Search

Fusion proteins comprising the Fc domain of human IgG and extracellular domains of receptor tyrosine kinases can neutralize the activity of their cognate ligands when administered in molar excess. We have generated a fusion protein using the ectodomain of TrkB (TrkB-Fc). Although the ability of TrkB-Fc to neutralize the activity of brain-derived neurotrophic factor (BDNF) in vitro has been demonstrated, there have been no conclusive demonstrations of its ability to neutralize the activity of BDNF in vivo. We co-infused TrkB-Fc with BDNF into the cortex and hippocampus of adult rats to determine whether TrkB-Fc would interfere with the ability of BDNF to upregulate neuropeptide Y (NPY). We report here that rather than neutralizing the activity of exogenous BDNF, co-infusion with the TrkB-Fc fusion protein greatly increased the volume of tissue in which neuropeptide Y immunostaining was upregulated. In addition, TrkB-Fc greatly enhanced BDNF's distribution through adult brain parenchyma. TrkB-Fc also markedly increased the otherwise limited diffusion of BDNF into brain parenchyma following intraventricular infusion. These results show that rather than neutralizing or sequestering BDNF, the TrkB-Fc, at close to molar equivalence to BDNF, can function as a carrier for BDNF and thus enhance the delivery or penetration of this polypeptide into the brain.

In addition to the known retrograde transport of neurotrophins, it is now evident that endogenous brain-derived neurotrophic factor (BDNF) is transported in the anterograde direction in peripheral and central neurons. We used a double-ligation procedure that distinguishes between anterograde and retrograde flow to quantify the anterograde transport of endogenous neurotrophins and neuropeptides in the peripheral nervous system before and after axotomy. BDNF accumulation proximal to the ligation (anterograde transport) was twice that distal to the ligation (retrograde direction). Anterograde transport of nerve growth factor and neurotrophin-3 was not evident. Furthermore, BDNF anterograde transport increased 3.5-fold within 24 hr after sciatic nerve injury or dorsal rhizotomy. Anterograde transport of substance P and calcitonin gene-related peptide decreased after peripheral nerve lesion, demonstrating that there was no generalized increase in anterograde transport. To determine the source of the anterogradely transported BDNF, we performedin situhybridization in a variety of tissues before and after axotomy. Expression of BDNF mRNA in proximal nerve segments did not change with treatment, showing that the increased accumulation of BDNF was not a result of increased local synthesis. BDNF mRNA and protein were expressed by dorsal root ganglion sensory neurons but not by motor neurons. BDNF mRNA expression was increased 1 d after nerve injury, and BDNF protein was also increased twofold to threefold, suggesting that sensory neurons are the major contributing source of the increased BDNF traffic in the sciatic nerve. Our results suggest that increased anterogradely transported BDNF plays a role in the early neuronal response to peripheral nerve injury at sites distal to the cell body.

The role of neurotrophins as target-derived proteins that promote neuron survival following their retrograde transport from the terminals to the cell bodies of neurons has been firmly established in the developing peripheral nervous system. However, neurotrophins appear to have more diverse functions, particularly in the adult central nervous system. Brain-derived neurotrophic factor (BDNF), for example, produces a variety of neuromodulatory effects in the brain that are more consistent with local actions than with long-distance retrograde signalling. Here we show that BDNF is widely distributed in nerve terminals, even in brain areas such as the striatum that lack BDNF messenger RNA, and that inhibition of axonal transport or deafferentation depletes BDNF. The number of striatal neurons that contain the calcium-binding protein parvalbumin was decreased in BDNF+/- and BDNF-/- mice in direct proportion to the loss of BDNF protein, which is consistent with anterogradely supplied BDNF having a functional role in development or maintenance. Thus the anterograde transport of BDNF from neuron cell bodies to their terminals may be important for the trafficking of BDNF in the brain.

