Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 283–293, 1998     

Cloning, expression during development, and evidence for release of a trophic factor for ciliary ganglion neurons.

Ciliary ganglion (CG) neurons undergo a period of cell death during development that may be regulated by the limited availability of trophic factor produced by their target tissues. We have previously reported the purification of a ciliary neurotrophic factor from adult chick sciatic nerve that we called growth promoting activity (GPA). Here we demonstrate that GPA can be purified and cloned from embryonic day 15 (E15) chick eyes, which contain all the target tissues of the CG. Our studies show the following: GPA mRNA is induced in embryonic chick eyes during the period of CG neuron cell death; GPA mRNA is expressed specifically in the layer of the eye that contains the targets of the CG and in primary cultures of smooth muscle cells isolated from the choroid layer of the eye; and biologically active GPA is released from cells transfected with a GPA cDNA.     

Depolarization promotes survival of ciliary ganglion neurons by BDNF-dependent and -independent mechanisms

Membrane activity upregulates brain derived neurotrophic factor (BDNF) expression to coordinately support neuronal survival in many systems. In parasympathetic ciliary ganglion (CG) neurons, activity mimicked by KCl depolarization provides nearly full trophic support. While BDNF has been considered unable to influence CG neuronal survival, we now document its expression during CG development and show that low concentrations do support survival via high-affinity TrkB receptors. Furthermore, a contribution of BDNF to activity-induced trophic support was demonstrated by showing that KCl depolarization increased BDNF mRNA and protein in, and release of BDNF from, CG neuron cultures. Application of anti-BDNF blocking antibody or mitogen activated protein kinase (MAPK) kinase inhibitor, attenuated depolarization-supported survival, implicating canonical BDNF/TrkB signaling. Ca2+-Calmodulin kinase II (CaMKII) was also required since its inhibition combined with anti-BDNF or MAPK kinase inhibitor abolished or greatly reduced the trophic effects of depolarization. Membrane activity may thus support CG neuronal survival both by stimulating release of BDNF that binds high-affinity TrkB receptors to activate MAPK and by recruiting CaMKII. This mechanism could have relevance late in development in vivo as ganglionic transmission and the effectiveness of BDNF over other growth factors both increase.     

Role of cell-cell interactions in the developmental regulation of Ca2+-activated K+ currents in vertebrate neurons

The functional expression of the Ca²⁺-activated K⁺ current (I_K[Ca]) is dependent on cell-cell interactions in developing chick autonomic neurons. In chick ciliary ganglion (CG) neurons, expression of macroscopic I_K[Ca] coincides with the formation of synapses with target tissues. CG neurons that develop in vivo in the absence of normal target tissues fail to express functional I_K[Ca], although voltage-activated Ca²⁺ currents and most other ionic currents are expressed at normal amplitudes and densities. CG neurons placed in cell culture prior to formation of synapses with target tissues also fail to express macroscopic I_K[Ca]. However, CG neurons cultured in the presence of a heat- and trypsin-sensitive extract of target tissues express I_K[Ca] at normal levels. Similarly, interactions with target tissue appear to regulate the expression of whole-cell I_K[Ca] in developing chick sympathetic ganglion neurons, although the relevant trophic factors appear to be different from those required by CG neurons. In addition to target tissue interactions, an intact preganglionic innervation is required for the normal in vivo development of I_K[Ca] in chick CG neurons. The trophic effects of the afferent innervation do not require synaptic activation of the CG neurons, indicating secretion of a trophic factor, possibly an isoform of β-neuregulin. The results are consistent with the hypothesis that target- and nerve terminal-derived trophic factors interact at a posttranslational level in the regulation of a functional I_K[Ca]. Together, this body of data demonstrates an essential role for cell-cell interactions in the differentiation of neuronal excitability. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 23–36, 1998     

Differential effects of ret and TRKB on axonal branching and survival of parasympathetic neurons

