Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations.     

Prenatally compromised neurons respond to brain-derived neurotrophic factor treatment in vitro

Prenatal hypoxia affects neuronal survival and process outgrowth. Brain-derived neurotrophic factor, which influences neural growth, is decreased in these conditions. We tested whether addition of brain-derived neurotrophic factor enhances growth of neurons cultured from guinea pig fetuses (n=7) compromised by chronic placental insufficiency from 30-52 days gestation (term approximately 67 days). Cultures were prepared from the olfactory bulb, hippocampus and cerebellum. Compared with controls (n=7), chronic placental insufficiency resulted in reduced total neurite length in olfactory bulb cultures. Brain-derived neurotrophic factor treatment for 5 days increased the total olfactory neurite length and somal size and number of primary neurites in all cultures from both control and compromised animals. Thus, brain-derived neurotrophic factor can influence the growth of compromised fetal neurons supporting its therapeutic use following chronic placental insufficiency.     

Reciprocal actions of interleukin-6 and brain-derived neurotrophic factor on rat and mouse primary sensory neurons

In low-density, serum-free cultures of neurons from embryonic rat dorsal root ganglia, interleukin-6 supports the survival of less than one third of the neurons yet virtually all of them bear interleukin-6 alpha-receptors. A finding that might explain this selectivity is that interleukin-6 acts on sensory neurons in culture through a mechanism requiring endogenous brain-derived neurotrophic factor. Antibodies or a trkB fusion protein that block the biological activity of brain-derived neurotrophic factor synthesized by dorsal root ganglion neurons also block the survival-promoting actions of interleukin-6 on these neurons. Two results indicate that interleukin-6 influences synthesis of brain-derived neurotrophic factor in adult dorsal root ganglion neurons. Intrathecal infusion of interleukin-6 in rats increases the concentration of brain-derived neurotrophic factor mRNA in rat lumbar dorsal root ganglia. The induction of brain-derived neurotrophic factor in dorsal root ganglion neurons that is seen after nerve injury in rats or wild-type mice is severely attenuated in mice with null mutation of the interleukin-6 gene. In brief, the ability of interleukin-6 to support the survival of embryonic sensory neurons in vitro depends upon the presence of endogenous brain-derived neurotrophic factor and the induction of brain-derived neurotrophic factor in injured adult sensory neurons depends upon the presence of endogenous interleukin-6.     

GABAergic drugs become neurotoxic in cortical neurons pre-exposed to brain-derived neurotrophic factor

A 24-h pretreatment with BNDF enhanced excitotoxic neuronal death in cultured mouse cortical cells challenged with NMDA in the presence of extracellular Mg(2+). The GABA(A) receptor antagonist, bicuculline, enhanced NMDA toxicity in control cultures but, unexpectedly, became neuroprotective in cultures pretreated with BDNF. In contrast, drugs that activate GABA(A) receptors (e.g. muscimol, benzodiazepines, or phenobarbital) or drugs that indirectly enhance GABAergic transmission were protective in control cultures but amplified NMDA toxicity after pretreatment with BDNF. The atypical behaviour of GABAergic drugs in cultures pretreated with BDNF depended on changes in the anion reversal potential because (i) increases in extracellular Cl(-) concentrations abolished the neurotoxic action of muscimol; (ii) muscimol stimulated (36)Cl(-) efflux after pretreatment with BDNF; and (iii) exposure to BDNF reduced the expression of the neuronal K(+)/Cl(-) co-transporter, KCC2. Our data raise the concern that GABAergic drugs may become neurotoxic under conditions associated with increases in brain BDNF levels.     