TIE2 is a receptor-like tyrosine kinase expressed almost exclusively in endothelial cells and early hemopoietic cells and required for the normal development of vascular structures during embryogenesis. We report the identification of a secreted ligand for TIE2, termed Angiopoietin-1, using a novel expression cloning technique that involves intracellular trapping and detection of the ligand in COS cells. The structure of Angiopoietin-1 differs from that of known angiogenic factors or other ligands for receptor tyrosine kinases. Although Angiopoietin-1 binds and induces the tyrosine phosphorylation of TIE2, it does not directly promote the growth of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates Angiopoietin-1 in endothelial developmental processes.

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Neurotrophic factors constitute a class of molecules that are now considered critical for the development, maintenance and regeneration of the nervous system. Much of the conceptual framework surrounding the suspected function of neurotrophic factors has emerged from studies of the prototypical neurotrophic factor—nerve growth factor (NGF). In this review we will compare established properties of NGF with recent studies on the biology of brain-derived neurotrophic factor (BDNF), an NGF-related neurotrophin, and an unrelated factor, ciliary neurotrophic factor (CNTF), not only in the context of the diverse roles of these factors in nervous system development and maintenance but also in terms of the therapeutic potential of neurotrophic factors.

Neurotrophin-4 (NT-4) is the most recently discovered member of the neurotrophin family. We have expressed, refolded, and purified recombinant human NT-4 from Escherichia coli and compared it with recombinant human NT-4 secreted into the culture medium of baculovirus-infected insect cells. Both preparations were characterized and determined to be indistinguishable according to several biochemical criteria. Recombinant NT-4 from E. coli was crystallized in a form suitable for x-ray analysis, and characterization of these crystals indicated that NT-4 was present as a dimer within the asymmetric unit. NT-4 was active in promoting the survival of rat TrkB receptor-expressing fibroblasts, but was inactive on embryonic chicken sensory neurons, unlike the other members of the neurotrophin family and in contrast to the reported activities of partially purified NT-4.

Polyclonal antibodies raised against mouse 2.5S NGF (mNGF) and against synthetic peptides made from hydrophilic portions of mNGF have been used to compare the immunological properties of mNGF, human recombinant brain-derived neurotrophic factor (hrBDNF), and human recombinant neurotrophin-3 (hrNT-3). Affinity-isolated antibodies raised against intact mNGF reacted with all three neurotrophins when tested by ELISA and totally or partially blocked the bioactivities of the proteins in survival assays of embryonic chicken sensory and sympathetic neurons. On Western blots, mNGF antibodies reacted with all three neurotrophins but less well with hrBDNF and hrNT-3 than with mNGF. Antibodies to hydrophilic peptides within NGF (amino acids 23–35, 59–67, 69–79, and 91–100) showed partial reactivity with some but not all of the neurotrophins when tested by ELISA and on Western blots. The peptide antibodies were also selectively effective in reducing the survival- promoting activity of the neurotrophins on sensory neurons. Results show that mNGF, hrBDNF, and hrNT-3 are immunologically related proteins and that mNGF antibodies react also with other members of the neurotrophin family.

Cholinergic properties are induced in sympathetic neurons by several factors applied to entire neurons in culture. Evidence from work with the rat sweat gland model indicates that factors located in target tissues can induce cholinergic differentiation in vivo. We now report that when leukemia inhibitory factor (LIF), heart cell-conditioned medium (HCCM), or dermal fibroblast-conditioned medium (DFCM) is applied to only distal neurites in compartmented cultures of rat sympathetic neurons, the neurons exhibit an increase in specific choline acetyltransferase activity and a concomitant decrease in levels of tyrosine hydroxylase. LIF, HCCM, and DFCM also induce neurite fasciculation, thus suggesting an additional role of cholinergic switching factors in regulating axon-axon and/or axon-substrate adhesion. These results demonstrate that rat sympathetic neurons have the cellular machinery to respond to cholinergic differentiation cues located in peripheral targets, analogous to the response to nerve growth factor.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.

