Levels Of GAP-43 Research Articles

Nerve growth factor stimulates GAP-43 expression in PC12 cell clones independently of neurite outgrowth.

Expression of the growth associated protein GAP-43 (B-50, F1, neuromodulin) increases with the onset of neuronal development as seen by the growth of axons. To investigate the relationship of the signaling events leading to GAP-43 expression and neurite outgrowth, we examined PC12 clones with different phenotypes. Three clones, PC12-N09, PC12-N15, and PC12-N21, responded to NGF with increased expression of GAP-43, but only two clones, PC12-N15 and PC12-N21, responded with growth of neurites. Similar increases in expression of GAP-43 were obtained when these clones were exposed to the phorbol ester PMA. Thus, NGF and PMA induced GAP-43 expression in PC12-N09 cells in the absence of neurite outgrowth. In contrast, all three clones, were able to respond to forskolin (FOR) by initiation of long neurites which had synaptophysin in the growth cones, but showed only low levels of GAP-43. Combined stimulation of PC12-N09 cells with FOR and PMA both initiated neurites and increased expression of GAP-43 as seen in normal PC12 cells. These results show that PC12-N09 cells, in response to either NGF or PMA, can express GAP-43, but without neurite outgrowth, and that all the PC12 clones were also able to respond to FOR with increased neurite outgrowth in the presence of low levels of GAP-43. The dissociation of GAP-43 expression and growth of neurites observed in PC12-N09 cells suggests that signaling mechanisms can independently regulate GAP-43 expression and neurite outgrowth during neuronal differentiation.

Ontogeny, sex dimorphism, and neonatal sex hormone determination of synapse-associated messenger RNAs in rat brain

Sex hormones influence neurite outgrowth and synaptogenesis in certain hormone-dependent areas of the rat brain during neonatal development. These alterations are thought to mediate changes in brain structure and function between the sexes. Growth-associated protein 43 kDa (GAP-43) gene expression is estrogen-regulated in the adult ventromedial hypothalamus (VMH) and sexually dimorphic (M:F = 1.8:1) in adult cortex (CTX). Such effects intimate hormonal regulation of synaptic plasticity. To investigate the nature of these dimorphisms, the present study examined the ontogeny of expression of mRNAs encoding 3 neural-specific proteins: GAP-43, SCG10, and synaptosomal-associated protein 25 kDa (SNAP-25); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the VMH and CTX; and also the effects of altering the neonatal sex hormonal milieu on the development of these adult dimorphisms. Levels of specific mRNAs in VMH and CTX were quantitated by slot-blot hybridization in rats of both sexes at different postnatal ages. To determine the involvement of neonatal sex hormones on the levels of these mRNAs, male neonatal rat pups were treated with an estrogen receptor antagonist or an aromatase inhibitor, and neonatal female pups were treated with testosterone or estrogen prior to slot-blot evaluations in adulthood. In VMH, GAP-43 mRNA levels were high on days P1 and P4 with a 3-fold decrease by day P23; in CTX, GAP-43 mRNA first increased by day P11, then fell to baseline by day P23. In VMH, SCG10 mRNA showed only small increases with time; but in CTX, there was a 5-fold drop from days P4 to P23. In VMH, SNAP-25 mRNA was low and changed only slightly; but in CTX there was a 5-fold increase between days P4 and P60. At birth, there was no sex dimorphism in either VMH or CTX, but the levels of all 3 neural-specific mRNAs were sexually dimorphic in adult CTX (M:F = 1.76 for GAP-43, 1.46 for SCG10, 1.44 for SNAP-25). GAPDH mRNA levels were regulated developmentally in VMH and CTX, but there was no sex dimorphism in either area. In male rats who received either an estrogen antagonist or aromatase inhibitor at birth, the CTX GAP-43 and SNAP-25 mRNA levels fell by 30%, to levels similar to untreated females. Conversely, in female rats, neonatal treatment with either testosterone or estrogen increased GAP-43 and SNAP-25 mRNA levels by about 30%, to levels similar to the untreated adult male. SCG10 levels did not demonstrate neonatal hormonal dependence. These results are consistent with other ontogenic studies, and show that synapse-associated mRNAs are differentially regulated, and are coincidental with synaptic ontogeny in these areas. They also confirm a sex dimorphism of these mRNAS in adult CTX, and show that these dimorphisms are not present at birth, but rather become apparent at adulthood. Their adult expression is determined, at least in part, by the availablity of testosterone substrate to be aromatized in situ to estradiol, and the ability of this estradiol to interact with CTX estrogen receptors present at birth. Thus, GAP-43 mRNA and SNAP-25 mRNA appear to be markers for sex-specific synaptic ontogeny, and are likely to contribute to both the structural and functional bases of rat brain sex differentiation.

