Regulation of TRKB surface expression by brain-derived neurotrophic factor and truncated TRKB isoforms

Brain-derived neurotrophic factor (BDNF) signaling through its receptor TRKB modulates survival, differentiation, and activity of neurons. BDNF activates TRKB on the cell surface, which leads to the initiation of intracellular signaling cascades and different biological responses in neurons. Neuronal activity has been shown to regulate TRKB levels on the plasma membrane of neurons, but little is known about other factors affecting TRKB surface expression levels. We report here that BDNF regulates the cell surface levels of transfected or endogenously expressed full-length TRKB, depending on the exposure time in neuroblastoma cells and primary hippocampal neurons. BDNF rapidly increases TRKB surface expression levels in seconds, whereas treatment of cells with BDNF for a longer time (minutes to hours) leads to decreased TRKB surface levels. Coexpression of the full-length TRKB together with the truncated TRKB.T1 isoform results in decreased levels of full-length TRKB on the cell surface. This effect is specific to the T1 isoform, because coexpression of a kinase-dead TRKB mutant or another kinase domain-lacking TRKB form, truncated T-Shc, leads to increased TRKB surface levels. Our results suggest that regulation of TRKB surface expression levels by different factors is tightly controlled by complex mechanisms in active neurons.     

Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons

Brain-derived neurotrophic factor (BDNF) has been shown to regulate neuronal survival and synaptic plasticity in the central nervous system (CNS) in an activity-dependent manner, but the underlying mechanisms remain unclear. Here we report that the number of BDNF receptor TrkB on the surface of hippocampal neurons can be enhanced by high frequency neuronal activity and synaptic transmission, and this effect is mediated by Ca(2+) influx. Using membrane protein biotinylation as well as receptor binding assays, we show that field electric stimulation increased the number of TrkB on the surface of cultured hippocampal neurons. Immunofluorescence staining suggests that the electric stimulation facilitated the movement of TrkB from intracellular pool to the cell surface, particularly on neuronal processes. The number of surface TrkB was regulated only by high frequency tetanic stimulation, but not by low frequency stimulation. The activity dependent modulation appears to require Ca(2+) influx, since treatment of the neurons with blockers of voltage-gated Ca(2+) channels or NMDA receptors, or removal of extracellular Ca(2+), severely attenuated the effect of electric stimulation. Moreover, inhibition of Ca(2+)/calmodulin-dependent kinase II (CaMKII) significantly reduced the effectiveness of the tetanic stimulation. These findings may help us to understand the role of neuronal activity in neurotrophin function and the mechanism for receptor tyrosine kinase signaling.     

Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.     

Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.     

Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer's disease

Brain-derived neurotrophic factor (BDNF) is critical for the function and survival of neurons that degenerate in the late stage of Alzheimer's disease (AD). There are two forms of BDNF, the BDNF precursor (proBDNF) and mature BDNF, in human brain. Previous studies have shown that BDNF mRNA and protein, including proBDNF, are dramatically decreased in end-stage AD brain. To determine whether this BDNF decrease is an early or late event during the progression of cognitive decline, we used western blotting to measure the relative amounts of BDNF proteins in the parietal cortex of subjects clinically classified with no cognitive impairment (NCI), mild cognitive impairment (MCI) or mild to moderate AD. We found that the amount of proBDNF decreased 21 and 30% in MCI and AD groups, respectively, as compared with NCI, consistent with our previous results of a 40% decrease in end-stage AD. Mature BDNF was reduced 34 and 62% in MCI and AD groups, respectively. Thus, the decrease in mature BDNF and proBDNF precedes the decline in choline acetyltransferase activity which occurs later in AD. Both proBDNF and mature BDNF levels were positively correlated with cognitive measures such as the Global Cognitive Score and the Mini Mental State Examination score. These results demonstrate that the reduction of both forms of BDNF occurs early in the course of AD and correlates with loss of cognitive function, suggesting that proBDNF and BDNF play a role in synaptic loss and cellular dysfunction underlying cognitive impairment in AD.     

