Mark E. Furth

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.

trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor

A variety of findings seem to functionally link brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), while distinguishing both of these factors from the third member of the neurotrophin family, nerve growth factor (NGF). Here we demonstrate that all three of these neuronal survival molecules bind similarly to the low affinity NGF receptor, but that BDNF and NT-3, unlike NGF, do not act via the high affinity NGF receptor. However, both BDNF and NT-3, but not NGF, bind to full-length and truncated forms of a receptor-like tyrosine kinase, trkB, for which no ligand had previously been identified. In addition to binding BDNF and NT-3, trkB can mediate functional responses to both of these neurotrophins when it is expressed in PC12 cells, although BDNF appears to be the more effective ligand. Thus trkB encodes an essential component of a functional receptor for BDNF and NT-3, but not for NGF. Further evidence predicts the existence of additional functional receptors for the neurotrophins.

Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations.

The development and maintenance of the vertebrate nervous system depends upon neuronal survival proteins known as neurotrophic factors. Nerve growth factor (NGF) remains the best characterized neurotrophic molecule. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. Here we describe the molecular cloning of the human and rat genes encoding BDNF, as well as the isolation of the human NT-3 gene. On the basis of comparison of our genomic and cDNA clones with those of previously isolated BDNF and NT-3 genes and cDNAs, we make inferences about the structures of processed transcripts derived from the neurotrophin genes and the protein precursors they encode. We demonstrate that the mature form of BDNF is identical in all mammals examined, and that the same is true of the mature form of NT-3. Furthermore, the respective tissue-distributions and neuronal specificities of NT-3 and BDNF are also conserved among mammals. Finally, we localize the gene encoding human BDNF (gene symbol designated BDNF) to chromosome 11, band p13, and the gene encoding human NT-3 (gene symbol designated NTF3) to chromosome 12, band p13.

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.

Recombinant Human and Rat Ciliary Neurotrophic Factors

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20–40% and 60–70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

The neurotrophin family of NGF-related neurotrophic factors

The recent molecular cloning of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) has established the existence of an NGF-related family of neurotrophic factors - the neurotrophins. Purification and recombinant production of BDNF and NT-3 has allowed the initiation or extension of in vitro studies of the neuronal specificity of each of these factors. We have found that NT-3, like NGF and BDNF, promotes survival and neurite outgrowth from certain populations of sensory neurons. There appear to be both distinct and overlapping specificities of the 3 neurotrophins towards peripheral neurons - sympathetic neurons and subpopulations of neural crest and neural placode-derived sensory neurons. Using cultures of central nervous system neurons, we have recently established that BDNF: (i) promotes the survival and phenotypic differentiation of rat septal cholinergic neurons, a property consistent with the discovery of high levels of BDNF mRNA expression within the hippocampus; (ii) promotes the survival of rat nigral dopaminergic neurons and furthermore protects these neurons from two dopaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and MPTP. Thus the neurotrophic effects of these factors towards peripheral neurons and neuronal populations known to degenerate in two of the major human neurodegenerative diseases - Alzheimers and Parkinsons disease - provokes the question of whether neurotrophic factors may have therapeutic potential in halting the progression and ameliorating the symptoms of devastating neurological disorders of the CNS or PNS, or improving regeneration of neurons of CNS or PNS after traumatic injury.

Neurotrophic factors, their receptors, and the signal transduction pathways they activate

Our studies of the spatiotemporal availability of neurotrophic factors, coupled with tagged ligand binding assays that identify cell bearing receptors for these factors, should lead toward defining the physiological roles of these molecules in the animal. The use of the tagged ligands to identify factor-responsive cell lines has also provided new model systems for the examination of ligand-receptor interactions, as well as for the study of the subsequent induction of intracellular response pathways. To obtain insights into such intracellular pathways, we have molecularly cloned genes encoding a family of serine-threonine protein kinases, most closely related to kinases involved in the yeast response to pheromones. These kinases may be crucial regulators of early steps in the response of mammalian cells to neurotrophic factors as well as other extracellular signals.