Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death.

Current ideas about the dependence of neurons on target-derived growth factors were formulated on the basis of experiments involving neurons with projections to the periphery. Nerve growth factor (NGF) and recently identified members of the NGF family of neuronal growth factors, known as neurotrophins, are thought to regulate survival of sympathetic and certain populations of sensory ganglion cells during development. Far less is known about factors that regulate the survival of spinal and cranial motor neurons, which also project to peripheral targets. NGF has not been shown to influence motor neuron survival, and whether the newly identified neurotrophins promote motor neuron survival is unknown. We show here that brain-derived neurotrophic factor (BDNF) is retrogradely transported by motor neurons in neonatal rats and that local application of BDNF to transected sciatic nerve prevents the massive death of motor neurons that normally follows axotomy in the neonatal period. These results show that BDNF has survival-promoting effects on motor neurons in vivo and suggest that BDNF may influence motor neuron survival during development.     

Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons

Nerve growth factor (NGF) is a trophic molecule essential for the survival of sympathetic and sensory neurons during ontogeny. The extent to which NGF is involved in the maintenance or regulation of the differentiated phenotypes of mature peripheral neurons is much less clear, however. Biochemical analysis of the actions of NGF upon peripheral neurons has been hampered by the lack of a preparation of neuronal cells that are responsive to NGF but do not require it for survival. We report here that in adult dorsal root ganglion neurons, which can be isolated, enriched and maintained in culture in the absence of neuronal growth factors, the expression of mRNAs encoding the precursors of two neuropeptides, substance P and calcitonin gene-related peptide is regulated by NGF. Our results provide the first direct evidence of a continuous dynamic role for NGF in regulation of peptide neurotransmitter/neuromodulator levels in mature sensory neurons.     

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries. I     

Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family

The survival and functional maintenance of vertebrate neurons critically depends on the availability of specific neurotrophic factors. So far, only two such factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been characterized and shown to have the typical features of secretory proteins. This characterization has been possible because of the extraordinarily large quantities of NGF in some adult tissues, and the virtually unlimited availability of brain tissue from which BDNF was isolated. Both NGF and BDNF promote the survival of distinct neuronal populations in vivo and are related in their primary structure, suggesting that they are members of a gene family. Although there is little doubt about the existence of other such proteins, their low abundance has rendered their identification and characterization difficult. Taking advantage of sequence identities between NGF and BDNF, we have now identified a third member of this family, which we name neurotrophin-3. Both the tissue distribution of the messenger RNA and the neuronal specificity of this secretory protein differ from those of NGF and BDNF. Alignment of the sequences of the three proteins reveals a remarkable number of amino acid identities, including all cysteine residues. This alignment also delineates four variable domains, each of 7-11 amino acids, indicating structural elements presumably involved in the neuronal specificity of these proteins.     

Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo

In Parkinson's disease, brain dopamine neurons degenerate most prominently in the substantia nigra. Neurotrophic factors promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. The most potent neurotrophic factor for dopamine neurons described so far is the glial-cell-line-derived neurotrophic factor (GDNF). Here we have identified a conserved dopamine neurotrophic factor (CDNF) as a trophic factor for dopamine neurons. CDNF, together with its previously described vertebrate and invertebrate homologue the mesencephalic-astrocyte-derived neurotrophic factor, is a secreted protein with eight conserved cysteine residues, predicting a unique protein fold and defining a new, evolutionarily conserved protein family. CDNF (Armetl1) is expressed in several tissues of mouse and human, including the mouse embryonic and postnatal brain. In vivo, CDNF prevented the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in a rat experimental model of Parkinson's disease. A single injection of CDNF before 6-OHDA delivery into the striatum significantly reduced amphetamine-induced ipsilateral turning behaviour and almost completely rescued dopaminergic tyrosine-hydroxylase-positive cells in the substantia nigra. When administered four weeks after 6-OHDA, intrastriatal injection of CDNF was able to restore the dopaminergic function and prevent the degeneration of dopaminergic neurons in substantia nigra. Thus, CDNF was at least as efficient as GDNF in both experimental settings. Our results suggest that CDNF might be beneficial for the treatment of Parkinson's disease.     