In contrast to all other molecules which are labelled 'growth factor', NGF is not a mitogen. It is a neurotrophic molecule essential for the development and maintenance of function of specific populations of peripheral and possibly also central neurons. The availability of NGF in large quantities from exocrine glands (e.g. male mouse submandibular gland), where NGF does not play a neurotrophic role, has allowed the purification of NGF, the production of specific antibodies, the determination of its amino acid sequence and finally the molecular cloning of NGF leading to the elucidation of its precursor structure and its genomic organization. Comparison of the biological activities and the immunological properties of NGF isolated from different sources demonstrated that the active centre of the molecule has been highly conserved during evolution, whereas other parts of the molecule determining immunological properties have undergone considerable changes. After a survey of the essential biological actions of NGF, this paper concentrates on two actual questions of NGF research, namely the regulation of NGF synthesis in the target tissues of NGF-responsive neurons, and the molecular mechanism(s) of action of NGF on these neurons.

Angiogenesis is required for tumor growth and metastasis, and inhibition of angiogenesis is a promising approach for anticancer therapy. Tie2 (a.k.a Tek) is an endothelium-specific receptor tyrosine kinase known to play a role in tumor angiogenesis. To explore the therapeutic potential of blocking the Tie2 pathway, an adenoviral vector was constructed to deliver a recombinant, soluble Tie2 receptor (AdExTek) capable of blocking Tie2 activation. Two days after i.v. injection of AdExTek, the plasma concentration of ExTek exceeded 1 mg/ml and was maintained for about 8 days. Administration of AdExTek to mice with two different well established primary tumors, a murine mammary carcinoma (4T1) or a murine melanoma (B16F10.9), significantly inhibited the growth rate of both tumors (64% and 47%, respectively). To study the effect of ExTek on tumor metastasis, both tumor cell lines were coinjected i.v. with either AdExTek or a control virus. Mice coinjected with control virus developed numerous large, well vascularized lung metastases. In contrast, mice coinjected with AdExTek virus developed few, if any, grossly apparent metastases, and histologic examination revealed only small avascular clusters of tumor cells. Administration of AdExTek also inhibited tumor metastasis when delivered at the time of surgical excision of primary tumors in a clinically relevant model of tumor metastasis. This study demonstrates the potential utility of gene therapy for systemic delivery of an antiangiogenic agent targeting an endothelium-specific receptor, Tie2.

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), members of distinct families of polypeptide growth factors, have been shown to support motoneurons under various in vitro and in vivo conditions. We used a model of motoneuron cell death induced by sciatic nerve section in newborn rats and compared the efficacy of BDNF and GDNF administered alone or simultaneously in order to determine whether combinations of neurotrophic proteins can produce more potent motoneuron rescue than individual factors. The factors were administered by different methods, including (i) a single dose on to the transected nerve, (ii) continuous delivery from implanted slow-release polymer rods (BDNF) or encapsulated cells (GDNF), and (iii) repeated systemic injections (BDNF). Irrespective of the method of administration, either factor alone produced rescue effects which dramatically declined at two weeks as compared to one week post-lesion. In contrast, this decrease was significantly reduced when BDNF and GDNF were used simultaneously provided that one factor was applied on to the nerve while the other was continuously released from the rods or capsules. Other combinations in which GDNF was replaced by ciliary neurotrophic factor or axokine-1 failed to reproduce such additive activity. Two conclusions can be made from these experiments. First, when BDNF and GDNF are administered simultaneously but by distinct routes of delivery, their survival-promoting effects on the injured developing motoneurons are potentiated; second, even continuous delivery of each of these trophic factors alone cannot completely abrogate the time-dependent decline in rescue effects in this model of motoneuron cell death.

Galactose intoxication of rats was used to disrupt metabolism of Schwann cells and skeletal muscle, two sites that contain the polyol-forming enzyme aldose reductase (AR). Galactose-fed rats develop a neuropathy characterized by nerve conduction deficits and axonal atrophy. To investigate the possibility that galactose metabolism by AR influences axonal function and structure by altering production of neurotrophic factors, the impact of galactose intoxication on nerve and muscle BDNF levels and the effects of exogenous BDNF treatment on galactose neuropathy were examined using biochemical, electrophysiologic and morphometric techniques. Galactose feeding increased BDNF protein in peripheral nerve and muscle. Exogenous BDNF treatment attenuated motor nerve conduction velocity deficits in the sciatic nerve of galactose-fed animals and myelin splitting of motor axons in the ventral root. In contrast, sensory nerve conduction velocity (SNCV) deficits in the sciatic nerve and myelin splitting in the central projections of sensory neurons were not prevented by BDNF treatment. BDNF treatment did not attenuate reduced axonal caliber in the sciatic nerve, but did ameliorate the diminution of the caliber of central sensory projections in the dorsal root. These findings point to the potential use of BDNF in the treatment of peripheral neuropathies.