Short-term response of postnatal rat vestibular neurons following brain-derived neurotrophic factor or neurotrophin-3 application

The effects of the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) neurotrophins on the intracellular calcium level ([Ca²⁺]_i) were studied in vestibular ganglion neurons (VGNs) from postnatal day 3 (P3) rats cultured for 50 hr. We first assessed the expression of trkB and trkC mRNA receptors in cultured VGNs. Immunobloting and immunocytochemistry confirmed the presence of the neurotrophin receptors on neurons. Both neurotrophins induced transient [Ca²⁺]_i elevations in VGNs: BDNF-treated neurons responded in 65% and NT-3-treated neurons in 56%. The responses could be inhibited by anti-BDNF or anti-NT-3 antibodies. The [Ca²⁺]_i elevation was dependent on extracellular calcium since it was abolished in calcium-free medium but also implicates the release of calcium from intracellular stores as tested by prior depletion with thapsigargin. Our results suggest the implication of a short-term calcium regulation in VGNs, which could reflect specific fast effects of neurotrophins in the early postnatal rat vestibular system. J. Neurosci. Res. 50:443–449, 1997. © 1997 Wiley-Liss, Inc.     

Serum brain-derived neurotrophic factor response to aerobic exercise in multiple sclerosis

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF)-1, support brain health through protective, regenerative and adaptive neural processes. In this study, we investigated whether exercise and exercise training would alter circulating BDNF and IGF-1 in people with multiple sclerosis (MS) and matched controls. Twenty-two volunteers (MS (n = 11) and controls (n = 11)) matched in age, weight, body fatness and aerobic capacity (VO_2peak) completed the study. Subjects cycled at 60% of VO_2peak, three times per week for 8 weeks. Serum was analyzed for BDNF and IGF-1 at rest and BDNF after a standardized exercise bout at weeks 0, 4 and 8. Resting BDNF levels were lower in MS compared to controls at week 0 (p = 0.03) and only tended to be lower at week 8 (p = 0.07). BDNF increased at week 4 in MS subjects (p = 0.04) and returned to baseline at week 8. With acute exercise, BDNF decreased in both groups during the 3-hour post-exercise recovery period. Resting IGF-1 concentration was not significantly different between groups before or after training. Our study provides preliminary evidence that exercise may influence BDNF regulation in humans. Further research is needed to elucidate the impact of exercise on neurotrophin production, secretion and target tissue responses in humans.     

Development of pontine noradrenergic A5 neurons requires brain-derived neurotrophic factor

Pontine noradrenergic A5 neurons play a pivotal role in maturation and regulation of the brainstem respiratory rhythm-generating network. Analysis of newborn brain-derived neurotrophic factor (BDNF)-null mice revealed a marked loss of tyrosine hydroxylase-positive A5 neurons compared to wildtype controls that was rescued by null mutation of the proapoptotic gene Bax. In cultures of the A5 region from E12.5 rat embryos, BDNF significantly increased the number and branching of tyrosine hydroxylase-positive neurons. Immunoneutralization of endogenous glial cell line-derived neurotrophic factor partially inhibited the BDNF-dependent increase in the number of tyrosine hydroxylase-positive cells without affecting neurite number. The A5 nucleus is the first brainstem cell group identified at which BDNF is required in vivo for development of neurons critical for cardiorespiratory control.     

The effect of brain-derived neurotrophic factor on sensory and autonomic function after lingual nerve repair

Brain-derived neurotrophic factor (BDNF) is important in the response to peripheral nerve injury and may enhance regeneration. We have assessed its role in the functional recovery of sensory afferents and autonomic efferents after repair of the chorda tympani and lingual nerves in the cat. Six months after entubulation repair, with or without the incorporation of BDNF at the repair site, the recovery of secretomotor and vasomotor efferents was determined by recording salivary flow from the submandibular gland and temperature changes on the tongue surface, each evoked by stimulation of the repaired nerve. Electrophysiological recordings from the lingual and chorda tympani nerves proximal to the repair were undertaken to characterise mechanosensitive, thermosensitive, and gustatory afferents. When compared with data from uninjured control animals, both repair groups showed changes in receptor sensitivity and spontaneous discharge, and persistent reductions in conduction velocity, proportion of gustatory and thermosensitive units, rate of salivary secretion, and vasomotor responses. Comparisons between the outcome of repair with or without BDNF revealed few differences. In the BDNF group, fewer units in the chorda tympani responded to gustatory or thermal stimuli and the sensitivity of the gustatory units was lower. The conduction velocity of afferents in the lingual nerve was also lower, but the mechanoreceptive field size was higher. Thus, despite its known trophic role in the gustatory system, BDNF had not enhanced recovery of these or other fibre populations. We conclude that the application of BDNF to a site of lingual nerve repair has a negative effect on the long-term outcome.     

