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.     

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.     

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.     

Retrograde neurotrophin signaling: Trk-ing along the axon

Target-derived neurotrophins are required for the growth and survival of innervating neurons. When released by postsynaptic targets, neurotrophins bind receptors (Trks) on nerve terminals. Activated Trks signal locally within distal axons and retrogradely through long axons to distant cell bodies in order to promote gene expression and survival. Although the mechanism of retrograde neurotrophin signaling is not fully elucidated, considerable evidence supports a model in which the vesicular transport of neurotrophin-Trk complexes transmits a survival signal that involves PI3K and Erk5. Other, non-vesicular modes of retrograde signaling are likely to function in parallel. Recent studies highlight the importance of the location of stimulation as a determinant of Trk signaling. Defects in signaling from distal axons to cell bodies may be causally related to neurodegenerative disorders.     

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.     

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.     

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.     

Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.     

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.     

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.     

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.     

Analysis of Ret knockin mice reveals a critical role for IKKs, but not PI 3-K, in neurotrophic factor-induced survival of sympathetic neurons

We analyzed the survival responses and downstream signaling elicited by GDNF on sympathetic neurons from different Ret knockin mice. Lack of tyrosine 1062, a multidocking site in Ret, completely prevented GDNF-mediated survival. Importantly, lack of tyrosine 981, although abrogating Akt phosphorylation, had no effect on neuronal survival, indicating that the PI 3-K/Akt pathway is not necessary for survival of sympathetic neurons. In contrast, silencing of B-Raf completely prevented not only GDNF-mediated but also NGF-mediated cell survival, independently of MEK-1/2. We identified IKKs as the main effectors of the protective effects of B-Raf. First, B-Raf interacted with and activated IKKs. Second, knockdown of IKKs reversed the protection afforded by a constitutively active form of B-Raf. Third, knockdown of IKKs prevented both NGF- and GDNF-mediated survival. In conclusion, our data delineate a novel survival pathway for sympathetic neurons linking B-Raf to IKKs, independently of both PI 3-K and MEK-1/2 pathways.     

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.     

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.     

Monokine induced by interferon-gamma acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons

We found that a monokine induced by interferon-gamma (Mig, CXCL9), which belongs to the CXC chemokine subfamily, acts as a neurotrophic factor on PC12 cells and rat primary sympathetic neurons. PC12 cells were shown to express a single class of high affinity binding sites for Mig (670 receptors/cell, Kd = 2.9 nm). Mig induced neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of extracellular signal-regulated kinase signaling pathways between Mig and nerve growth factor (NGF) revealed that these pathways are crucial for Mig action as well as NGF. K252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks) did not inhibit the action of Mig, suggesting that Mig action occurs via a different receptor from that of NGF. Furthermore, Mig as well as NGF promoted PC12 survival under serum-free conditions and activated Akt/protein kinase B downstream from phosphatidylinositol 3-kinase (PI3K). Because the PI3K inhibitor LY294002 prevented the Mig- and NGF-induced survival effect, this effect is probably mediated by the PI3K signaling pathway. Mig also promoted survival of rat primary sympathetic neurons that die when deprived of NGF. These results suggest that chemokines, including Mig (CXCL9) have neurotrophic effects on the nervous system.     

Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.     

NGF augments the autophosphorylation of Ret via inhibition of ubiquitin-dependent degradation

Nerve growth factor (NGF) is required for the trophic maintenance of postnatal sympathetic neurons. A significant portion of the growth-promoting activity of NGF is from NGF-dependent phosphorylation of the heterologous receptor tyrosine kinase, Ret. We found that NGF applied selectively to distal axons of sympathetic neurons maintained in compartmentalized cultures activated Ret located in these distal axons. Inhibition of either proteasomal or lysosomal degradation pathways mimicked the effect of NGF on Ret activation. Likewise, NGF inhibited the degradation of Ret induced by glial cell line-derived neurotrophic factor-dependent activation, a process that requires ubiquitination and proteasomal degradation. NGF induced the accumulation of autophosphorylated Ret predominantly in the plasma membrane, in contrast to GDNF, which promoted the internalization of activated Ret. An accretion of monoubiquitinated, but not polyubiquitinated, Ret occurred in NGF-treated neurons, in contrast to glial cell line-derived neurotrophic factor that promoted the robust polyubiquitination of Ret. Thus, NGF stimulates Ret activity in mature sympathetic neurons by inhibiting the ongoing ubiquitin-mediated degradation of Ret before its internalization and polyubiquitination.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

Uptake of lipoproteins for axonal growth of sympathetic neurons

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.