Interactions between neurons and their targets of innervation influence many aspects of neural development. To examine how synaptic activity interacts with neurotrophic signaling, we determined the effects of blocking neuromuscular transmission on survival and axonal outgrowth of ciliary neurons from the embryonic chicken ciliary ganglion. Ciliary neurons undergo a period of cell loss due to programmed cell death between embryonic Days (E) 8 and 14 and they innervate the striated muscle of the iris. The nicotinic antagonist d‐tubocurarine (dTC) induces an increase in branching measured by counting neurofilament‐positive voxels (NF‐VU) in the iris between E14‐17 while reducing ciliary neuron survival. Blocking ganglionic transmission with dihyro‐β‐erythroidin and α‐methyllycacontine does not mimic dTC. At E8, many trophic factors stimulate neurite outgrowth and branching of neurons placed in cell culture; however, at E13, only GDNF stimulates branching selectively in cultured ciliary neurons. The GDNF‐induced branching at E13 could be inhibited by BDNF. Blocking ret signaling in vivo with a dominant negative (dn)ret decreases survival of ciliary and choroid neurons at E14 and prevents dTC induced increases in NF‐VU in the iris at E17. Blocking TRKB signaling with dn TRKB increases NF‐VU in the iris at E17 and decreases neuronal survival at E17, but not at E14. Thus, RET promotes survival during programmed cell death in the ciliary ganglion and contributes to promoting branching when synaptic transmission is blocked while TRKB inhibits branching and promotes maintenance of neuronal survival. These studies highlight the multifunctional nature of trophic molecule function during neuronal development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Interactions between neurons and their targets during in vivo synaptogenesis.

In vivo synaptogenesis is described in a simple vertebrate system, the chick ciliary ganglion, a parasympathetic autonomic ganglion. An attempt is made to integrate anatomical, physiological and biochemical observations during synapse formation in the ganglion and in the peripheral target structures; the iris, ciliary muscle, and smooth muscle of the choroid coat. The relationship between synaptogenesis and neuron survival is explored, and it is shown that a critically timed interaction between the neuron and target organ is necessary for full neuronal maturation and survival. The existence of an active competition between neurons for survival is documented, and the possible relationship between neuronal cell death and specificity of connections is discussed.     

Cell death and neuronal replacement during formation of the avian ciliary ganglion

Programmed cell death is a prominent feature of embryonic development and is essential in matching the number of neurons to the target tissues that are innervated. Although a decrease in neuronal number which coincides with peripheral synaptogenesis has been well documented in the avian ciliary ganglion, it has not been clear whether cell death also occurs earlier. We observed TUNEL-positive neurons as early as stage 24, with a large peak at stage 29. This cell death at stage 29 was followed by a statistically significant (P < 0.0001) decrease in total neuron number at stage 31. The total number of neurons was recovered by stage 33/34. This suggested that dying neurons were replaced by new neurons. This replacement process did not involve proliferation because bromodeoxyuridine applied at stages 29 and 31 was unable to label neurons harvested at stage 33/34. The peak of cell death at stage 29 was increased 2.3-fold by removal of the optic vesicle and was reduced by 50% when chCNTF was overexpressed. Taken together, these results suggest that the regulation of neuron number in the ciliary ganglion is a dynamic process involving both cell death and neural replacement from postmitotic precursors prior to differentiation and innervation of target tissues.     

Avian parasympathetic neurotrophic factors: Age-related increases and lack of regional specificity

Parasympathetic neurons from 8-day-old chick embryo ciliary ganglia were grown in culture for 24 hr in the presence of extracts of chick heart from animals aged 7 days in ovo to 8 days posthatching. The biological activity of these cardiac extracts with respect to both neuronal survival and nerve fiber production increased with age of the donor animal to reach a plateau around hatching. Following polyacrylamide gel isoelectric focusing of posthatching chick heart and eye, a peak in activity that supports parasympathetic neuronal survival was found associated with eluates of gel slices of pH between 4.5 and 5.5 for both tissues. Our results suggest that the factor responsible for parasympathetic neuronal survival is common to at least two parasympathetic target organs.     

