A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.     

A muscle-derived substrate-bound factor that promotes neurite outgrowth from neurons of the central and peripheral nervous systems

The development and survival of spinal motor neurons depends upon muscle-derived trophic factors. Some circumstantial evidence suggested to us that the regulatory subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase (cAMP-dPK)-type II might be involved in neuritic outgrowth from spinal neurons. In the present study, we tested a commercial preparation of cAMP-dPK for neurite-promoting activity. Commercial cAMP-dPK-type II from skeletal and cardiac muscles elicited a significant neurite outgrowth from cultured embryonic chicken neurons when the enzyme preparation was bound to polylysine-coated substrata; type I cAMP-dPK from skeletal muscle was ineffective. Neither cAMP-dPK-type I nor -type II had a significant effect on the survival of spinal neurons in culture. Type II cAMP-dPK also stimulated neurite outgrowth from chicken cerebral hemisphere neurons, dorsal root ganglionic neurons, ciliary ganglionic neurons, and rat sympathetic ganglionic neurons in culture. The neurite-promoting activity appears to reside in a contaminant of the preparation since neither the purified regulatory nor catalytic subunits of cAMP-dPK-type II had an effect on neurite outgrowth per se from cultured neurons and since neurite-promoting activity did not correlate with [3H]cAMP binding or cAMP-dependent kinase activity. The neurite-promoting protein was then partially purified from commercial cAMP-dPK-type II by gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DE-52 cellulose. Sodium dodecyl sulfate gel electrophoresis of the active protein peak revealed a major protein band (MW 50 kDa) and several minor bands (e.g., MW 200 kDa, 52 kDa, 45 kDa). Also, immunoblot analysis and immunoprecipitation revealed that the partially purified neurite-promoting protein was distinct from laminin, heparan sulfate proteoglycan, nerve growth factor, neural cell adhesion molecule, and fibronectin. Furthermore, the neurite-promoting activity was not diminished by treatment with heparinase nor was it bound to heparin conjugated to Sepharose. Our results demonstrate that a protein unrelated to laminin or its associated macromolecules and which copurifies with the type II cAMP-dPK of striated muscle stimulates neurite outgrowth from neurons of the central and peripheral nervous systems.     

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).     

Characterization of a neuronal growth factor from mouse heart-cell-conditioned medium

A fraction of medium conditioned by embryonic mouse heart cells in culture promotes the growth of sympathetic and parasympathetic neurons in vitro. The factor stimulates neurite outgrowth, elevates specific activities of tyrosine hydroxylase and choline acetyltransferase in sympathetic ganglion explants, and enhances survival of dissociated sympathetic neurons in culture. The growth-promoting activity, which has a profound effect on survival of mouse sympathetic and parasympathetic neurons but little effect on mouse sensory neuron survival, is sensitive to trypsin and elevated temperature, suggesting association with a polypeptide or protein. Unlike nerve growth factor (NGF), the conditioned medium fraction is insensitive to anti-NGF antiserum, and fosters growth of mouse parasympathetic neurons. Consequently, the conditioned medium appears to contain a new nerve growth-promoting factor.     

Neurite-promoting activities for embryonic spinal neurons and their developmental changes in the chick

Spinal motoneurons may depend upon muscle-derived factors for axon outgrowth and stabilization at two principal stages of their development: during the initial invasion of the differentiating muscle masses in the embryo and during the perinatal regression of multiple innervation. Using a bioassay involving the measurement of neurite outgrowth from 4.5-day embryonic chick spinal neurons in dissociated cell culture, neurite-promoting activities were detected both in medium conditioned over embryonic chicken myotubes in vitro (embryonic muscle-conditioned medium) and in soluble extracts of chick leg muscle prepared 3-5 days after hatching (postnatal muscle extract). The molecules responsible for these two activities had physicochemical properties that distinguished them both from each other and from some other reported neurite-promoting factors. The factor in embryonic muscle-conditioned medium, although active on uncoated tissue culture wells, bound with only low affinity to tissue culture plastic under cell culture conditions. It was inactivated by incubation with trypsin, and was essentially found only in media conditioned by muscle and liver cells. The factor in PNME, on the other hand, bound to plastic culture wells and was found in extracts of a variety of tissues. Its concentration in postnatal leg muscle was developmentally regulated: the specific activity increased approximately 10-fold between hatching and Day 3 (maximum value: 3200 units/mg protein) and then fell back to nearly its original levels by Day 7. Evidence is presented that the observed effects of these two neurite-promoting factors did not result from differential survival in vitro of different cell subpopulations. Possible roles for the two active factors during motoneuron development are discussed.     

Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor

The response of embryonic chick nodose ganglion (neural placode-derived) and dorsal root ganglion (neural crest-derived) sensory neurons to the survival and neurite-promoting activity of brain-derived neurotrophic factor (BDNF) was studied in culture. In dissociated, neuron-enriched cultures established from chick embryos between Day 6 (E6) and Day 12 (E12) of development, both nodose ganglion (NG) and dorsal root ganglion (DRG) neurons were responsive on laminin-coated culture dishes to BDNF. In the case of NG, BDNF elicited neurite outgrowth from 40 to 50% of the neurons plated at three embryonic ages; E6, E9, and E12. At the same ages, nerve growth factor (NGF) alone or in combination with BDNF, had little or no effect upon neurite outgrowth from NG neurons. The response of NG neurons to BDNF was dose dependent and was sustainable for at least 7 days in culture. Surprisingly, in view of a previous study carried out using polyornithine as a substrate for neuronal cell attachment, on laminin-coated dishes BDNF also sustained survival and neurite outgrowth from a high percentage (60-70%) of DRG neurons taken from E6 embryos. In marked contrast to NG neurons, the combined effect of saturating levels of BDNF and NGF activity on DRG neurons was greater than the effect of either agent alone at all embryonic ages studied. Under similar culture conditions, BDNF did not elicit survival and neurite outgrowth from paravertebral chain sympathetic neurons or parasympathetic ciliary ganglion neurons. We propose that primary sensory neurons, regardless of their embryological origin, are responsive to a "central-target" (CNS) derived neurotrophic factor--BDNF, while they are differentially responsive to "peripheral-target"-derived growth factors, such as NGF, depending on whether the neurons are of neural crest or placodal origin.     