Differential sorting of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons.

Nerve growth factor (NGF) is released through the constitutive secretory pathway from cells in peripheral tissues and nerves where it can act as a target-derived survival factor. In contrast, brain-derived neurotrophic factor (BDNF) appears to be processed in the regulated secretory pathway of brain neurons and secreted in an activity-dependent manner to play a role in synaptic plasticity. To determine whether sorting differences are intrinsic to the neurotrophins or reflect differences between cell types, we compared NGF and BDNF processing in cultured hippocampal neurons using a Vaccinia virus expression system. Three independent criteria (retention or release from cells after pulse-chase labeling, depolarization-dependent release, and immunocytochemical localization) suggest that the bulk of newly synthesized NGF is sorted into the constitutive pathway, whereas BDNF is primarily sorted into the regulated secretory pathway. Similar results occurred with AtT 20 cells, including those transfected with cDNAs encoding neurotrophin precursor-green fluorescent protein fusions. The NGF precursor, but not the BDNF precursor, is efficiently cleaved by the endoprotease furin in the trans-Golgi network (TGN). Blocking furin activity in AtT 20 cells with alpha1-PDX as well as increasing the expression of NGF precursor partially directed NGF into the regulated secretory pathway. Therefore, neurotrophins can be sorted into either the constitutive or regulated secretory pathways, and sorting may be regulated by the efficiency of furin cleavage in the TGN. This mechanism may explain how neuron-generated neurotrophins can act both as survival factors and as neuropeptides.     

Prophylactic lithium response and polymorphism of the brain-derived neurotrophic factor gene

INTRODUCTION: Brain-derived neurotrophic factor (BDNF) has been involved in the pathogenesis of bipolar mood disorder and in the mechanism of mood-normalizing action of lithium. The aim of this study was to find a possible association between lithium prophylactic effect in bipolar patients and two polymorphisms of BDNF gene. METHODS: Eighty-eight patients (35 males, 53 females) with bipolar illness were studied. Duration of lithium prophylaxis ranged between 5-27 years (mean 15 years). Three categories of prophylactic lithium response were delineated: excellent responders (ER), partial responders (PR) and non-responders (NR). All patients were genotyped for two polymorphisms of BDNF gene: Val66Met and -270C/T. RESULTS: The Val/Met genotype of Val66Met polymorphism occurred more frequently (p = 0.037) and there was a trend for a higher incidence of Met allele (p = 0.076), in ER than in NR. A trend for C/T genotype and T allele of -270C/T polymorphism was observed to occur more frequently in ER than in NR (p = 0.057 and p = 0.065, respectively). CONCLUSION: The data obtained suggest that polymorphism of BDNF gene may be connected with a quality of lithium prophylaxis.     

Human immunodeficiency virus type 1 alters brain-derived neurotrophic factor processing in neurons

