Functional roles of neurotrophin 3 in the developing and mature sympathetic nervous system

Nerve growth factor (NGF) is a potent regulator of sympathetic neuronal function in both developing and adult animals. This article reviews the evidence published in recent years indicating that another member of the NGF family, neurotrophin 3 (NT3), plays both a complementary and overlapping role in the development and maturation of sympathetic neurons. In migratory neural crest cells, expression of the high-affinity receptor, trkC, and promotion of mitosis by NT3 suggest an involvement in gangliogenesis, since sympathetic neuroblasts express both NT3 and trkC and require NT3 for their proliferation, differentiation, and survival, it has been proposed that the factor acts at this developmental stage as an autocrine or paracrine factor. However, NT3 also acts in parallel with NGF to promote the survival of postmitotic neurons during late development. Both trkC and trkA are expressed in sympathetic neurons and function as high-affinity receptors for NT3. NT3 is synthesized in sympathetic effector tissues and the endogenous factor is retrogradely transported to accumulate within the cell soma. Thus, in addition to its role in the differentiation of sympathetic neurons, NT3, like NGF, is also an effector tissue-derived neurotrophic factor for these neurons in maturity.     

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.     

Reduced age-related plasticity of neurotrophin receptor expression in selected sympathetic neurons of the rat

Selective vulnerability of particular groups of neurons is a characteristic of the aging nervous system. We have studied the role of neurotrophin (NT) signalling in this phenomenon using rat sympathetic (SCG) neurons projecting to cerebral blood vessels (CV) and iris which are, respectively, vulnerable to and protected from atrophic changes during old age. RT-PCR was used to examine NT expression in iris and CV in 3- and 24-month-old rats. NGF and NT3 expression in iris was substantially higher compared to CV; neither target showed any alterations with age. RT-PCR for the principal NT receptors, trkA and p75, in SCG showed increased message during early postnatal life. However, during mature adulthood and old age, trkA expression remained stable while p75 declined significantly over the same period. In situ hybridization was used to examine receptor expression in subpopulations of SCG neurons identified using retrograde tracing. Eighteen to 20 h following local treatment of iris and CV with NGF, NT3 or vehicle, expression of NT receptor protein and mRNA was higher in iris- compared with CV-projecting neurons from both young and old rats. NGF and NT3 treatment had no effect on NT receptor expression in CV-projecting neurons at either age. However, similar treatment up-regulated p75 and trkA expression in iris-projecting neurons from 3-month-old, but not 24-month-old, rats. We conclude that lifelong exposure to low levels of NTs combined with impaired plasticity of NT receptor expression are predictors of neuronal vulnerability to age-related atrophy.     

BDNF is essentially required for the early postnatal survival of nociceptors

Neurotrophins promote the survival of specific types of neurons during development and ensure proper maintenance and function of mature responsive neurons. Significant effects of BDNF (Brain-Derived Neurotrophic Factor) on pain physiology have been reported but the contribution of this neurotrophin to the development of nociceptors has not been investigated. We present evidence that BDNF is required for the survival of a significant fraction of peptidergic and non-peptidergic nociceptors in dorsal root ganglia (DRG) postnatally. Bdnf homozygous mutant mice lose approximately half of all nociceptive neurons during the first 2 weeks of life and adult heterozygotes exhibit hypoalgesia and a loss of 25% of all nociceptive neurons. Our in vitro analyses indicate that BDNF-dependent nociceptive neurons also respond to NGF and GDNF. Expression analyses at perinatal times indicate that BDNF is predominantly produced within sensory ganglia and is more abundant than skin-derived NGF or GDNF. Function-blocking studies with BDNF specific antibodies in vitro or cultures of BDNF-deficient sensory neurons suggest that BDNF acts in an autocrine/paracrine way to promote the early postnatal survival of nociceptors that are also responsive to NGF and GDNF. Altogether, the data demonstrate an essential requirement for BDNF in the early postnatal survival of nociceptive neurons.     

