The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

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.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Defining Responsiveness of Avian Cochlear Neurons to Brain-Derived Neurotrophic Factor and Nerve Growth Factor by HSV-1-Mediated Gene Transfer

Abstract: The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV-1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV-1-mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF-responsive by transducing them with the high-affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.     

An immunoglobulin-like domain determines the specificity of neurotrophin receptors.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.     

The neurotrophins and CNTF: specificity of action towards PNS and CNS neurons.

The availability of relatively large amounts of nerve growth factor (NGF) has allowed extensive in vitro and in vivo characterization of the neuronal specificity of this neurotrophic factor. The restricted neuronal specificity of NGF (sympathetic neurons, neural crest-derived sensory neurons, basal forebrain cholinergic neurons) has long predicted the existence of other neurotrophic factors possessing different neuronal specificities. Whereas there have been many reports of "activities" distinct from NGF, full characterization of such molecules has been hampered by their extremely low abundance. The recent molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that this protein is closely related to NGF and suggested that these two factors might be members of an even larger gene family. A PCR cloning strategy based on homologies between NGF and BDNF has allowed us to identify and clone a third member of the NGF family which we have termed neurotrophin-3 (NT-3). The establishment of suitable expression systems has now made available sufficient quantities of these proteins to allow us to begin to establish the neuronal specificity of each member of the neurotrophin family, and the role of each in development, maintenance and repair of the PNS and CNS. Using primary cultures of various PNS and CNS regions of the developing chick and rat, and Northern blot analysis, we describe novel neuronal specificities of BDNF, NT-3 and an unrelated neurotrophic factor-ciliary neurotrophic factor (CNTF).     

Effects of the neurotrophins nerve growth factor,neurotrophin-3, and brain-derived neurotrophic factor (BDNF) on neurite growth from adult sensory neurons in compartmented cultures

We used compartmented cultures to study the regulation of adult sensory neurite growth by neurotrophins. We examined the effects of the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT3), and BDNF on distal neurite elongation from adult rat dorsal root ganglion (DRG) neurons. Neurons were plated in the center compartments of three-chambered dishes in the absence of neurotrophin, and neurite extension into the distal (side) compartments containing NGF, BDNF, or NT3 was quantitated. Initial proximal neurite growth did not require any of the neurotrophins, while subsequent elongation into distal compartments required NGF. After neurites had extended into NGF-containing distal compartments, removal of NGF by treatment with anti-NGF resulted in the cessation of growth with minimal neurite retraction. In contrast to the effects of NGF, no distal neurite elongation was observed into compartments with BDNF or NT3. To examine possible additive influences, neurite extension into compartments containing BDNF plus NGF or NT3 plus NGF was quantitated. There was no increased neurite extension into NGF plus NT3 compartments, while the combination of BDNF plus NGF resulted in an inhibition of neurite extension compared with NGF alone. We then investigated whether the regrowth of neurites that had originally grown into NGF subsequent to in vitro axotomy still required NGF. The results demonstrated that unlike adult sensory nerve regeneration in vivo, the in vitro regrowth did require NGF, and neither BDNF nor NT3 was able to substitute for NGF. Since the initial growth from neurons after dissociation (which is also a regenerative response) did not require NGF, it would appear that neuritic growth and regrowth of adult DRG neurons in vitro includes both NGF-independent and NGF-dependent components. The compartmented culture system provides a unique model to further study aspects of this differential regulation of neurite growth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 395–410, 1997     

Brain-Derived Neurotrophic Factor Is More Highly Conserved in Structure and Function than Nerve Growth Factor During Vertebrate Evolution

Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fish Xiphophorus, with the mammalian homologues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purified recombinant BDNF. However, the activity of recombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng/ml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF had reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates from a common ancestral gene about 600 million years ago. By RNA gel blot analysis NGF mRNA was detected during late embryonic development; BDNF was present in adult brain.     

In Vivo Regulation of Brain-Derived Neurotrophic Factor in Dorsal Root Ganglia Is Mediated by Nerve Growth Factor-Triggered Akt Activation during Cystitis

The role of brain-derived neurotrophic factor (BDNF) in sensory hypersensitivity has been suggested; however the molecular mechanisms and signal transduction that regulate BDNF expression in primary afferent neurons during visceral inflammation are not clear. Here we used a rat model of cystitis and found that the mRNA and protein levels of BDNF were increased in the L6 dorsal root ganglia (DRG) in response to bladder inflammation. BDNF up-regulation in the L6 DRG was triggered by endogenous nerve growth factor (NGF) because neutralization of NGF with a specific NGF antibody reduced BDNF levels during cystitis. The neutralizing NGF antibody also subsequently reduced cystitis-induced up-regulation of the serine/threonine kinase Akt activity in L6 DRG. To examine whether the NGF-induced Akt activation led to BDNF up-regulation in DRG in cystitis, we found that in cystitis the phospho-Akt immunoreactivity was co-localized with BDNF in L6 DRG, and prevention of the endogenous Akt activity in the L6 DRG by inhibition of phosphoinositide 3-kinase (PI3K) with a potent inhibitor LY294002 reversed cystitis-induced BDNF up-regulation. Further study showed that application of NGF to the nerve terminals of the ganglion-nerve two-compartmented preparation enhanced BDNF expression in the DRG neuronal soma; which was reduced by pre-treatment of the ganglia with the PI3K inhibitor LY294002 and wortmannin. These in vivo and in vitro experiments indicated that NGF played an important role in the activation of Akt and subsequent up-regulation of BDNF in the sensory neurons in visceral inflammation such as cystitis.     

