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.     

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.     

Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.     

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.     

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.     

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.     

Regulation of trafficking of activated TrkA is critical for NGF-mediated functions

Upon activation by nerve growth factor (NGF), TrkA is internalized, trafficked and sorted through different endosomal compartments. Proper TrkA trafficking and sorting are crucial events as alteration of these processes hinders NGF-mediated functions. However, it is not fully known which proteins are involved in the trafficking and sorting of TrkA. Here we report that Nedd4-2 regulates the trafficking of TrkA and NGF functions in sensory neurons. Depletion of Nedd4-2 disrupts the correct sorting of activated TrkA at the early and late endosome stages, resulting in an accumulation of TrkA in these compartments and, as a result of the reduced trafficking to the degradative pathway, TrkA is either reverted to the cell surface through the recycling pathway or retrogradely transported to the cell body. In addition, Nedd4-2 depletion enhances TrkA signaling and the survival of NGF-dependent dorsal root ganglion neurons, but not those of brain-derived neurotrophic factor-dependent neurons. Furthermore, neurons from a knock-in mouse expressing a TrkA mutant that does not bind Nedd4-2 protein exhibit increased NGF-mediated signaling and cell survival. Our data indicate that TrkA trafficking and sorting are regulated by Nedd4-2 protein.     

Rapid Retrograde Tyrosine Phosphorylation of trkA and Other Proteins in Rat Sympathetic Neurons in Compartmented Cultures

According to the current theory of retrograde signaling, NGF binds to receptors on the axon terminals and is internalized by receptor-mediated endocytosis. Vesicles with NGF in their lumina, activating receptors in their membranes, travel to the cell bodies and initiate signaling cascades that reach the nucleus. This theory predicts that the retrograde appearance of activated signaling molecules in the cell bodies should coincide with the retrograde appearance of the NGF that initiated the signals. However, we observed that NGF applied locally to distal axons of rat sympathetic neurons in compartmented cultures produced increased tyrosine phosphorylation of trkA in cell bodies/ proximal axons within 1 min. Other proximal proteins, including several apparently localized in cell bodies, displayed increased tyrosine phosphorylation within 5–15 min. However, no detectable ¹²⁵I-NGF appeared in the cell bodies/proximal axons within 30–60 min of its addition to distal axons. Even if a small, undetectable fraction of transported ¹²⁵I-NGF was internalized and loaded onto the retrograde transport system immediately after NGF application, at least 3–6 min would be required for the NGF that binds to receptors on distal axons just outside the barrier to be transported to the proximal axons just inside the barrier. Moreover, it is unlikely that the tiny fraction of distal axon trk receptors located near the barrier alone could produce a measurable retrograde trk phosphorylation even if enough time was allowed for internalization and transport of these receptors. Thus, our results provide strong evidence that NGF-induced retrograde signals precede the arrival of endocytotic vesicles containing the NGF that induced them. We further suggest that at least some components of the retrograde signal are carried by a propagation mechanism.     

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.     

Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons.

Ceramide inhibits axonal growth of cultured rat sympathetic neurons when the ceramide content of distal axons, but not cell bodies, is increased (Posse de Chaves, E. I., Bussiere, M. Vance, D. E., Campenot, R. B., and Vance, J.E. (1997) J. Biol. Chem. 272, 3028-3035). We now report that inhibition of growth does not result from cell death since although ceramide is a known apoptotic agent, C(6)-ceramide given to the neurons for 24 h did not cause cell death but instead protected the neurons from death induced by deprivation of nerve growth factor (NGF). We also find that a pool of ceramide generated from sphingomyelin in distal axons, but not cell bodies, inhibits axonal growth. Analysis of endogenous sphingomyelinase activities demonstrated that distal axons are rich in neutral sphingomyelinase activity but contain almost no acidic sphingomyelinase, which is concentrated in cell bodies/proximal axons. Together, these observations are consistent with the idea that generation of ceramide from sphingomyelin by a neutral sphingomyelinase in axons inhibits axonal growth. Furthermore, we demonstrate that treatment of distal axons with ceramide inhibits the uptake of NGF and low density lipoproteins by distal axons by approximately 70 and 40%, respectively, suggesting that the inhibition of axonal growth by ceramide might be due, at least in part, to impaired endocytosis of NGF. However, inhibition of endocytosis of NGF by ceramide could not be ascribed to decreased phosphorylation of 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.     

NGF and the local control of nerve terminal growth

It is generally believed that the mechanism of action of neurotrophic factors involves uptake of neurotrophic factor by nerve terminals and retrograde transport through the axon and back to the cell body where the factor exerts its neurotrophic effect. This view originated with the observation almost 20 years ago that nerve growth factor (NGF) is retrogradely transported by sympathetic axons, arriving intact at the neuronal cell bodies in sympathetic ganglia. However, experiments using compartmented cultures of rat sympathetic neurons have shown that neurite growth is a local response of neurites to NGF locally applied to them which does not directly involve mechanisms in the cell body. Recently, several NGF-related neurotrophins have been identified, and several unrelated molecules have been shown to act as neurotrophic or differentiation factors for a variety of types of neurons in the peripheral and central nervous systems. It has become clear that knowledge of the mechanisms of action of these factors will be crucial to understanding neurodegenerative diseases and the development of treatments as well as the means to repair or minimize neuronal damage after spinal injury. The concepts derived from work with NGF suggest that the site of exposure of a neuron to a neurotrophic factor is important in determining its response. 1994 John Wiley & Sons, Inc.     

NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Target-derived NGF promotes the phenotypic maintenance of mature dorsal root ganglion (DRG) nociceptive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of endosomes containing NGF, activated TrkA, and signaling proteins of the Rap1/Erk1/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erk1/2, p-p38, and pAkt in these membranes. Conversely, NGF antibody injections decreased the retrograde transport of p-Erk1/2 and p-p38. Our results are evidence that signaling endosomes, with the characteristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

Role of nerve growth factor in plasticity of forebrain cholinergic neurons

Neuronal plastic rearrangements during the development and functioning of neurons are largely regulated by trophic factors, including nerve growth factor (NGF). NGF is also involved in the pathogenesis of Alzheimer’s disease. In the brain, NGF is produced in structures innervated by basal forebrain cholinergic neurons and retrogradely transported along the axons to the bodies of cholinergic neurons. NGF is essential for normal development and functioning of the basal forebrain; it affects formation of the dendritic tree and modulates the activities of choline acetyltransferase and acetylcholinesterase in basal forebrain neurons. The trophic effect of NGF is mediated through its interactions with TrkA and p75 receptors. Experimental and clinical studies have shown that brain levels of NGF are altered in various pathologies. However, the therapeutic use of NGF is limited by its poor ability to penetrate the blood–brain barrier, adverse side effects that are due to the pleiotropic action of this factor, and the possibility of immune response to NGF. For this reason, the development of gene therapy methods for treating NGF deficit-associated pathologies is of particular interest. Another approach is creation of low molecular weight NGF mimetics that would interact with the corresponding receptors and display high biological activity but be free of the unfavorable effects of NGF.