Promotion of survival and neurite outgrowth of cultured peripheral neurons by exogenous lipids and detergents

Gangliosides, in particular the monosialoglycosphingolipids Gtet 1 (GM1), have previously been implicated in the mediation of neuronal rescue and restitutional axonal growth, both in vitro and subsequent to brain and peripheral nerve lesions. In the present study it is shown that the bis-sialosyl gangliosides Gtet2b and Gtet3b, but not the gangliosides Gtet2a and Gtet1, promote the survival of dissociated dorsal root ganglion (DRG) neurons cultured from Embryonic Day (E) 8 chicks (DRG8) almost to the same extent as nerve growth factor (NGF). Ciliary ganglion (CG) neurons from E8 chicks (CG8) and DRG10 neurons were virtually not supported suggesting considerable specificity in terms of neuronal targets and developmental stages being addressed. Moreover, a variety of other lipids including cerebroside (Cb), dipalmitoylphosphatidylcholine (DPPC) and -serine (DPPS), sulfatide (Sf), and sphingomyelin (Sm) were tested for putative survival promoting activity toward chick CG, DRG, and lumbar sympathetic ganglion (SG11) neurons. At the highest concentration employed (2.5 x 10(-5) M), Sm, DPPC, and DPPS maintained between 45 and 65% of the plateau survival with CG8 (maximally supported by ciliary neuronotrophic factor (CNTF], DRG8, and DRG10 neurons, and 30 to 40% with SG11 neurons. Cb supported CG8 neurons at about 55% of the plateau value achieved with CNTF, but had hardly any effect on the other neuron populations tested. Control experiments using highly enriched neurons and serum-free conditions assured that the effects were unlikely to be mediated by serum components or nonneuronal cells. A variety of detergents, in particular Triton X-100, also promoted the survival of CG8 and DRG10 neurons. Ganglioside Gtet1, Sm, and Triton X-100 shifted the NGF titration curve for DRG10 neurons between 6- and 15-fold in a dose-dependent manner suggesting synergisms between NGF and lipids for neuronal maintenance. These results document the neuronotrophic potency of certain gangliosides, a heterogeneous group of structurally unrelated lipids, and detergents. The mechanisms by which these agents modulate neuronal survival still await clarification.     

TGF-β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-β expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-β2 and -β3 as well as the TGF-β receptor TβR-II, but are not ir for TGF-β1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-β3 mRNA and TGF-β biological activity in cultures of E8 CG neurons. None of the TGF-β isoforms—β1, β2, or β3—has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-β significantly promotes CG neuron survival. However, TGF-β does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-β released from CG neurons using an antibody to TGF-β1/-β2/-β3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti–pan-TGF-β antibody could be rescued by adding exogenous TGF-β. Together, these data suggest that para-/autocrine TGF-β signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 563–572, 1998     

GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRalpha1 and Ret, but not of GFRalpha2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.     

Purification of the Chick Eye Ciliary Neuronotrophic Factor

Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons.     

Neuronal survival in intact ciliary ganglia in vivo and in vitro: Ciliary neuronotrophic factor as a target surrogate

Ciliary neuronotrophic factor (CNTF) requirements for neuronal survival in the intact ciliary ganglion (CG) have been investigated in organ culture. Exogenous CNTF was not essential for neuronal survival until embryonic Day 8. Three-day cultures from 5-day ganglia were similar with or without CNTF, showing numerous neurons and extensive neuritic development. In 3-day cultures from 8-day-old ganglia, however, no neurons survived without CNTF, and the ganglia contained only nonneuronal cells and cell debris. Similar ganglia cultured with CNTF contained many neurons, surrounded by nonneuronal cells, and abundant neuritic processes. Morphologic maturation of the neurons was less advanced in CNTF-supported ganglia than in their in vivo counterparts.     

Antiserum Against Neurite Outgrowth Factor in Chick Gizzard Extract and Its Inhibitory Effect on Neuritic Response in Cultured Ciliary Neurons