The impact of exaggerated polyol pathway flux on ciliary neurotrophic factor (CNTF)-like bioactivity and expression of CNTF in rat sciatic nerve was examined after 2 months of galactose intoxication. Polyol content was elevated (P < 0.001) and motor nerve conduction velocity reduced (P < 0.05) in galactose-fed rats compared with control animals or control and galactose-fed rats treated with the aldose reductase inhibitor (ARI) Ponalrestat. CNTF-like bioactivity in the galactose-fed group was reduced to 30% of that assayed in the control group (P < 0.001). ARI treatment significantly increased CNTF-like bioactivity by 60% compared with the untreated galactose group (P < 0.05) but did not restore it to control levels. Unexpectedly, bioactivity in ARI-treated control animals was increased by nearly 250% compared with untreated controls (P < 0.005). In addition to the deficit in CNTF bioactivity in untreated galactose rats, the expression of protein, but not of mRNA, was reduced (P < 0.05). In ARI-treated control and galactose-fed rats, the expression of CNTF peptide was significantly enhanced above control levels (both P < 0.05). Concomitant with the reduction in CNTF levels, there was a shift in the axonal size-frequency distribution of myelinated fibers toward smaller axons in galactose-fed rats that was prevented by ARI treatment. Since galactose feeding has little impact on levels of CNTF mRNA, these observations suggest that deficits in CNTF-like bioactivity may result from a posttranscriptional modification of neurotrophic protein expression or turnover. Unlike other functional and structural disorders in galactose neuropathy, factors other than polyol accumulation may contribute to the deficit in CNTF-like bioactivity.

By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

During the initial phase of their development, sensory neurons of the dorsal root ganglion (DRG) require target-derived trophic support for their survival, but as they mature they lose this requirement. Because many of these neurons express BDNF (brain-derived neurotrophic factor) messenger RNA, we hypothesized that BDNF might act as an autocrine survival factor in adult DRG neurons, thus explaining their lack of dependence on exogenous growth factors. When cultured adult DRG cells were treated with antisense oligonucleotides to BDNF, expression of BDNF protein was reduced by 80%, and neuronal survival was reduced by 35%. These neurons could be rescued by exogenous BDNF or neurotrophin-3, but not by other growth factors. Similar results were obtained with single-neuron microcultures, whereas microcultures derived from mutant mice lacking BDNF were unaffected by antisense oligonucleotides. Our results strongly support an autocrine role for BDNF in mediating the survival of a subpopulation of adult DRG neurons.

Human and rat ciliary neurotrophic factors (CNTF), which share 85% sequence identity, promote the survival of chicken embryo ciliary ganglia neurons in vitro, but display a 4-5-fold difference in specific activity. To explore the origin of this difference and gain insight into the structural organization of CNTF, we created chimeric proteins of these two species. Surprisingly, we found that the differences in two apparently unrelated properties, gel mobility and specific activity, resided in a single amino acid. Substituting arginine residue 63 of rat CNTF into the human sequence created a protein with the properties of rat CNTF. Conversely, substituting the human CNTF glutamine residue 63 into rat CNTF generated a protein with the properties of human CNTF. Binding experiments confirmed that the distinct specific activities of human and rat CNTF and their chimeras reside in structural differences among these ligands rather than species differences in their receptors. Alanine substitution (Q63A) had no effect on the properties of human CNTF, whereas the R63A substitution reduced both the gel mobility and the specific activity of rat CNTF. Finally, a Q95R substitution at a different position of human CNTF had no effect on its properties. These results demonstrate that Arg-63 is both specific and critical in determining the structural differences of human and rat CNTF.