Distinct modulatory actions of TGF-β and LIF on neurotrophin-mediated survival of developing sensory neurons

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are important for the regulation of survival and differentiation of distinct, largely non-overlapping populations of embryonic sensory neurons. We show here that the multifunctional cytokine transforming growth factor-β (TGF-β) fails to maintain sensory neurons cultured from embryonic day (E) 8 chick dorsal root ganglia (DRG), although DRG neurons are immunoreactive for the TGF-β receptor type II, which is essential for TGF-β signaling. However, in combination with various concentrations of NT-3 and NT-4, but not NGF, TGF-β3 causes a further significant increase in neuron survival. In DRG cell cultures treated with NGF, NT-3, and NT-4, a neutralizing antibody to TGF-β decreases neuron survival suggesting that endogenous TGF-β in these cultures affects the efficacies of neurotrophins. Consistent with this notion and a modulatory role of TGF-β in neurotrophin functions is the observation that TGF-β2 and-β3 immunoreactivities and TGF-β3 mRNA are located in embryonic chick DRG in close association with neurons from E5 onwards. We also show that leukemia inhibitory factor (LIF) significantly decreases NGF-mediated DRG neuron survival. Together, these data indicate that actions and efficacies of neurotrophins are under distinct control by TGF-β and LIF in vitro, and possibly also in vivo. Special issue dedicated to Dr. Hans Thoenen.     

Neurotrophin-4: a survival factor for adult sensory neurons

The nerve growth factor (NGF) family of neurotrophins provides a substantial part of the normal trophic support for sensory neurons during development. Although these neurotrophins, which include Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4), continue to be expressed into adulthood, there is little evidence that they are survival factors for adult neurons. Here we have examined the age-dependent neurotrophic requirements of a specialized type of mechanoreceptive neuron, called a D-hair receptor, in the dorsal root ganglion (DRG). Studies using knockout mice have demonstrated that the survival of D-hair receptors is dependent upon both NT-3 and NT-4. Here, we show that the time period when D-hair receptors require these two neurotrophins is different. Survival of D-hair receptors depends on NT-3 early in postnatal development and NT-4 later in the mature animal. The age-dependent loss of D-hair neurons in older NT-4 knockout mice was accompanied by a large reduction (78%) in neurons positive for the NT-4 receptor (trkB) together with neuronal apoptosis in the DRG. This is the first evidence that sensory neurons have a physiological requirement for a single neurotrophin for their continued survival in the adult.     

The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors —trkA binds only NGF, the relatedtrkB receptor binds BDNF and NT-3, andtrkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to thetrk gene products.     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Decrease in the levels of NGF and BDNF in brains of mice fed a tryptophan-deficient diet

The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.     

Growth factor synergism and antagonism in early neural crest development.

This review article focuses on data that reveal the importance of synergistic and antagonistic effects in growth factor action during the early phases of neural crest development. Growth factors act in concert in different cell lineages and in several aspects of neural crest cell development, including survival, proliferation, and differentiation. Stem cell factor (SCF) is a survival factor for the neural crest stem cell. Its action is neutralized by neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) through apoptotic cell death. In contrast, SCF alone does not support the survival of melanogenic cells (pigment cell precursors). They require the additional presence of a neurotrophin (NGF, BDNF, or NT-3). Fibroblast growth factor-2 (FGF-2) is an important promoter of proliferation in neuronal progenitor cells. In neural crest cells, fibroblast growth factor treatment alone does not lead to cell expansion but also requires the presence of a neurotrophin. The proliferative stimulus of the fibroblast growth factor - neurotrophin combination is antagonized by transforming growth factor beta-1 (TGFbeta-1). Moreover, TGFbeta-1 promotes the concomitant expression of neuronal markers from two cell lineages, sympathetic neurons and primary sensory neurons, indicating that it acts on a pluripotent neuronal progenitor cell. Moreover, the combination of FGF-2 and NT3, but not other neurotrophins, promotes expression or activation of one of the earliest markers expressed by presumptive sympathetic neuroblasts, the norepinephrine transporter. Taken together, these data emphasize the importance of the concerted action of growth factors in neural crest development at different levels and in several cell lineages. The underlying mechanisms involve growth-factor-induced dependence of the cells on other factors and susceptibility to growth-factor-mediated apoptosis.     

Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

Neurotrophin-3 (NT-3) has low-affinity (Kd = 8 x 10(-10) M), as well as high-affinity receptors (Kd = 1.8 x 10(-11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), NT-3 also binds to the low-affinity NGF receptor, a molecule that we suggest to designate low-affinity neurotrophin receptor (LANR). NT-3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT-3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT-3 to its high-affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand-fold higher than those corresponding to their Kd to their high-affinity neuronal receptors. Thus, specific high-affinity NT-3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.