Injury‐associated induction of GAP‐43 expression displays axon branch specificity in rat dorsal root ganglion neurons

Peripheral nerve injury results in the increased synthesis and axonal transport of the growth-associated protein GAP-43 in dorsal root ganglion (DRG) neurons, coincident with regenerative growth of the injured peripheral axon branches. To determine whether the injury-associated signalling mechanism which leads to GAP-43 induction also operates through the central branches of DRG axons, we used immunocytochemistry to compare the expression of GAP-43 in adult rat DRG neurons 2 weeks after dorsal root crush lesions (central axotomy) or peripheral nerve crush lesions (peripheral axotomy). In uninjured ganglia, a subpopulation of smaller DRG neurons expresses moderate levels of GAP-43, whereas larger neurons generally do not. At 2 weeks following peripheral axotomy, virtually all axotomized neurons, large and small, express high levels of GAP-43. At 2 weeks following dorsal root lesions, no increase in GAP-43 expression is detected. Thus, the injury-associated up-regulation of GAP-43 expression in DRG neurons is triggered by a mechanism that is responsive to injury of only the peripheral, and not the central, axon branches. These findings support the hypothesis that GAP-43 induction in DRG neurons is caused by disconnection from peripheral target tissue, not by axon injury per se.

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction.

Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells.

Mn2+ has been shown to promote cell-substrate adhesion and cell spreading in many cell culture systems. In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells. In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes. These morphological effects of Mn2+ were both time- and dose-dependent. In the presence of cycloheximide, a protein synthesis inhibitor, both Mn(2+)-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place. Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+. Although Mn(2+)-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth. The neurite outgrowth-promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites. Mn2+ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells. In cells grown in serum-free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata. Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only. The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells. These findings are consistent with a hypothesis that Mn(2+)-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects.

Antisense GAP‐43 Inhibits the Evoked Release of Dopamine from PC12 Cells

To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K+ (56 mM) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.

Dexamethasone-Induced Effects on B-50/GAP-43 Expression and Neurite Outgrowth in PC 12 Cells.

Undifferentiated PC 12 cells contain detectable levels of the nervous-specific protein B-50/GAP-43. Upon treatment with NGF or change of culture medium, B-50/ GAP-43 levels remained unchanged during the first 12 hours while neuritogenesis starts. Both, B-50/GAP-43 levels and neurite outgrowth peak at 24 hours. These results suggest that in PC 12 cells the amount of B-50 already present is sufficient to support the start of NGF-induced neuritogenesis, presumably by translocation from cytosolic compartments to the membrane. Addition of DEX reversed the rise in B-50/GAP-43 levels induced by either the change of medium or by NGF. In contrast, neurite outgrowth was inhibited to a lesser extent, although after 36 hours of pretreatment with DEX neurite length was lower than control. NGF was capable of enhancing B-50/GAP-43 levels both in the presence and absence of DEX. This corroborates data from others, who concluded that DEX and NGF exert their effects through different mechanisms, e.g., transcription versus mRNA stabilization, respectively. The inhibitory effect of DEX under various conditions on both B-50 expression and neurite outgrowth in the normal PC 12 cell line demonstrates the tight coupling of these parameters that might be indicative of a threshold effect of B-50 levels on neurite outgrowth.

The expression of growth-associated protein GAP-43 mRNA in the rat hippocampus in response to adrenalectomy and aging

GAP-43 is expressed in axonal growth cones during development and regeneration. High levels of GAP-43 persist in the adult hippocampus, reflecting perhaps the ongoing neurogenesis and synaptic plasticity characteristic of this brain region. Since the morphology and survival of hippocampal neurons are altered by adrenal steroids and aging, we have investigated the effects of adrenalectomy and of aging on the expression of hippocampal GAP-43 mRNA, as a molecular marker for these events. Adrenalectomy for 7 days results in an increase in GAP-43 mRNA expression, as assessed by in situ hybridization, in the pyramidal cell layers of the hippocampus. Both male and female aged rats exhibit levels of GAP-43 mRNA in hippocampal pyramidal cells lower than those of their younger counterparts.