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.

Primary cultures of bovine adrenal chromaffin cells provide large quantities of a homogeneous population of target cells for nerve growth factor (NGF) and, thus, are a suitable system for studying the molecular mechanism of action of NGF. In this study, we have shown that NGF mediates the specific induction of the key enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). Acetylcholinesterase (AChE), an enzyme which catalyzes the breakdown of acetylcholine, is also induced by NGF. We have compared NGF-mediated TH and AChE induction and have provided pharmacological evidence that TH induction involves a post-transcriptional, polyadenylation-dependent event (blockable by 9-beta-arabinofuranosyladenine but not by alpha- amanitin), whereas AChE induction requires transcription (blockable by alpha-amanitin). DBH and PNMT appear to be regulated via the same mechanism as TH. The time course of TH induction is such that NGF must be continuously present for at least the first 36 hr (during which time TH levels remain unchanged), and then the entire increase takes place during the subsequent 12 hr. In contrast, AChE induction proceeds linearly with time of NGF exposure. These data suggest that there may be multiple mechanisms by which NGF regulates enzyme induction. We have also compared the effects of cAMP with those of NGF. As compared to NGF, cAMP produces a different pattern of enzyme induction (in addition to TH, DBH, PNMT, and AChE, dopa decarboxylase (DDC) is also induced), it acts rapidly (a 12-hr exposure produces the full effect), and it acts only at the transcriptional level (its effects are blocked by alpha-amanitin). These data provide evidence that cAMP does not act as a second messenger for NGF with regard to enzyme induction.

We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5–60 min) results in TH activation, which involves a change in the Fmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC 12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5′-deoxy-5′-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.

The neural cell adhesion molecule (NCAM) can influence a number of diverse intercellular events, including junctional communication, the association of axons with pathways and targets, and signals that alter levels of neurotransmitter enzymes. These pleiotropic effects appear to reflect the ability of NCAM to regulate membrane-membrane contact required to initiate specific interactions between other molecules. Such regulation can occur through changes in either NCAM expression or the molecule's content of polysialic acid (PSA). When NCAM with a low PSA content is expressed, adhesion is increased and contact-dependent events are triggered. In contrast, the large excluded volume of NCAM PSA can inhibit cell-cell interactions through hindrance of overall membrane apposition.

Elevated concentrations of K+ (35 mM) have previously been shown to support the survival of most embryonic chick sympathetic neurons in vitro (Wakade et al., Exp cell res 144 (1983) 377, [23]) and to be interchangeable with nerve growth factor (NGF) as a survival-promoting agent for these cells (Wakade & Thoenen, Neurosci lett 45 (1984) 71 [21]). In the present study, we show that dorsal root ganglion (DRG) neurons from embryonic day 6 do not survive in the presence of high K+, although both NGF and brain-derived neurotrophic factor (BDNF) each support the survival of more than 50% of the cells at this developmental stage. At E6, high K+ appears to have a cytotoxic effect on BDNF-dependent neurons, and there is also considerable inhibition of neurite outgrowth. At a later developmental stage (E12), high K+ supports the survival of about 40% of DRG cells. This subpopulation of neurons is distinct from that supported by NGF (as evidenced by the additivity of these two agents), but partially overlaps with that supported by BDNF (i.e., the two agents are less than additive). At E12, only approx. 20% of the cells can be supported by either NGF or BDNF, with the rest depending exclusively on one or the other of these factors. This is in contrast to the situation at E6, where there is considerable overlap between NGF- and BDNF-dependent populations.

Nerve growth factor (NGF) is a well-defined protein which is necessary for the development and maintainance of differentiated properties of peripheral sympathetic and neural crest-derived sensory neurons (for review see Thoenen et al., 1985 and refs therein). Many morphological and biochemical effects of NGF have been described in both the physiological target cells of NGF and a clonal cell line (PC12) derived from a rat pheochromocytoma. NGF is known to bind to specific cell-surface receptors, an event which is followed by internalization of the ligand-receptor complex; however, the mechanism of signal transduction after NGF’s interaction with its receptor is unknown.