Core Protein of Chondroitin Sulfate Proteoglycan Promotes Neurite Outgrowth from Cultured Neocortical Neurons

Chondroitin sulfate proteoglycan (CS-PG) was purified from rat brain and examined for its effect on neurite outgrowth in primary cultures of embryonic rat neocortical neurons. Neurite outgrowth was increased in culture wells coated with CS-PG. The core protein and glycosaminoglycan (GAG) prepared from the CS-PG were also examined for neurite-promoting activity. The activity was observed in culture wells coated with the core protein but not with GAG. These results suggest that CS-PG stimulates neurite outgrowth from the cultured neurons via its core protein.     

Laminin promotes neuritic regeneration from cultured peripheral and central neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co- purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.     

A glia-derived neurite-promoting factor with protease inhibitory activity.

Brain cells and glioma cells in culture release a protein which induces neurite outgrowth in neuroblastoma cells. This neurite-promoting factor (NPF), which has been purified from serum-free glioma conditioned medium, has an apparent mol. wt. of 43 000. NPF inhibits urokinase as well as plasminogen activator-dependent caseinolysis or fibrinolysis. NPF and urokinase form an SDS-resistant complex. The fact that this glia-derived NPF is a potent protease inhibitor indicates that glial cells modulate the proteolytic activity associated with neuronal cells and suggests that this phenomenon is one of the biochemical events involved in the regulation of neurite growth.     

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.     

Thrombospondin promotes process outgrowth in neurons from the peripheral and central nervous systems.

Thrombospondin (TSP) is a prominent constituent of the extracellular matrix of the developing nervous system. We have examined the effects of TSP on the morphological differentiation of neurons. In short-term cultures (less than or equal to 24 hr) of embryonic rat sympathetic neurons, TSP stimulated neurite outgrowth, causing significant increase in the number of processes and their length. Similar effects were observed in cultures of rat dorsal root ganglion, hippocampal, and cerebral cortical neurons. Moreover, in cultures of central neurons, TSP was more effective than laminin in enhancing process extension. Analysis of long-term (5-7 days) cultures of sympathetic neurons indicated that processes formed in the presence of TSP had the cytochemical characteristics of axons. Thus, TSP can influence neuronal development by selectively enhancing axonal growth. The neurite-promoting region of the molecule was identified using a panel of monoclonal antibodies targeted to different regions of the protein. Process outgrowth could be totally inhibited with antibody A4.1, which recognizes the stalk region of TSP. These data suggest that the neurite-promoting activity is localized to a single region of the TSP molecule.     

Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex.

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.     

Rescue of motoneurons from cell death by a purified skeletal muscle polypeptide: effects of the ChAT development factor, CDF

Rat skeletal muscle contains a 22 kd polypeptide that increases the level of choline acetyltransferase (ChAT) activity in cultures of embryonic rat spinal cord neurons and has been purified to homogeneity. The application of this factor, ChAT development factor or CDF, to developing chick embryos during the period of naturally occurring motoneuron cell death significantly increased the survival of motoneurons but did not affect the survival of dorsal root ganglion neurons or sympathetic preganglionic neurons (column of Terni). These results provide the first demonstration that an isolated, skeletal muscle-derived molecule can selectively enhance the survival of motoneurons in vivo and suggest that CDF may function in vivo to regulate the survival and development of motoneurons.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

An 82-Kilodalton Membrane Protein that Inhibits the Activity of Neurite Outgrowth Factor

Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P-40 of chicken gizzard muscle membranes inhibited the neurite-promoting activity of NOF in a dose-dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite-promoting activity and may modulate NOF activity during development in vivo.     

Regulation of neurotransmitter synthesis: from neuron to gene.

We are interested in the molecular mechanisms of the regulation of neurotransmitter related gene expression by neurotrophic factors and neuronal activity. Previous work has shown that conditioned medium of muscle cells induces choline acetyltransferase (CAT) activity and represses tyrosine hydroxylase, dopamine-beta-hydroxylase and aromatic L-amino acid decarboxylase (AADC) activities in cultured sympathetic neurons. Here, we show that a new muscle-derived cell line secretes two factors which induce CAT activity in spinal cord cultures; one of those is related to LIF, a CAT inducing factor for sympathetic neurons. Preliminary data are presented on the structure of the human AADC and CAT genes. Putative promoter regions have been coupled to reporter genes; transient transfection experiments will allow us to determine the promoter elements responsible for the regulation by neurotrophic factors. We also summarize the distribution of AADC-immunoreactive cells in rat and cat brain, which will be used as a reference for the study of the specificity of expression of AADC promoter in transgenic mice.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Excess K+ and Phorbol Ester Activate Protein Kinase C and Support the Survival of Chick Sympathetic Neurons in Culture

The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.     

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.