A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling

A fundamental question in developmental biology is how a limited number of growth factors and their cognate receptors coordinate the formation of tissues and organs endowed with enormous morphological complexity. We report that the related neurotrophins NGF and NT-3, acting through a common receptor, TrkA, are required for sequential stages of sympathetic axon growth and, thus, innervation of target fields. Yet, while NGF supports TrkA internalization and retrograde signaling from distal axons to cell bodies to promote neuronal survival, NT-3 cannot. Interestingly, final target-derived NGF promotes expression of the p75 neurotrophin receptor, in turn causing a reduction in the sensitivity of axons to intermediate target-derived NT-3. We propose that a hierarchical neurotrophin signaling cascade coordinates sequential stages of sympathetic axon growth, innervation of targets, and survival in a manner dependent on the differential control of TrkA internalization, trafficking, and retrograde axonal 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.     

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.     

P53 Is Essential for Developmental Neuron Death as Regulated by the TrkA and p75 Neurotrophin Receptors

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/− or p53−/− mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.     

TrkA receptor "hot spots" for binding of NT-3 as a heterologous ligand

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.     

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.     

SHP-1 negatively regulates neuronal survival by functioning as a TrkA phosphatase

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.     

The p75 Neurotrophin Receptor Mediates Neuronal Apoptosis and Is Essential for Naturally Occurring Sympathetic Neuron Death

Abstract. To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.     

The Protein Phosphatase 2A Regulatory Subunits B′β and B′δ Mediate Sustained TrkA Neurotrophin Receptor Autophosphorylation and Neuronal Differentiation

Nerve growth factor (NGF) is critical for the differentiation and maintenance of neurons in the peripheral and central nervous system. Sustained autophosphorylation of the TrkA receptor tyrosine kinase and long-lasting activation of downstream kinase cascades are hallmarks of NGF signaling, yet our knowledge of the molecular mechanisms underlying prolonged TrkA activity is incomplete. Protein phosphatase 2A (PP2A) is a heterotrimeric Ser/Thr phosphatase composed of a scaffolding, catalytic, and regulatory subunit (B, B′, and B" gene families). Here, we employ a combination of pharmacological inhibitors, regulatory subunit overexpression, PP2A scaffold subunit exchange, and RNA interference to show that PP2A containing B′ family regulatory subunits participates in sustained NGF signaling in PC12 cells. Specifically, two neuron-enriched regulatory subunits, B′β and B′δ, recruit PP2A into a complex with TrkA to dephosphorylate the NGF receptor on Ser/Thr residues and to potentiate its intrinsic Tyr kinase activity. Acting at the receptor level, PP2A/ B′β and B′δ enhance NGF (but not epidermal growth factor or fibroblast growth factor) signaling through the Akt and Ras-mitogen-activated protein kinase cascades and promote neuritogenesis and differentiation of PC12 cells. Thus, select PP2A heterotrimers oppose desensitization of the TrkA receptor tyrosine kinase, perhaps through dephosphorylation of inhibitory Ser/Thr phosphorylation sites on the receptor itself, to maintain neurotrophin-mediated developmental and survival signaling.     

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.     

APP Regulates NGF Receptor Trafficking and NGF-Mediated Neuronal Differentiation and Survival

β-amyloid precursor protein (APP) is a key factor in Alzheimer''s disease (AD) but its physiological function is largely undetermined. APP has been found to regulate retrograde transport of nerve growth factor (NGF), which plays a crucial role in mediating neuronal survival and differentiation. Herein, we reveal the mechanism underlying APP-mediated NGF trafficking, by demonstrating a direct interaction between APP and the two NGF receptors, TrkA and p75NTR. Downregulation of APP leads to reduced cell surface levels of TrkA/p75NTR and increased endocytosis of TrkA/p75NTR and NGF. In addition, APP-deficient cells manifest defects in neurite outgrowth and are more susceptible to Aβ-induced neuronal death at physiological levels of NGF. However, APP-deficient cells show better responses to NGF-stimulated differentiation and survival than control cells. This may be attributed to increased receptor endocytosis and enhanced activation of Akt and MAPK upon NGF stimulation in APP-deficient cells. Together, our results suggest that APP mediates endocytosis of NGF receptors through direct interaction, thereby regulating endocytosis of NGF and NGF-induced downstream signaling pathways for neuronal survival and differentiation.     

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.     

Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation

The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75–TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.     

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.     

Expression of nerve growth factors in pancreatic neural tissue and pancreatic cancer.

One of the characteristics of pancreatic cancer is its tendency to invade neural tissue. We hypothesized that the affinity of cancer cells for nerve tissue is related to the presence of growth factors in neural tissue and their receptors in cancer cells. Sections of pancreatic cancer and normal pancreatic tissue were examined by immunohistochemistry for the expression of the neurotrophins NGF, BDNF, NT-3, NT-4, and their receptors TrkA, TrkB, and TrkC, as well as the low-affinity receptor, p75NTR. TrkA expression was found in duct, islet, and cancer cells; TrkB was found in the alpha-cells of the islet only. The anti-pan-Trk antibody (TrkB3), which is presumed to recognize all three receptors, immunoreacted with duct and acinar cells in normal tissue and with cancer cells. The staining with TrkC was similar to that of TrkA. The low-affinity receptor p75NTR was expressed in the neural tissue and in scattered duct cells of the normal tissue only. Duct and acinar cells, as well as neural tissue and cancer cells, showed weak to strong immunoreactivity with NGF. NT-3 expression was noted in capillary endothelia and erythrocytes. NT-4 showed specific staining for ductule cells. The expression and distribution of neurotrophins and their receptors suggest their role in the potential of pancreatic cancer cells for neural invasion.     

神经生长因子及其受体相关信号传导通路的研究进展

神经生长因子是神经营养因子家族成员之一,对不同时期神经元的存活、分化、生长及损伤后的修复和再生都有着十分重要的作用。不仅在神经系统中,随着人类的其他正常和肿瘤组织中同样也检测得到了NGF,神经生长因子在各方面的应用也得到了重视并均已得到了证实。NGF功能的发挥离不开与其受体的结合,根据NGF表面糖蛋白与凝集素结合能力的不同,其受体可被分为高亲和力受体酪氨酸激酶A和低亲和力受体p75。Trk A与NGF结合后所介导的信号通路主要有:1MAPK通路;2PLC-γ通路;3PI3K/PKB通路。而p75与NGF结合介导的信号传导通路主要包括:1NF-κB通路;2JNK-p53-Bax凋亡通路;3神经酰胺通路。Trk A一般介导的是正性信号,如促进神经细胞生长、维持神经细胞的存活等;而p75既可促进神经细胞存活,也可诱导神经细胞凋亡,但以后者为主。当Trk A与p75同时表达时,Trk A可抑制p75诱导细胞凋亡,使受损神经细胞大量增殖,所以其生物学总效应是促进神经细胞的生长和存活。