Brain-derived neurotrophic factor prevents neuronal death in vivo

Developing vertebrate neurons are thought to depend for their survival on specific neurotrophic proteins present in their target fields. The limited availability of these proteins does not allow the survival of all neurons initially innervating a target, resulting in the widely observed phenomenon of naturally occurring neuronal death. Although a variety of proteins have been reported to promote the survival of neurons in tissue culture, the demonstration that these proteins increase neuronal numbers and/or decrease neuronal death in vivo has only been possible with nerve growth factor (NGF). The generalization of the concept that neurotrophic proteins regulate neuronal survival during normal development critically depends on the demonstration that the survival of neurons in vivo can be increased by the administration of a neurotrophic protein different from NGF. We report here that this is the case with brain-derived neurotrophic factor, a protein of extremely low abundance purified from the central nervous system.     

Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF.

Nerve growth factor (NGF) is a neurotrophic factor responsible for the differentiation and survival of sympathetic and sensory neurons as well as selective populations of cholinergic neurons. NGF binds to specific cell-surface receptors but the mechanism for transduction of the neurotrophic signal is unknown. Several experiments using the NGF-responsive pheochromocytoma cell line, PC12, have implicated tyrosine phosphorylation in NGF-mediated responses, although no NGF-specific tyrosine kinases have been identified. Here we show that NGF induces tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product, a tyrosine kinase receptor whose expression is restricted in vivo to neurons of the sensory spinal and cranial ganglia of neural crest origin. Tyrosine phosphorylation of trk by NGF is rapid, specific and occurs with picomolar quantities of factor, indicating that the response is mediated by physiological amounts of NGF. Activation of the trk tyrosine kinase receptor provides a possible mechanism for signal transduction by NGF.     

Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival and differentiation and to guide axon extension both in vitro and in vivo. The BDNF-induced chemo-attraction of axonal growth cones requires Ca2+ signalling, but how Ca2+ is regulated by BDNF at the growth cone remains largely unclear. Extracellular application of BDNF triggers membrane currents resembling those through TRPC (transient receptor potential canonical) channels in rat pontine neurons and in Xenopus spinal neurons. Here, we report that in cultured cerebellar granule cells, TRPC channels contribute to the BDNF-induced elevation of Ca2+ at the growth cone and are required for BDNF-induced chemo-attractive turning. Several members of the TRPC family are highly expressed in these neurons, and both Ca2+ elevation and growth-cone turning induced by BDNF are abolished by pharmacological inhibition of TRPC channels, overexpression of a dominant-negative form of TRPC3 or TRPC6, or downregulation of TRPC3 expression via short interfering RNA. Thus, TRPC channel activity is essential for nerve-growth-cone guidance by BDNF.     

Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve

The Schwann cells and fibroblast-like cells of the intact sciatic nerve of adult rats synthesize very little nerve growth factor (NGF). After lesion, however, there is a dramatic increase in the amounts of both NGF-mRNA and NGF protein synthesized by the sciatic non-neuronal cells. This local increase in NGF synthesis partially replaces the interrupted NGF supply from the periphery to the NGF-responsive sensory and sympathetic neurons, whose axons run within the sciatic nerve. Macrophages, known to invade the site of nerve lesion during wallerian degeneration, are important in the regulation of NGF synthesis. Here we demonstrate that the effect of macrophages on NGF-mRNA levels in cultured explants of sciatic nerve can be mimicked by conditioned media of activated macrophages, and that interleukin-1 is the responsible agent.     

Activin is a nerve cell survival molecule

The structures of five neurotrophic molecules have so far been published. Nerve growth factor, fibroblast growth factor and purpurin, have been identified as nerve-cell survival molecules. More recently, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor have been cloned and sequenced. As all these proteins stimulate the survival of ciliary or sensory neurons, a new cell survival assay is required if novel neurotrophic molecules are to be discovered. P19 teratoma cells differentiate to nerve-like cells in the presence of 5 x 10(-7) M retinoic acid (RA). But when P19 cells are plated in N2 synthetic medium without being exposed to RA, they die within 48 h. In an attempt to identify a molecule(s) that can substitute for RA in promoting P19 survival, we assayed serum-free growth-conditioned media for their ability to promote P19 survival. One cell line from the rat eye secreted a molecule that promoted the survival of P19 cells and some types of nerve cell. We identified this molecule as activin, better known for its role in hormone secretion.     