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

Activation of ETa receptors is partially responsible for the rapid increase in haematocrit induced by bacterial lipopolysaccharide in the rat

It is believed that changes in the production and release of endothelin-1 (ET-1) are mediated over prolonged periods, and, therefore, that it is unlikely that ET-1 mediates rapid responses within the circulation. Here we show that ET-1 is involved in the rapid changes produced by injection of LPS in vivo. In anaesthetised rats, a bolus of LPS induced an increase in haematocrit and a fall in blood pressure within 10 min. The increase in haematocrit was reduced by administration of antagonists selective for endothelin ETa receptors, while the accompanying decrease in MAP was potentiated. Thus, activation of ETa receptors is partially responsible for the rapid increase in haematocrit seen in this model. This clearly demonstrates that ET-1 can act as a rapid responder to acute cardiovascular challenges.     

Cell density regulates the number of cell surface receptors for fibroblast growth factor

Previous studies have shown that the binding of fibroblast growth factor to several different non-transformed cell lines decreases as cell density increases. However, it was not determined whether this is due to a reduction in receptor number or to a decrease in receptor affinity. In this study, we demonstrate that the reduction in fibroblast growth factor binding is due to a reduction in receptor number. In addition, flow cytometric analysis indicated little or no difference in the cell cycle distribution of the cells at low and high cell densities, yet the binding of fibroblast growth factor was reduced substantially at high cell densities. Thus, the reduction in growth factor binding observed in this study does not appear to result from cell cycle-dependent regulation of growth factor receptors.     

Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system

BACKGROUND: The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS: We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS: BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.     

Involvement of Rac1 in macrophage activation by brain-derived neurotrophic factor

Molecular Biology Reports - Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation, which directs the quality of...     

Receptive-field modification in rat visual cortex induced by paired visual stimulation and single-cell spiking

Experience-dependent plasticity of visual cortical receptive fields (RFs) involves synaptic modifications in the underlying neural circuits, but the site and mechanism of these modifications remain to be elucidated. Using in vivo whole-cell recordings, we show that pairing visual stimulation at a given retinal location with spiking of a single neuron in developing rat visual cortex induces rapid RF modifications. The time course of the response to the visual stimulus at the paired RF location is altered, with an enhancement of the response preceding the spike time and a reduction following the spike. Such bidirectional modification is consistent with spike timing-dependent plasticity. Response modification also occurs at nearby locations, the direction and magnitude of which are correlated with the change at the paired location. In addition, changes at unpaired locations show a negative correlation with the initial strength of the response, which may facilitate rapid modification of the spatial RF profile.     

Maternal low-protein diet decreases brain-derived neurotrophic factor expression in the brains of the neonatal rat offspring

Prenatal exposure to a maternal low-protein (LP) diet has been known to cause cognitive impairment, learning and memory deficits. However, the underlying mechanisms have not been identified. Herein, we demonstrate that a maternal LP diet causes, in the brains of the neonatal rat offspring, an attenuation in the basal expression of the brain-derived neurotrophic factor (BDNF), a neurotrophin indispensable for learning and memory. Female rats were fed either a 20% normal protein (NP) diet or an 8% LP 3 weeks before breeding and during the gestation period. Maternal LP diet caused a significant reduction in the Bdnf expression in the brains of the neonatal rats. We further found that the maternal LP diet reduced the activation of the cAMP/protein kinase A/cAMP response element binding protein (CREB) signaling pathway. This reduction was associated with a significant decrease in CREB binding to the Bdnf promoters. We also show that prenatal exposure to the maternal LP diet results in an inactive or repressed exon I and exon IV promoter of the Bdnf gene in the brain, as evidenced by fluxes in signatory hallmarks in the enrichment of acetylated and trimethylated histones in the nucleosomes that envelop the exon I and exon IV promoters, causing the Bdnf gene to be refractory to transactivation. Our study is the first to determine the impact of a maternal LP diet on the basal expression of BDNF in the brains of the neonatal rats exposed prenatally to an LP diet.     

miR-210 inhibits cell migration and invasion by targeting the brain-derived neurotrophic factor in glioblastoma

Recently, there is increasing evidence that microRNAs are related to the development, diagnosis, treatment, and prognosis of glioblastoma. microRNA-210 (miR-210) had been identified in many human cancers, but the specific function of miR-210 remains unclear in glioblastoma. The present study mainly focused on exploring its biological role and potential molecular mechanisms in glioblastoma. We found that miR-210 expression was decreased in glioblastoma, and downregulation of miR-210 was related to worse prognosis in glioblastoma patients. In addition, miR-210 overexpression inhibited the migration and invasion of human glioblastoma cells. At the same time, we found that miR-210 directly targets the brain-derived neurotrophic factor (BDNF) and reduces BDNF expression level. Consistently, BDNF silencing had the same effects as miR-210 overexpression in glioblastoma, and upregulation of BDNF counteracted the inhibitory effect of miR-210 in glioblastoma. In conclusion, miR-210 suppressed the migration and invasion of glioblastoma cells by targeting BDNF.     