The downregulation of growth-associated proteins in motoneurons at the onset of synapse elimination is controlled by muscle activity and IGF1

Axonal growth during development and regeneration coincides with the expression of growth-associated proteins (GAPs), including GAP-43 and tubulin-alpha 1. Following contact with the target region and synaptogenesis, GAPs are downregulated. However, high levels of GAP-43 are expressed in some neurons in the adult CNS, indicating that its function may not be restricted to axonal growth. To define the type of signals that regulate GAP levels during development, we have determined whether GAP downregulation coincides with a defined phase of synapse development and whether it is controlled by the postsynaptic target. Levels of GAP-43 mRNA in spinal motoneurons and protein at the neuromuscular junction were analyzed in the developing neuromuscular system of chick and rat. In both species, GAP-43 mRNA declined rapidly at a time corresponding to the onset of synapse elimination, and nerve terminal GAP-43 immunoreactivity became undetectable with a delay of 2-3 d. In rat motoneurons, GAP-43 and tubulin-alpha 1 mRNA levels declined with a similar time course, indicating that at least two GAPs are downregulated at the same time. Blockade of neuromuscular transmission with either a pre- or a postsynaptically acting toxin prevented GAP downregulation, indicating that target activity affects GAP mRNA levels in motoneurons. Finally, counteracting the activity-dependent decline of muscle insulin-like growth factors during synapse elimination by local subcutaneous injections prevented motoneuron GAP downregulation, suggesting that these growth factors may be involved in the regulation of motoneuron GAPs by the muscle. These results demonstrate that motoneurons undergo target-sensitive changes in GAPs at the onset of synapse elimination. In addition, these results raise the possibility that termination of a presynaptic growth mode may be a prerequisite for synapse elimination.

Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons.

In situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexed's lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of nerve growth factor-induced [formula omitted] expression by antisense oligomers interferes with neurite outgrowth of PC12 cells

Substantial evidence has now been gathered for the involvement of B-50 GAP-43 in neuronal development and regeneration. The precise role of this protein, however, is still debated. In an earlier study, a linear correlation between NGF-induced neurite outgrowth and B- 50 GAP-43 levels was observed in PC12 cells. To establish the involvement of B-50 GAP-43 expression in neurite outgrowth in these cells, we interfered with the expression by antisense oligomers and measured the outgrowth. In the present study, a B-50 GAP-43 antisense 5′-oligomer interfered both with the NGF-induced increase in B-50 GAP-43 and with neurite outgrowth, whereas an antisense 3′-oligomer was ineffective. We conclude, that in PC12 cells under normal conditions B-50 GAP-43 expression and neurite outgrowth are or become coupled upon NGF-induction, in contrast to the situation in PC12 clones with no or very low B-50 GAP-43 expression.

GAP-43 distribution is correlated with development of growth cones and presynaptic terminals.

GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.

The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study

GAP43 is a developmentally regulated phosphoprotein which is almost exclusively found in neurons. Numerous correlative studies have shown that GAP43 is expressed at high levels during neurite extension, axonal elongation and synaptogenesis. In this study we used in situ hybridization to examine GAP43 expression during late embryonic and early postnatal development. The highest relative levels of GAP43 at all stages were present in the neocortex. Levels in this and other regions peaked between postnatal days 5 and 10. These results indicate that high levels of GAP43 mRNA correlate most highly with the latter stages of axon outgrowth and with the early stages of synapse formation.