Nerve growth factor is a mitogen for cultured chromaffin cells

Nerve growth factor (NGF) is essential for the survival and differentiation of a number of neural crest derivatives, including sympathetic and sensory neurones. While early studies suggested that NGF might also have a mitogenic effect on these neurones, subsequent work has favoured the interpretation that NGF promotes cell survival or differentiation rather than proliferation. We have addressed the issue of a mitogenic effect of NGF using adrenal chromaffin cells, which are endocrine cells derived from the neural crest, and are closely related to sympathetic neurones. Adrenal chromaffin cells respond to NGF in vitro by expressing neuronal traits. We now report that NGF elicits a mitotic response in cultured chromaffin cells from young rats, and that this response is blocked by an antiserum to 2.5S NGF. The chromaffin cells that divided in response to NGF can subsequently become neuronal in the continued presence of NGF.     

Molecular cloning and expression of brain-derived neurotrophic factor

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.     

Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor

We show that nerve growth factor (NGF) synthesis in developing skin begins with sensory innervation and that sensory neurons do not express NGF receptors until their fibres reach their cutaneous targets. Both cutaneous epithelium and mesenchyme synthesize NGF, the concentration of messenger RNA for NGF being higher in the more densely innervated epithelium.     

Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival

Primary sensory neurones in cranial and dorsal root ganglia (DRG) of adult animals are generally thought to be maintained through connections with their peripheral (but not central) targets by trophic factor(s) other than nerve growth factor (NGF). Damage to the peripheral process of sensory neurones results in a dramatic response or even death of the neurones, whereas axotomy (cutting) of the central process does not initiate profound reaction in these neurones. The development and maintenance of neurones are highly dependent on a supply of trophic agents produced by targets and retrogradely transported via the peripheral process to the cell body. NGF deprivation in fetal rodents produced either by exogenously administered antibodies or by those of maternal origin, results in death of DRG and of some cranial sensory neurones. However, as chronic NGF deprivation in neonatal or adult rodents produces little or no cell death, it has been assumed that some other trophic factor(s) derived from the peripheral target sustains sensory neurones in postnatal life. By inducing NGF deprivation by autoimmunizing guinea pigs with mouse NGF and/or by cutting the central root (process) of a DRG, we demonstrate here that under certain conditions DRG neurones require NGF and centrally derived trophic support. Our results indicate that sensory neurones are maintained by the trophic support provided by both peripheral and central targets. This support is mediated by NGF and other as yet unidentified trophic factors. The relative importance of the two target fields and NGF compared with other trophic factors changes during development.     

Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex

Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks.     

Immunohistochemical localization of endogenous nerve growth factor

Nerve growth factor (NGF) has been proposed as a trophic molecule essential for the development of sympathetic and primary sensory neurones. In newborn mice and rats, administration of nerve growth factor results in an increase in the number of surviving neurones, whereas administration of antiserum to NGF decreases neuronal survival. Thus it has been proposed that the factor is produced and secreted by the relevant target tissues to provide trophic support for the ingrowing nerves. The site of synthesis of nerve growth factor is still unknown, and it has been emphasized that a precise physiological role for the molecule cannot be ascribed until the cell types that produce it are known. I report here the use of immunohistochemistry to localize endogenous NGF in the rat iris, a tissue in which there is sound biochemical evidence for the production of NGF activity. Surprisingly, the results reveal that NGF can be detected readily in Schwann cells, but not in smooth muscle cells of the iris when it is sympathetically denervated or cultured.     

Neurotrophin-evoked rapid excitation through TrkB receptors.

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.     

BDNF controls dopamine D3 receptor expression and triggers behavioural sensitization

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, is a polypeptidic factor initially regarded to be responsible for neuron proliferation, differentiation and survival, through its uptake at nerve terminals and retrograde transport to the cell body. A more diverse role for BDNF has emerged progressively from observations showing that it is also transported anterogradely, is released on neuron depolarization, and triggers rapid intracellular signals and action potentials in central neurons. Here we report that BDNF elicits long-term neuronal adaptations by controlling the responsiveness of its target neurons to the important neurotransmitter, dopamine. Using lesions and gene-targeted mice lacking BDNF, we show that BDNF from dopamine neurons is responsible for inducing normal expression of the dopamine D3 receptor in nucleus accumbens both during development and in adulthood. BDNF from corticostriatal neurons also induces behavioural sensitization, by triggering overexpression of the D3 receptor in striatum of hemiparkinsonian rats. Our results suggest that BDNF may be an important determinant of pathophysiological conditions such as drug addiction, schizophrenia or Parkinson's disease, in which D3 receptor expression is abnormal.