Development of GABAA receptors in the central visual structures of rat brain. Effect of visual pattern deprivation

The postnatal development of high-affinity 3H-muscimol binding to GABAA receptors was studied in the lateral geniculate nucleus, superior colliculus, frontal and visual cortex of the rat brain. In the lateral geniculate nucleus 3H-muscimol binding rises from day 10 through day 37 reaching the highest value during the entire development followed by a slight decrease until adulthood. In the superior colliculus 3H-muscimol binding increased continuously from day 10 through day 37, and then decreased until day 50 reaching the adult value. In the visual and frontal cortex, binding reached the highest levels on days 14 and 25, respectively, persisted until day 37 followed by a slight decrease until adulthood. The ontogeny of 3H-muscimol binding sites in the visual regions does not essentially differ from that in other brain regions, suggesting that the appearance of 3H-muscimol binding sites in the visual system is not correlated with the functional maturation of the visual system. Unilateral eyelid closure from day 11 until day 25 did not affect the development of GABAA receptors in any of the central visual regions examined, indicating the lack of environmentally controlled mechanism.     

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels

A high level of hippocampal brain-derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (gamma-hexachlorocyclohexane); and L-type voltage-dependent calcium channel agonist, BAY-K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY-K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12--24 h, 8--16 h and 6 h following administration of kainic acid, PTZ and BAY-K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY-K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY-K 8644, but not PTZ, effects. omega-Conotoxin GVIA and DCG-IV partially blocked kainic acid-induced enhancement of BDNF, indicating involvement of L-type and N-type voltage-dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in D-myo-inositol-1,4,5-triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY-K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant-dependent stimulation of BDNF production and involvement of region-specific voltage-dependent calcium channels.     

The excitoprotective effect of N-methyl-D-aspartate receptors is mediated by a brain-derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N-methyl-D-aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre-incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor-mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time- and concentration-dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain-derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF-dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 microM) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4-specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre-incubation with a BDNF-blocking antibody and TrkB-IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA-mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.     

In vivo induction of glial cell proliferation and axonal outgrowth and myelination by brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of neuronal cell survival and differentiation factors but is thought to be involved in neuronal cell proliferation and myelination as well. To explore the role of BDNF in vivo, we employed the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis as a model system. These cells mediate background adaptation of the animal by producing high levels of the prohormone proopiomelanocortin (POMC) when the animal is black adapted. We used stable X. transgenesis in combination with the POMC gene promoter to generate transgenic frogs overexpressing BDNF specifically and physiologically inducible in the melanotrope cells. Intriguingly, an approximately 25-fold overexpression of BDNF resulted in hyperplastic glial cells and myelinated axons infiltrating the pituitary, whereby the transgenic melanotrope cells became located dispersed among the induced tissue. The infiltrating glial cells and axons originated from both peripheral and central nervous system sources. The formation of the phenotype started around tadpole stage 50 and was induced by placing white-adapted transgenics on a black background, i.e. after activation of transgene expression. The severity of the phenotype depended on the level of transgene expression, because the intermediate pituitaries from transgenic animals raised on a white background or from transgenics with only an approximately 5-fold BDNF overexpression were essentially not affected. In conclusion, we show in a physiological context that, besides its classical role as neuronal cell survival and differentiation factor, in vivo BDNF can also induce glial cell proliferation as well as axonal outgrowth and myelination.     

Single eight-hour shift of light-dark cycle increases brain-derived neurotrophic factor protein levels in the rat hippocampus

We previously reported that an eight hour phase advance in the light-dark (LD) cycle increases sleep in rats. Brain-derived neurotrophic factor (BDNF) is suggested to be one of the sleep and circadian regulating factors. We have therefore observed the responses of BDNF protein in the hippocampus, cerebellum and brainstem under conditions of LD change. BDNF protein was quantitatively measured using an ELISA kit. Under an 8-h LD phase advance, the levels of hippocampal BDNF were significantly increased on the day of the phase change, while the levels in the cerebellum and brainstem remained constant. Plasma corticosterone levels were not largely affected. Thus, a single LD shift acutely affects hippocampal BDNF metabolism with no large stress response.