The role of neurotrophins in the developing nervous system

Neurotrophins were originally identified by their ability to promote the survival of developing neurons. However, recent work on these proteins indicates that they may also influence the proliferation and differentiation of neuron progenitor cells and regular several differentiated traits of neurons throughout life. Moreover, the effects of neurotrophins on survival have turned out to be more complex than originally thought. Some neurons switch their survival requirements from one set of neurotrophins to another during development, and several neurotrophins may be involved in regulating the survival of a population of neurons at any one time. Much of our understanding of the developmental physiology of neurotrophins has come from studying neurons of the peripheral nervous system. Because these neurons and their progenitors are segregated into anatomically discrete sites, it has been possible to obtain these cell for in vitro experimental studies from the earliest stage of their development. The recent generation of mice having null mutations in the neurotrophin and neurotrophin receptor genes has opened up an unparalleled opportunity to assess the physiological relevance of the wealth of data obtained from these in vitro studies. Here I provide a chronological account of the effects of members of the NGF family of neurotrophins on cells of the neural lineage with special reference to the peripheral nervous system. 1994 John Wiley & Sons, Inc.     

Neurotrophin-regulated signalling pathways

Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB and TrkC). In addition, each neurotrophin activates p75 neurotrophin receptor (p75NTR), a member of the tumour necrosis factor receptor superfamily. Through Trk receptors, neurotrophins activate Ras, phosphatidyl inositol-3 (PI3)-kinase, phospholipase C-gamma1 and signalling pathways controlled through these proteins, such as the MAP kinases. Activation of p75NTR results in activation of the nuclear factor-kappaB (NF-kappaB) and Jun kinase as well as other signalling pathways. Limiting quantities of neurotrophins during development control the number of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. The neurotrophins also regulate cell fate decisions, axon growth, dendrite growth and pruning and the expression of proteins, such as ion channels, transmitter biosynthetic enzymes and neuropeptide transmitters that are essential for normal neuronal function. Continued presence of the neurotrophins is required in the adult nervous system, where they control synaptic function and plasticity, and sustain neuronal survival, morphology and differentiation. They also have additional, subtler roles outside the nervous system. In recent years, three rare human genetic disorders, which result in deleterious effects on sensory perception, cognition and a variety of behaviours, have been shown to be attributable to mutations in brain-derived neurotrophic factor and two of the Trk receptors.     

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.     

Development of trophic interactions in the vertebrate peripheral nervous system

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of thetrk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.     

Neurotrophins and lung disease

Neurotrophins are growth factors that exert multiple actions on neuronal and nonneuronal cells. Neurotrophin receptors are expressed on central and peripheral neurons, lymphocytes, monocytes, mast cells, and fibroblasts. In accordance with the distribution of their receptors, neurotrophins control the development and function of neurons and regulate inflammatory processes. Production of neurotrophins is altered in asthma, lung cancer, and pulmonary fibrosis. Evidence from animal models has implicated nerve growth factor (NGF) as a mediator of pulmonary inflammation, bronchoconstriction, and airway hyperreactivity, all of which are hallmarks of asthma. NGF regulates the growth of lung tumor cells and cultured lung fibroblasts. Thus neurotrophins, particularly NGF, are candidate molecules for regulating disease processes in asthma, lung cancer, and pulmonary fibrosis.     

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.     

Neurotrophins improve synaptic transmission in the adult rodent diaphragm

Neurotrophins are usually viewed as secreted proteins that control long-term survival and differentiation of neurons. However, recent studies have established that among the most important functions of neurotrophins is their capacity to regulate synaptic functions and plasticity. When altering synaptic function, neurotrophins are able to produce two types of outcomes, an immediate effect on synaptic transmission and long-term control of synaptic structure and function. The first effect occurs within seconds or minutes after the neurotrophic factor has been applied and usually involves acute modification of synaptic transmission. The second effect takes hours and days, as protein synthesis is required to complete the structural changes. Neurotrophins and their receptors are expressed within the neuromuscular system, making these agents ideal candidates for the short-and long-term regulation of skeletal muscle function. For instance, neurotrophins can alter neuromuscular function acutely, by modulating the amount of neurotransmitter released with each nerve impulse, or chronically, by changing postsynaptic properties or the content and size of synaptic vesicles. It is obvious that the effects of neurotrophins depend on the specific neurotrophin involved (four neurotrophins have been found in mammals; these are nerve growth factor, brain-derived neurotrophic factor, and neurotrophins-3 and-4) and on the specific synapse being studied. Growing evidence highlights the role of neurotrophins in the development and function of neuromuscular synapses. This review will examine the role of neurotrophins in the regulation of neuromuscular transmission. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 327–337, July–October, 2007.     

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons.

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.     

Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons.

Neurotrophins promote neuronal survival by signalling through Trk receptor tyrosine kinases: nerve growth factor signals through TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)4 through TrkB and NT3 through TrkC. Although studies in some, but not all, cell lines indicate that NT3 can also signal through TrkA and TrkB, it is not known if such signalling can occur in neurons. We show that NT3 can promote the in vitro survival of sensory and sympathetic neurons isolated from embryos that are homozygous for a null mutation in the trkC gene. During the mid-embryonic period, NT3 promoted the survival of as many trigeminal and nodose neurons as the preferred neurotrophins, NGF and BDNF. However, later in development, these neurons lost their ability to respond to NT3. NT3 also promoted the survival of almost all sympathetic neurons, but no decrease in effectiveness was observed during development. Trigeminal neurons from trkC-/- trkA-/- embryos did not respond to NT3 and nodose neurons from trkB-/- embryos likewise failed to respond to NT3. These results show that NT3 can signal through TrkA and TrkB in neurons at certain stages of development and may explain why the phenotype of NT3-/- mice is more severe than that of trkC-/- mice.     

