Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons

The mechanism by which target-derived Nerve Growth Factor (NGF) signaling is propagated retrogradely, over extremely long distances, to cell bodies to support survival of neurons is unclear. Here we show that survival of sympathetic neurons supported by NGF on distal axons requires the kinase activity of the NGF receptor, TrkA, in both distal axons and cell bodies. In contrast, disruption of TrkA activity exclusively in proximal axonal segments affects neither retrograde NGF-TrkA signaling in cell bodies nor neuronal survival. Ligand-receptor internalization is necessary for survival of neurons supported by NGF on distal axons. Furthermore, antibody neutralization experiments indicate that retrogradely transported NGF, within cell bodies, is critical for neuronal survival but not for growth of distal axons. Taken together, our results indicate that retrogradely transported NGF-TrkA complexes promote sympathetic neuron survival.     

Spatially and functionally distinct roles of the PI3-K effector pathway during NGF signaling in sympathetic neurons

NGF is a target-derived growth factor for developing sympathetic neurons. Here, we show that application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement for PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling.     

Going the distance, or not, with neurotrophin signals

NGF and NT-3 both signal through TrkA receptors on the axons of developing sympathetic neurons, but while NGF supports survival and differentiation, NT-3 does not. In this issue of Cell, the difference is explained as the ability of NGF, but not NT-3, to induce internalization and retrograde transport of activated TrkA.     

Colloquium 10: 3

Previous work has shown that neurotrophins bind to and activate Trk receptors on distal axons, and that neurotrophin‐Trk complexes are internalized and retrogradely transported to cell bodies. Whether retrograde transport of neurotrophins and retrograde neurotrophin‐Trk signalling are necessary for survival remains unclear, and recently published findings are controversial. We are using compartmentalized cultures of sympathetic neurons to address the mechanism of retrograde NGF signalling and survival. We performed survival experiments using either the Trk kinase inhibitor K252a to inhibit TrkA activity in different cellular compartments, or a dominant‐negative form of dynamin, K44A dynamin, to block internalization of NGF‐TrkA complexes. We found that sympathetic neurons supported by NGF acting on distal axons undergo apoptosis when TrkA activity in either cell bodies or distal axons is inhibited by K252a, or when internalization is blocked by K44A dynamin. Results of experiments employing three‐compartment chambers indicate that TrkA signalling is required within cell bodies and distal axons, but not in proximal axons, for retrograde support of survival. Likewise, TrkA activity within distal axons, but not in proximal axons, is required for retrograde transport of [125I] NGF. Finally, peptide‐mediated delivery of affinity‐purified anti‐NGF into cell bodies results in apoptosis of neurons. Taken together, our results support a model in which NGF internalization and retrograde transport and retrograde TrkA signalling are necessary for survival of sympathetic neurons. This work is supported by the NIH and HHMI.     

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.     

Dose and age-dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target-derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF-dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax-/- mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA-positive, and TrkA-/- TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose- and age-dependent fashion, mediated by p75NTR signaling through TrkA expressing axons.     

NGF and Neurotrophin-3 Both Activate TrkA on Sympathetic Neurons but Differentially Regulate Survival and Neuritogenesis

In this report we examine the biological and molecular basis of the control of sympathetic neuron differentiation and survival by NGF and neurotrophin-3 (NT-3). NT-3 is as efficient as NGF in mediating neuritogenesis and expression of growth-associated genes in NGF-dependent sympathetic neurons, but it is 20–40fold less efficient in supporting their survival. Both NT-3 and NGF induce similar sustained, long-term activation of TrkA, while NGF is 10-fold more efficient than NT-3 in mediating acute, short-term TrkA activity. At similar acute levels of TrkA activation, NT-3 still mediates neuronal survival two- to threefold less well than NGF. However, a mutant NT-3 that activates TrkC, but not TrkA, is unable to support sympathetic neuron survival or neuritogenesis, indicating that NT3–mediated TrkA activation is necessary for both of these responses. On the basis of these data, we suggest that NGF and NT-3 differentially regulate the TrkA receptor both with regard to activation time course and downstream targets, leading to selective regulation of neuritogenesis and survival. Such differential responsiveness to two ligands acting through the same Trk receptor has important implications for neurotrophin function throughout the nervous system.     