The molecular mechanisms leading to synaptic simplification and neuronal apoptosis in human immunodeficiency virus type 1 (HIV-1)-positive subjects are unknown. The HIV protein gp120 reduced the length of neuronal processes similarly to the proneurotrophin pro-brain-derived neurotrophic factor (proBDNF). Intriguingly, the effects of both proBDNF and gp120 were blocked by inhibitors of the p75 neurotrophin receptor, suggesting that proBDNF and gp120 share a similar mechanism of neurotoxicity. Therefore, we tested the hypothesis that gp120 affects the release of proBDNF. Using rat primary neurons, we observed that gp120 promotes a time-dependent intracellular and extracellular accumulation of proBDNF concomitantly with a decrease in mature BDNF. A similar imbalance in the ratio proBDNF/mature BDNF was confirmed in postmortem brains of HIV-positive subjects cognitively impaired and motor impaired. Therefore, it is conceivable to formulate the hypothesis that HIV neurotoxicity includes a gp120-mediated alteration of BDNF processing. To determine the cellular mechanism whereby gp120 produces an accumulation of proBDNF, we examined the levels of intracellular and extracellular enzymes that proteolytically cleave proBDNF furin and tissue plasminogen, respectively. In rat neurons exposed to gp120, intracellular furin levels decreased before cell death, whereas tissue plasminogen changed only during apoptosis. Our data suggest that HIV, through gp120, reduces proBDNF processing by affecting furin levels, and therefore causes an altered balance between antiapoptotic and proapoptotic neurotrophins. Our studies identify a new mechanism that may explain how HIV promotes neuronal injury.     

Localization of brain-derived neurotrophic factor mRNA to neurons in the brain by in situ hybridization

Brain-derived neurotrophic factor (BDNF) which supports survival of and fiber outgrowth from sensory neurons and retinal ganglion cells has recently been cloned. It is closely related to nerve growth factor (NGF), which we had demonstrated earlier by in situ hybridization to be expressed in the brain by cholinergic target neurons. Using oligonucleotide probes we now describe aspects of the distribution of BDNF mRNA in the adult pig and rat brain. Similar to NGF, BDNF expression in vivo in the central nervous system appeared to be mostly confined to neurons. In the hippocampal formation, strongly labeled neurons were found in and around the pyramidal layer, as well as in the granular layer and the hilus of the dentate gyrus of both pig and rat. Other neurons in these areas were more weakly labeled or unlabeled. High BDNF expression was also shown in scattered neurons in cortex cerebri, in many neurons in claustrum and in certain other areas. These results demonstrate that the similarity in structure between NGF and BDNF is paralleled by a similarity in cellular expression, suggesting that BDNF, like NGF, may act via a direct neuron-to-neuron interaction. It is possible that some neurons, for example, in the hippocampal formation, express both NGF and BDNF. Finally, the localization of BDNF expression in cortical areas and in claustrum suggests that the target neurons for this factor extend beyond the sensory ganglion and retinal ganglion cells.     

Dual actions of brain-derived neurotrophic factor on GABAergic transmission in cerebellar Purkinje neurons

The ability to regulate inhibitory synapses is a critical feature of the nervous system and a growing body of evidence indicates that brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of GABA synaptic transmission. Although the neuronal potassium-chloride cotransporter 2 (KCC2) has been implied in this BDNF-induced ionic plasticity, the reports about actions of BDNF on GABA signaling remain conflicting. Here we show dual effects of BDNF on GABAergic synaptic transmission in Purkinje neurons in rat cerebellar slices. BDNF decreased the amplitude of evoked outward IPSCs postsynaptically. It induced a depolarizing shift in the reversal potential (E(IPSC)), which reduced the driving force for outward IPSCs. However, in the absence of KCC2 activity, BDNF directly potentiated rather than inhibited GABA(A) receptor, which was reflected by an increase in the amplitude of outward IPSCs. This action of BDNF coincided with its effect in increasing the amplitude of inward IPSCs. Furthermore, an interaction between GABA(A) receptor and KCC2 was revealed by co-immunoprecipitation. The effects of BDNF on both GABA(A) receptor and KCC2 were dependent on TrkB and also activation of cyclin-dependent kinase 5 (Cdk5). However, only the effect of BDNF on KCC2 activity was dependent on a rise of intracellular calcium. Taken together, these data highlight distinct actions of BDNF on KCC2 and GABA(A) receptor in the regulation of GABAergic synaptic transmission.     