GAP-43 immunoreactivity is widespread in the autonomic neurons and sensory neurons of the rat

GAP-43 is a membrane-bound phosphoprotein generally associated with axon growth during development and regeneration. Using immunohistochemical and immunoblotting techniques this study shows that GAP-43 is expressed extensively in the unperturbed adult autonomie nervous system. Strong immunoreactivity was seen in the developing and mature enteric subdivision of the autonomie nervous system and in nerves of the iris and various blood vessels. The presence of GAP-43 immunoreactivity in varicose nerve fibres, and a comparison of the labelling pattern of GAP-43 with the nerve associated marker PGP 9.5 suggests that GAP-43 is present in most or all autonomie nerve fibres in these organs. Immunoblotting of gut samples on 10% polyacrylamide gels revealed a single band of approximately 45,000 mol. wt that co-migrated with pure central nervous system GAP-43. Surgical sympathectomy experiments resulting in almost complete elimination of sympathetic fibres did not markedly affect the pattern of GAP-43 immunoreactivity in the iris, indicating that GAP-43 is expressed not only in sympathetic nerves but also in parasympathetic and sensory fibres. These findings show that GAP-43 is expressed extensively in autonomie nerves of the adult rat, at levels comparable to those seen during development. High levels of GAP-43 are not therefore restricted to development and regeneration in this part of the nervous system.

Growth-associated protein (GAP-43)-like immunoreactivity in primary and permanent tooth pulp nerve fibers of the cat

GAP-43-like immunoreactivity in developing and mature incisor and canine tooth pulp nerve fibers in the cat was examined with fluorescence immunohistochemistry and pre-embedding immunogold electron microscopy. As expected, pulpal and periodontal nerve fibers in primary teeth aged 2–3 weeks showed strong immunoreactivity. Double-labeling experiments demonstrated that 50–70% of primary pulpal GAP-43-positive nerve fibers showed CGRP-like immunoreactivity. However, in adult permanent teeth the vast majority of pulpal nerve fibers also displayed intense GAP-43-like immunoreactivity both when surrounding pulpal blood vessels and in the subodontoblast/odontoblast region. There was a high degree (90–95%) of simulataneous expression of GAP-43-like immunoreactivity and CGRP-like immunoreactivity in adult permanent pulps. Immunogold GAP-43 labeling was mainly associated with the cytoplasmic side of axonal membranes. However, occasional examples of immunolabeled Schwann cells were also found. High levels of GAP-43 in normal mature permanent pulpal nerves may facilitate neural plasticity after dental wear or injury.

GAP-43 and 5B4-CAM immunoreactivity during the development of transplanted fetal mesencephalic neurons

Developing neurons contain high levels of several proteins which are absent or relatively scarce in mature neurons. GAP-43 is a cytoplasmic protein primarily found within neurons; high levels of this protein are correlated with axonal elongation or regeneration. 5B4-CAM, a glycosylated transmembrane protein, is a member of the NCAM family present in growth cones and in plastic CNS structures. Antibodies directed against these two developmentally regulated proteins were used to characterize the time-course of maturation of transplanted fetal mesencephalic neurons. For our experiments unilateral injections of 6-hydroxydopamine were made into the nigrostriatal bundle in Sprague-Dawley rats. The effectiveness of the lesion was verified by apomorphine-induced rotation and by postmortem examination of the substantia nigra. Following behavioral testing, pieces of ventral mesencephalon obtained from E15 fetuses were transplanted into the caudoputamen ipsilateral to the lesion. Immunocytochemistry revealed high levels of GAP-43 and 5B4-CAM at 5, 11, and 15 days post-transplant but relatively lower levels by 3 weeks. At 13 weeks the immunoreactivity present within the transplant tissue was approximately equal to that found within the host striatal neuropil. This time-course of higher GAP-43 and 5B4-CAM immunoreactivities coincides with the time-course of neuritic outgrowth of dopamine containing cell populations within the ventral mesencephalon in situ as well as within ventral mesencephalic transplants. This implies that axon elongation occurs over a period similar to that which occurs during normal development. These data suggest that the effects of transplantation surgery and the altered environment of the host striatum do not significantly affect the time-course of development of ventral mesencephalic neurons.