Abstract: Antiserum against a neurite outgrowth factor (NOF) of gizzard extract that promotes neurite outgrowth from dissociated ciliary ganglionic neurons (CG neurons) of 8-day-old chick embryo was prepared to determine whether or not the antiserum inhibits neurite outgrowth from cultured neurons or explants of chick and murine tissues. When CG neurons were cultured on a polyornithine-coated well exposed to NOF (NOF-bound POR well), marked neurite outgrowth was observed. When NOF-bound POR wells were exposed to antiserum, neurite outgrowth from CG neurons was gradually inhibited with increasing amounts of antiserum, while exposure to preimmune serum did not prevent neurite outgrowth. Antiserum had no effect on neuronal survival during a 48-h incubation. The diluted antiserum, which produced nearly 100% inhibition of the NOF activity, was almost equally active in suppressing the activity of NOFs in conditioned media (CM) of various chick embryo tissues, but showed much less inhibitory effects on NOFs in CM of murine tissues. The appearance of neurites from explants of spinal cord, dorsal root ganglion, or retina of chick embryo was also inhibited by the antiserum. These results indicate that antiserum against NOF from gizzard extract suppressed the activity of NOFs from various sources, and that there are species differences in NOFs, at least between chick and murine.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Cholinergic neuronotrophic factors: VI. Age-dependent requirements by chick embryo ciliary ganglionic neurons

During development, ciliary ganglionic neurons become postmitotic and extend neurites in apparent independence of the presence of their future intraocular innervation targets. After reaching their peripheral innervation territory, however, these neurons become target dependent and about half of them die. We have previously reported that chick embryo intraocular target tissues contain a ciliary neuronotrophic factor (CNTF), which can be extracted and partially purified in a soluble form and which ensures near-total survival of 8-day chick embryo ciliary ganglionic neurons in monolayer cultures. In this study we have dissociated and cultured ciliary ganglia from embryonic Day (ED) 5 through 14, and examined dependence and responsiveness of their neurons to exogenously added CNTF. Two cell classes (dark and bright) could be distinguished by phase microscopy and differentially counted in cell dissociates from ED7–14, but not in ED5–6 ones. Dark cell number per ganglion increased from 6000 to 78,000 over this developmental time period. In contrast, bright cells (putative neurons) declined from a maximum of about 10,000 to 6000, suggesting a correlation with the expected neuronal cell death in vivo. Dissociated cells from ED5–14 ganglia were seeded on a polyornithine substratum coated with neurite promoting factor, cultured for 24 hr with or without added CNTF, and numerically examined for survival and neuritic development. Cultures from ED7–14 ganglia showed two cell categories: (i) flat nonneuronal elements dramatically increased in number with ganglionic age (thereby correlating with the increasing number of dark cells in the dissociates) and (ii) large, bright cells (often displaying neurite outgrowth) decreased in number in parallel with bright cell number in the dissociate. The survival of these neuronal elements was strictly dependent on exogenously added CNTF between ED7 and 10, but became progressively independent with older ages. ED14 neurons (fully capable of surviving for 24 hr without added CNTF) continued to require CNTF for neurite extension, thus displaying retained sensitivity to this factor. Although the ED5–6 cultures contained well-recognizable flat cells, the dominant category comprised cells with variable morphology, practically all of which exhibited neurite-like processes. Both the survival and neurite extension of these cells, which we tentatively interpret as immature neurons were independent of the presence of added CNTF.     

Inhibition of protein synthesis prevents cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro

Shortly after neurons begin to innervate their targets in the developing vertebrate nervous system they become dependent on the supply of a neurotrophic factor, such as nerve growth factor (NGF) for survival. Recently, Martin et al. (1988) have shown that inhibiting protein synthesis prevents the death of NGF-deprived sympathetic neurons, suggesting that NGF promotes neuronal survival by suppressing an active cell death program. To determine if other neurotrophic factors may regulate neuronal survival by a similar mechanism we examined the effects of inhibiting protein and RNA synthesis in other populations of embryonic neurons that require different neurotrophic factors, namely: 1) trigeminal mesencephalic neurons, a population of proprioceptive neurons that are supported by brain-derived neurotrophic factor; 2) dorsomedial trigeminal ganglion neurons, a population of cutaneous sensory neurons that are supported by NGF; 3) and ciliary ganglion neurons, a population of parasympathetic neurons that are supported by ciliary neuronotrophic factor. Blocking either protein or RNA synthesis rescued all three populations of neurons from cell death induced by neurotrophic factor deprivation in vitro. Thus, at least three different neurotrophic factors appear to promote survival by a similar mechanism that may involve the suppression of an endogenous cell death program.     

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.     

Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2

The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present.