Expression disparity of brain-derived neurotrophic factor immunoreactivity and mRNA in ischemic hippocampal neurons

We studied the spatial and temporal expression of BDNF immunoreactivity and mRNA in the hippocampal formation after transient forebrain ischemia in gerbils. Our study demonstrated that in the vulnerable CA1 neurons, there was a prolonged expression disparity between BDNF immunoreactivity and mRNA and the BDNF level was reduced, in contrast to the ischemia-resistant dentate gyrus neurons that showed transient expression disparity and maintained the BDNF level. This expression disparity of the neurotrophic factor may be related to delayed neuronal death. Double immunostaining showed that reactive astroglia and microglia could express BDNF, suggesting a possible involvement of these cells in the mechanism of neuronal survival after ischemia.     

Selective acid vulnerability of dopaminergic neurons and its recovery by brain-derived neurotrophic factor

Among the pathogenetic phenomena of Parkinson's disease, the character of the selective degeneration of nigrostriatal system with severe gliosis is not fully understood. Here, we have shown that dopaminergic neurons may be exclusively sensitive to elevated acidity elicited after the addition of glial mitogenic factors such as epidermal growth factor and basic fibroblast growth factor or after the direct treatment with hydrochloric acid. The acid sensitivity was specific to dopaminergic neurons. The neurons other than dopaminergic neurons in culture from the ventral mesencephalon were not sensitive to acidity and the neurons from several brain areas were the same as above, except for the hippocampal neurons which had slight acid vulnerability. Choline acetyltransferase assay studies demonstrated that the cholinergic neuronal population in the septum and corpus striatum had no acid sensitivity. The vulnerability of dopaminergic neurons either elicited by glial mitogenic factor or derived from the direct acid exposure was inhibited by the addition of brain-derived neurotrophic factor (BDNF), but not by neurotrophin-3 or nerve growth factor. These findings suggest that dopaminergic neurons have selective acid vulnerability on which BDNF has a pronounced protective effect.     

The response of human and rat fetal ventral mesencephalon in culture to the brain-derived neurotrophic factor treatment

Brain-derived neurotrophic factor (BDNF) has been shown to increase the survival of dopaminergic neurons in rodent mesencephalic cultures. The mRNAs of BDNF and trk B receptor have been found to be expressed in the substantia nigra of rat. In this study, the action of BDNF was studied on the survival and transmitter-specific differentiation of dopaminergic neurons of fetal human CNS aged 9–10-week in vitro. Dopaminergic neuron viability and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunohistochemistry and measurement of dopamine (DA) content with HPLC, respectively. After seven days of treatment with BDNF there were 2.2-fold greater number of TH⁺ neurons surviving than in untreated cultures. Although very low levels of DA were detectable in human tissue, considerable amounts of DA was found in the culture medium from around 13 days in vitro (DIV), indicating that DA in human fetal tissue tended to be synthesised and released into the incubation medium more readily than from cultured rat fetal tissue during the same period. The content of DA in the BDNF-treated cultures was approximately double that of untreated cultures after 7 days. In rat fetal tissue, the capacity of each TH⁺ neuron to produce DA was not changed in the BDNF-treated cultures (7 DIV) compared with control cultures, suggesting that BDNF does not up-regulate the production of DA but rather acts to reduce cell death rates. Ciliary neurotrophic factor (CNTF) treatment of rat mesencephalic culture failed to improve the period of survival of fetal dopaminergic neurons and had no effect on the production of DA in cultures. Taken together, our results suggest that BDNF has potent trophic effect on both rat and human fetal mesencephalic dopaminergic neurons in culture and has a potential application in the treatment of Parkinson's disease.     

Lack of association between two polymorphisms of brain-derived neurotrophic factor and response to typical neuroleptics

Summary. Several studies have connected brain-derived neurotrophic factor (BDNF) with treatment response to neuroleptics. In recent studies, the BDNF expression was reduced by typical neuroleptics. We conducted a retrospective study on 94 patients with schizophrenia and 98 controls. The BDNF G196A and C270T polymorphisms are not associated with treatment response to typical neuroleptics or with age at first hospitalization. Moreover, these polymorphisms of the BDNF gene are not associated with the risk of schizophrenia.