Post-transcriptional regulation of GAP-43 rnRNA levels during neuronal differentiation and nerve regeneration

The expression of GAP-43 is a well-characterized correlate of the growth and reorganization of neural connections. Although the expression of this protein is known to depend on the levels of the mRNA, the mechanisms that control the latter are poorly understood. Using Northern blots and nuclear run-on assays, we measured the levels of synthesis and accumulation of GAP-43 mRNA in (a) developing rat cortical neurons, (b) NGF-induced PC12 cells, and (c) regenerating goldfish optic nerve. In all of these instances, significant levels of the nascent transcript were detected even when cells were not growing and this changed little during differentiation, though the steady-state levels of GAP-43 mRNA increased 6- to 20-fold under these conditions. The lack of correlation between the levels of transcription and accumulation of GAP-43 mRNA suggested that the levels of this mRNA are regulated via a post-transcriptional mechanism. To investigate the nature of the mechanism involved, we measured the turnover of the mRNA in control and NGF-treated PC12 cells. Experiments using pulse-chase labeling or actinomycin D treatment showed that NGF induced a 2-fold increase in the half-life of GAP-43 mRNA, which is consistent with a 7-fold increase in the accumulation of the mRNA within 24 h. Thus, changes in the rate of degradation of the mRNA appear to be the major mechanism that controls GAP-43 levels during neurite outgrowth. The presence of some highly conserved putative instability-conferring motifs in the 3′ untranslated region (3′ UTR) of GAP-43 mRNA lends further support to this possibility.

Phospholipid-mediated delivery of anti-GAP-43 antibodies into neuroblastoma cells prevents neuritogenesis

The neuronal growth-associated protein GAP-43 is expressed during axonal outgrowth and regeneration (for review, see Benowitz and Routtenberg, 1987). In the present study, we demonstrate that GAP-43 is constitutively expressed by NB2a/d1 neuroblastoma cells. The initial, most rapid outgrowth period of neuritogenesis [0-4 hr after dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) treatment] is accompanied by intense GAP-43 immunoreactivity along the entire length of most neurites. However, this immunoreactivity declined nearly to background levels within hours during continued neurite outgrowth and persisted only at varicosities and growth cones. GAP-43 was detectable by metabolic labeling and immunoblot analysis in undifferentiated cells, and synthetic rates and steady-state levels of GAP-43 underwent only a modest (approximately twofold) increase during dbcAMP-induced differentiation. Unlike levels observed in neurites, perikarya of undifferentiated and differentiated cells contained similar, intense levels of GAP-43 immunoreactivity. Neurite elaboration and GAP-43 immunoreactivity were unaffected by treatment with cycloheximide, suggesting that translocation of perikaryal GAP-43 pools, rather than de novo synthesis, contributes to the transient burst of GAP-43 observed in developing neurites. Phosphatidylcholine-mediated delivery of anti-GAP-43 antibodies (alpha GAP) into cells immediately before dbcAMP treatment arrested neuritogenesis but did not induce the retraction of existing neurites. These results indicate that, while GAP-43 expression is insufficient to induce neuritogenesis in NB2a/d1 cells, GAP-43 is nevertheless essential for the initial, dynamic phase of neurite outgrowth.

Quantitation of the growth‐associated protein B‐50/GAP‐43 and neurite outgrowth in PC12 cells

A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.

Distribution of GAP-43 mRNA in the brain stem of adult rats as evidenced by in situ hybridization: localization within monoaminergic neurons

Using in situ hybridization, we examined the distribution of the mRNA encoding for the growth-associated protein GAP-43 in the brain stem of adult rats. GAP-43 was expressed at the highest level in the nucleus raphe dorsalis (NDR), nucleus centralis superior (NCS), substantia nigra compacta (SNc), ventral tegmental area (VTA), and locus coeruleus (LC). An intermediate level of signal was detected over the periaque-ductal gray, superior colliculi, and thalamic region, and no significant signal was detected in the substantia nigra pars reticulata and red nucleus. The hybridization signals of GAP-43 mRNA and tryptophan hydroxylase mRNA completely overlapped in the NDR and NCS, and signals for GAP-43 mRNA and tyrosine hydroxylase mRNA overlapped in the SNc, VTA, and LC. The disappearance of the hybridization signal for GAP-43 mRNA after intracerebroventricular injections of the neurotoxins 5,7-dihydroxytryptamine (5,7-DHT) or 6-hydroxydopamine (6-OHDA) indicated that high levels of GAP-43 are synthesized in the serotonergic neurons of the raphe nuclei and in the catecholaminergic neurons of the SNc, VTA, and LC. In light of the role of GAP-43 in axonal outgrowth, modulation of signal transduction, and release of different neurotransmitters in the adult CNS, this phosphoprotein might be involved in the functional plasticity and synaptic transmission of monoaminergic neurons.