Making and breaking the innervation of the ear: neurotrophic support during ear development and its clinical implications

Analyses of single and double mutants of members of the neurotrophin family and their receptors are reviewed. These data demonstrate that the two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), and their high-affinity receptors trkB and trkC, are the sole support for the developing afferent innervation of the ear. Neurotrophins are first expressed in the otocyst around the time afferent sensory neurons become postmitotic. They are crucial for the survival of certain topologically distinct populations of sensory neurons. BDNF supports all sensory neurons to the semicircular canals, most sensory neurons to the saccule and utricle, and many sensory neurons to the apex and middle turn of the cochlea. In contrast, NT-3 supports few sensory neurons to the utricle and saccule, all sensory neurons to the basal turn of the cochlea and most sensory neurons to the middle and apical turn. Some topologically restricted effects reflect the pattern of neurotrophin distribution as revealed by in situ hybridization (e.g., loss of all innervation to the semicircular canal sensory epithelia in BDNF or trkB mutants). However, other topologically restricted effects cannot be explained on the basis of current knowledge of neurotrophin or neurotrophin receptor distribution. Data on mutants also support the notion that BDNF may play a role in neonatal plastic reorganization of the pattern of innervation in the ear and possibly the brainstem. In contrast, data obtained thus far on the ability of neurotrophins to rescue adult sensory neuron after insults to cochlear hair cells are less compelling. The ear is a model system to test the interactions of the two neurotrophins, BDNF and NT-3, with their two high-affinity receptors, trkB and trkC.     

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.     

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases.     

Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB.

In vertebrates, the formation and maintenance of neuronal connections are subject to regulation by multiple target-derived, diffusible (neurotrophic) factors. Here we describe the identification and characterization of a novel neurotrophic factor designated neurotrophin-5 (NT-5). NT-5 is structurally related to nerve growth factor and is expressed in embryonic as well as adult tissues. Recombinant NT-5 promotes the survival of peripheral sensory and sympathetic neurons and induces differentiation of the pheochromocytoma cell line PC12. NT-5 activates two trk-related tyrosine kinase receptors and shares these receptors with other neurotrophins. Activation of multiple receptors may permit a single neurotrophin to control target innervation by distinct neuronal populations. Receptor sharing could enable neurotrophic factors emanating from distinct targets to cooperate in regulating neurons with multiple connections.     

The survival and growth of embryonic proprioceptive neurons is promoted by a factor present in skeletal muscle

To date, the neurotrophic factor requirements of developing sensory neurons have been studied using heterogeneous populations of neurons that innervate a wide variety of different sensory structures. To ascertain the particular neurotrophic factor requirements of different kinds of sensory neurons and to determine whether these requirements are related to the type of sensory receptors innervated, it is necessary to study homogeneous preparations of functionally distinct sensory neurons. For this reason I have studied the influence of a soluble extract of skeletal muscle on the survival and growth of proprioceptive neurons isolated from the trigeminal mesencephalic nucleus (TMN) of the embryonic chick. Explants of the TMN and dissociated glia-free cultures of TMN neurons were established from chick embryos of 10 to 18 days incubation (E10 to E18). Skeletal muscle extract prepared from E18 chick pectoral muscle and enriched for neurotrophic activity by ammonium sulfate fractionation promoted marked neurite outgrowth from explants and substantial survival in dissociated cultures established during the period of natural neuronal death in the TMN. In these latter cultures 70 to 80% of the neurons survived and grew in the presence of the extract compared with less than 2% in control cultures. At later ages, following the period of natural neuronal death, these effects were less marked. The neurotrophic activity of extracts prepared from muscle of different ages increased steadily from E10 to E20 (the oldest muscle studied). The active factor is heat labile, trypsin sensitive, and non-dialyzable, it is neither functionally nor immunochemically related to NGF and it has negligible neurotrophic effect on the predominantly cutaneous sensory neuron population of the trigeminal ganglion. These findings demonstrate that skeletal muscle contains a neurotrophic factor which supports the survival and growth of proprioceptive neurons and suggest that this factor has some specificity among functionally distinct kinds of sensory neurons.     

Neurotrophins: the biological paradox of survival factors eliciting apoptosis

Neurotrophins are target-derived soluble polypeptides required for neuronal survival. Binding of neurotrophins to Trk receptor tyrosine kinases initiate signaling cascades that promote cell survival and differentiation. All family members bind to another receptor (p75NTR), which belongs to the tumor necrosis factor superfamily. Hence, nerve growth factor (NGF) and related trophic factors are unique in that two separate receptor types are utilized. Although the biological function of p75NTR has been elusive, it has been suggested to mediate apoptosis of developing neurons in the absence of Trk receptors. This presents a tantalizing paradigm, in which life-death decisions of cells are dependent upon the expression and action of two different receptors with distinctive signaling mechanisms. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced NGF responsiveness leading to a survival signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. Here we discuss the unique features and implications of this unusual signal transduction system.     

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.     

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.