The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA

The TrkA receptor is activated primarily by nerve growth factor (NGF), but it can also be activated by high concentrations of neurotrophin 3 (NT-3). The pan-neurotrophin receptor p75(NTR) strongly inhibits activation of TrkA by NT-3 but not by NGF. To examine the role of p75(NTR) in regulating the specificity of TrkA signaling, we expressed both receptors in Xenopus oocytes. Application of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of (45)Ca(2+) by a phospholipase C-gamma-dependent pathway. Coexpression of p75(NTR) with TrkA inhibited (45)Ca(2+) efflux in response to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA increased with increasing expression of p75(NTR). Coexpression of a truncated p75(NTR) receptor lacking all but the first 9 amino acids of the cytoplasmic domain inhibited NT-3 stimulation of (45)Ca(2+) efflux, whereas coexpression of an epidermal growth factor receptor/p75(NTR) chimera (extracellular domain of epidermal growth factor receptor with transmembrane and cytoplasmic domains of p75(NTR)) did not inhibit NT-3 signaling through TrkA. These studies demonstrated that the extracellular domain of p75(NTR) was necessary to inhibit NT-3 signaling through TrkA. Remarkably, p75(NTR) binding to NT-3 was not required to prevent signaling through TrkA, since occupying p75(NTR) with brain-derived neurotrophic factor or anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate (45)Ca(2+) efflux. These data suggested a physical association between TrkA and p75(NTR). Documenting this physical interaction, we showed that p75(NTR) and TrkA could be coimmunoprecipitated from Xenopus oocytes. Our results suggest that the interaction of these two receptors on the cell surface mediated the inhibition of NT-3-activated signaling through TrkA.     

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that?a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.     

NGF signaling from clathrin-coated vesicles: evidence that signaling endosomes serve as a platform for the Ras-MAPK pathway.

The target-derived neurotrophic factor "nerve growth factor" (NGF) signals through TrkA to promote the survival, differentiation, and maintenance of neurons. How the NGF signal in axon terminals is conveyed to the cell body is unknown. The "signaling endosome hypothesis" envisions that NGF-TrkA complexes are internalized at the axon terminal and retrogradely transported to the cell body. Following NGF treatment, we found that clathrin-coated vesicles contained NGF bound to TrkA together with activated signaling proteins of the Ras-MAP kinase pathway. Evidence that these vesicles could signal was their ability in vitro to activate Elk, a downstream target of Erk1/2. Our results point to the existence of a population of signaling endosomes derived from clathrin-coated membranes in NGF-treated cells.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

Dose and age‐dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptor

Nerve growth factor (NGF) and related neurotrophins are target‐derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF‐dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax^−/− mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA‐positive, and TrkA^−/− TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75^NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose‐ and age‐dependent fashion, mediated by p75^NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

The tyrosine phosphatase inhibitor orthovanadate mimics NGF-induced neuroprotective signaling in rat hippocampal neurons

Activation of the high affinity neurotrophin receptor tropomyosin-related kinase A (TrkA) by nerve growth factor (NGF) leads to phosphorylation of intracellular tyrosine residues of the receptor with subsequent activation of signaling pathways involved in neuronal survival such as the phosphoinositide-3-kinase (PI3-K)/protein kinase B (PKB/Akt) pathway and the mitogen-activated protein kinase (MAPK) cascade. In the present study, we tested whether inhibition of protein-tyrosine phosphatases (PTP) by orthovanadate could enhance tyrosine phosphorylation of TrkA thereby stimulating NGF-like survival signaling in embryonic hippocampal neurons. We found that the PTP inhibitor orthovanadate (1 microM) enhanced TrkA phosphorylation and protected neurons against staurosporine (STS)-induced apoptosis in a time-and concentration-dependent manner. Inhibition of PTP enhanced TrkA phosphorylation also in the presence of NGF antibodies indicating that NGF binding to TrkA was not required for the effects of orthovanadate. Moreover, orthovanadate enhanced phosphorylation of Akt and the MAPK Erk1/2 suggesting that the signaling pathways involved in the protective effect were similar to those activated by NGF. Accordingly, inhibition of PI3-K by wortmannin and MAPK-kinase (MEK) inhibition by UO126 abolished the neuroprotective effects. In conclusion, the results indicate that orthovanadate mimics the effect of NGF on survival signaling pathways in hippocampal neurons. Thus, PTP inhibition appears to be an appropriate strategy to trigger neuroprotective signaling pathways downstream of neurotrophin receptors.     

TrkA mediates developmental sympathetic neuron survival in vivo by silencing an ongoing p75NTR-mediated death signal.

Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/- mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/- sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.     

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.     

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.