Neurotrophin-3 and TrkC-immunoreactive neurons in rat dorsal root ganglia correlate by distribution and morphology

Previous studies have shown that a subpopulation of large dorsal root ganglion neurons contains neurotrophin-3 (NT3)-like immunoreactivity. It is not known, however, whether these NT3 immunoreactive neurons also express the high affinity receptor for NT3, trkC. In the present study, the distribution and morphology of trkC immunoreactive neurons have been correlated with those of NT3 immunoreactive neurons in the dorsal root ganglia. Size and segmental distributions of both antigens indicate that they are present in the same group of large sensory neurons. Almost twice the number of these neurons are present in the cervical and lumbar spinal ganglia than in the thoracic. Co-localization study indicates that 94% of NT3 immunoreactive neurons express trkC. Our findings support the proposal that NT3 in these neurons is derived from their peripheral targets rather than synthesized in situ. Special issue dedicated to Dr. Hans Thoenen.     

Dynamic expression of neurotrophin receptors during sensory neuron genesis and differentiation

To identify potential functions for neurotrophins during sensory neuron genesis and differentiation, we determined the temporal and spatial protein expression patterns of neurotrophin receptors throughout the process of sensory neurogenesis in the dorsal root ganglia (DRG). We show that neurotrophin receptors are expressed early, being first detected on subsets of migrating neural crest cells, and that trkC is among the earliest markers of neural lineage specification. In the immature DRG, we find that both trkC and p75(NTR) are expressed on subsets of dividing progenitor cells in vivo. Furthermore, our data directly reveal distinct patterns of trk receptor expression by individual sensory neurons from the time of their inception with all early arising cells initially being trkC(+), some subsets of whom also coexpress either trkA or trkB or both. As sensory neurons innervate their targets and establish their mature identities, the spectrum of trk receptors expressed by individual neurons is altered. The stereotyped trk receptor expression profiles identified here may potentially correspond to distinct lineages of sensory neurons. These data, in conjunction with other studies, argue for multiple functions for neurotrophins during the process of sensory neuron differentiation, including effects on both neural crest and DRG mitotically active progenitor cells, in addition to possibly influencing the establishment of sensory neuron identity.     

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.     

Subpopulations of rat B2(+) neuroblasts exhibit differential neurotrophin responsiveness during sympathetic development

Sympathetic neurons comprise a population of postmitotic, tyrosine hydroxylase expressing cells whose survival is dependent upon nerve growth factor (NGF) both in vivo and in vitro. However, during development precursors to rat sympathetic neurons in the thoracolumbar region are not responsive to NGF because they lack the signal transducing NGF receptor, trkA. We have previously shown that acquisition of trkA expression is sufficient to confer a functional response to NGF. Here we describe four subpopulations of thoracolumbar sympathetic neuroblasts which are mitotically active and unresponsive to NGF at E13.5 of rat gestation, but differ based upon their neurotrophic responsiveness in vitro. The survival in culture of the largest sympathetic subpopulation is mediated by neurotrophin-3 (NT-3) or glial-derived neurotrophic factor (GDNF), whereas the cell survival of two smaller subpopulations of neuroblasts are mediated by either solely GDNF or solely NT-3. Finally, we identify a subpopulation of sympathetic neuroblasts in the thoracolumbar region whose survival, exit from the cell cycle, induction of trkA expression, and consequent acquisition of NGF responsiveness in culture appear to be neurotrophin independent and cell autonomous. These subpopulations reflect the diversity of neurotrophic actions that occur in the proper development of sympathetic neurons.     

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.     

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target‐derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret‐expressing neurons in the DRG complete developmental cell death prior to TrkA‐expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701–717, 2018     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

Differential regulation of survival and growth in adult sympathetic neurons: An invitro study of neurotrophin responsiveness

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long‐term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti‐NGF antibodies (anti‐NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti‐NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth‐promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 295–305, 2001     

Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.