effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor (BDNF) gene expression in rat hippocampus. The findings indicated that the activities of DNA methyltransferase (DNMT), methylated reelin and BDNF genes were up-regulated, whereas, the activities of histone acetylases (HAT), the levels of global acetylated histone 3 (H3) and global acetylated histone 4 (H4), and acetylated H3, acetylated H4 at reelin promoter and at BDNF gene promoter for exon II were down-regulated in the hippocampus at the developmental stage of the hypothyroid animals. These results suggest that epigenetic modification of chromatin might underlie the mechanisms of hypothyroidism-induced down-regulation of reelin and BDNF gene expression in developmental rat hippocampus.     

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF^+/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

Introduction

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.

Methods

Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.

Results

Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.

Conclusion

Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.     

DNA methylation and expression profiles of the brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in patients with schizophrenia

Methylation and expression profile of CpG islands were examined in the promoters of the brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes. These are well known to be involved in the pathophysiology of psychiatric disorders such as schizophrenia. Genomic DNA was extracted from peripheral blood of 80 patients with schizophrenia and 71 healthy controls. Methylation pattern was studied by Methylation-Specific PCR. RNA expression analysis was done on extracted RNA from blood samples from patients suffering from schizophrenia (n?=?17) and healthy controls (n?=?17). Frequency of the BDNF gene methylation was highlighted as a statistically significant relationship between cases and controls regarding decreased risk of disease in comparison to unmethylated patterns (OR?=?0.24; 95?% CI?=?1.11–0.50; P?=?0.00007). For the DAT1 gene, this relationship was insignificant in 61 cases (76.25?%) and 52 controls (73.23?%) (OR?=?1.17; 95?% CI?=?0.53–2.61). Estimates of relative gene expression revealed a statistically significant association of the BDNF gene between schizophrenic patients and healthy controls (Mean?±?SD: 13.3920?±?15.19 and 0.437?±?0.328, P?=?0.0001) respectively; however, it was not significant for the DAT1 gene. This first hand evidence, regarding BDNF and DAT1 gene methylation and their expression profile with risk of schizophrenia, indicated a significant function for the BDNF gene in the development of schizophrenia. However, further populations with large sample sizes need to be studied to verify the exact role of BDNF in mental disorders such as schizophrenia.     

BDNF Val66Met Variant and Smoking in a Chinese Population

Several recent studies have supported the hypothesis that brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, might be associated with nicotine addiction. Association studies have also suggested that the BDNF gene might play a role in the susceptibility to nicotine dependence but results appear contradictory. The present work was therefore undertaken to examine the association of smoking with the BDNF Val66Met gene polymorphism in Chinese population. The BDNF Val66Met gene polymorphism was examined in 628 healthy male volunteers including 322 smokers and 306 non-smokers. Also, the BDNF serum levels were measured in 136 smokers and 97 nonsmokers. Our results showed no significant association between the BDNF Val66Met polymorphism or serum levels among smokers and non-smokers. Smokers with the Met allele however started smoking significantly earlier than those with the Val/Val genotype (mean age at smoking initiation of 17.4, 17.9 and 21.2 years for Met/Met, Met/Val, and Val/Val, respectively; both p<0.05). No other significant differences between other variables such as number of cigarettes per day, smoking severity as measured by the Fagerstrom Test for Nicotine Dependence (FTND) score and carbon monoxide (CO) levels (all p>0.05). In addition, there was no main effect of genotype on serum BDNF levels. Our findings suggest that the BDNF Val66Met polymorphism may not be involved in susceptibility to smoking among the Chinese male population, but may influence the age at which smoking is initiated. However, the findings must be interpreted with caution because of the relatively small sample size for an association study. Results should be confirmed in a larger cohort.