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.     

Expression levels of B-50/GAP-43 in PC12 cells are decisive for the complexity of their neurites and growth cones

To study the role of the protein B-50/GAP-43 in NGF-induced neurite outgrowth, a number of stable PC12 subclones with either very low or considerably enhanced expression levels of the protein were selected. Cell bodies of subclones with suppressed B-50 expression (−B2, −B5, or −B12) possessed a relative small spherical shape and, on NGF-treatment for 7 d, developed processes that were virtually devoid of branches and that mostly bore short or blunt-ended growth cones. Cells of subclones with overexpression of B-50 (+B3, +B4, or +B11), on NGF treatment, acquired a flattened, spiky appearance with highly branched neurites possessing extended and complex growth cones. Confocal microscopy with immunofluorescence for B-50 and F-actin revealed that in neurites and growth cones of the B-50-deficient subclone −B2, no detectable B-50 and reduced amounts of filamentous F-actin were present, whereas in overexpressing +B3 cells, cell membranes, neurites, and complex growth cones were intensively stained for B-50 and exhibited numerous spikes, in which B-50 was strikingly colocalized with F-actin. These data suggest that, under normal conditions of neuritogenesis, the expression level of B-50 in PC12 cells is decisive for the complexity of neurites and growth cones.     

B-50/GAP-43 in neuronal development and repair

The protein kinase C substrate B-50 is identical to the growth-associated protein GAP-43. Although as yet no causal relationship has been established between B-50/GAP-43 and neurite outgrowth, evidence accumulates that the function of the protein relates to neuronal plasticity. Stimulation of PC12 cells by NGF results in translocation of the protein from cytosolic stores to the membrane of newly formed neurite-like extensions. The protein is associated with the inner leaflet of the growth cone membrane isolated from neonatal rat brain and was used as a marker to study the development of the rat pyramidical tract and olfactory system. In the adult rat, crush lesion of the sciatic nerve results in a rapid expression of B-50/GAP-43 mRNA followed by synthesis of B-50/GAP-43 protein in dorsal root ganglia and transport of the protein into the newly formed sprouts. Presumably, the neurotrophic effect of melanocortins on peripheral nerve repair is not brought about by enhancement of B-50/GAP-43 synthesis per se. Bulbectomy (central) or Triton X-100 lesioning (peripheral) of the olfactory epithelium results in a differential expression of B-50/GAP-43 and the olfactory marker protein characterizing two stages in the regeneration of this epithelium. Evidence that the degree of phosphorylation may co-determine the role of B-50/GAP-43 in neurite outgrowth is discussed.     

TrkA Tyrosine Residues Involved in NGF-induced Neurite Outgrowth of PC12 Cells

The proto-oncogene product gp 140^prototrk (TrkA) is the receptor tyrosine kinase that mediates nerve growth factor-induced neuronal survival and differentiation. In receptor tyrosine kinases, specific intracellular tyrosine residues become phosphorylated after ligand binding and the phosphorylated tyrosines induce the cascade of signal transduction. Here we have identified intracellular tyrosine residues of TrkA involved in nerve growth factor-induced neurite outgrowth of PC12 cells, using site-directed mutagenesis and a PC12 cell line expressing very low levels of endogenous TrkA (PC12^nnr5 cells). We analysed eight conserved intracellular tyrosine residues of TrkA while the three putative autophosphorylation sites conferring tyrosine kinase activity were left intact. Five tyrosine residues, Y499, Y643, Y704, Y760 and Y794, in rat TrkA were involved in nerve growth factor-induced neurite outgrowth. None of these tyrosines mediated the full activity of wild-type TrkA, and a pair of these tyrosines, Y760 and Y794, promoted neurite outgrowth in an additive manner. These data indicate that no single tyrosine is sufficient to induce complete neurite outgrowth but the five tyrosine residues Y499, Y643, Y704, Y760 and Y794 cooperate to exhibit the full activity of wild-type TrkA.     

Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native B-50/GAP-43 expression in immature olfactory neurons

The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth. During maturation these neurons migrate into the upper portion of the epithelium, upregulate expression of olfactory marker protein (OMP) and concomitantly downregulate the expression of B-50/GAP-43. Transgenic mice that exhibit OMP-promoter directed expression of B-50/GAP-43 in mature olfactory neurons display an unexpected decrease in the complement of B-50/GAP-43-positive cells in the lower region of the olfactory epithelium [A.J.G.D. Holtmaat, P.A. Dijkhuizen, A.B. Oestreicher, H. J. Romijn, N.M.T. Van der Lugt, A. Berns, F.L. Margolis, W.H. Gispen, J. Verhaagen, Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular growth, J. Neurosci. 15 (1995) 7953-7965]. We have investigated whether the decrement in B-50/GAP-43-positive cells in this region was due to a dislocation of the immature neurons to other regions of the olfactory epithelium or to a downregulation of B-50/GAP-43 synthesis in these immature neurons. In eight of nine independent transgenic mouse lines that express the transgene in different numbers of olfactory neurons, a decline in the number of B-50/GAP-43-expressing neurons in the basal portion of the olfactory neuroepithelium was observed, both at the protein level and the mRNA level. An alternative marker for immature cells, a juvenile form of tubulin, was normally expressed in this location, indicating that the olfactory epithelium of OMP-B-50/GAP-43 transgenic mice contains a normal complement of immature olfactory neurons and that most of these neurons display a downregulation of B-50/GAP-43 expression.     

B-50/GAP43 Expression Correlates with Process Outgrowth in the Embryonic Mouse Nervous System

The hypothesis that B-50/GAP43, a membrane-associated phosphoprotein, is involved in process outgrowth has been tested by studying the developmental pattern of expression of B-50/GAP43 mRNA and protein during mouse neuroembryogenesis. B-50/GAP43 mRNA is first detectable at embryonic day 8.5 (E8.5) in the presumptive acoustico-facialis ganglion. Subsequently, both B-50/GAP43 mRNA and protein were co-expressed in a series of neural structures: in the ventral neural tube (from E9.5) and dorsal root ganglia (from E10.5), in the marginal layer of the neuroepithelium surrounding the brain vesicles and in the cranial ganglia (from E9.5), in the autonomic nervous system (from E10.5), in the olfactory neuroepithelium and in the mesenteric nervous system (from E11.5), in a continuum of brain regions (from E12.5) and in the retina (from E13.5). Immunoreactive fibers were always seen arising from these regions when they expressed B-50/GAP43 mRNA. The spatial and temporal pattern of B-50/GAP43 expression demonstrates that this protein is absent from neuroblasts and consistently appears in neurons committed to fiber outgrowth. The expression of the protein in immature neurons is independent of their embryological origin. Our detailed study of B-50/GAP43 expression during mouse neuroembryogenesis supports the view that this protein is involved in a process common to all neurons elaborating fibers.     

B-50/GAP-43 potentiates cytoskeletal reorganization in raft domains.

B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.     

Oxygen Tension Modulates Neurite Outgrowth in PC12 Cells Through A Mechanism Involving HIF and VEGF

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1α stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair.     

Estrogen Effects on Neurite Outgrowth and Cytoskeletal Gene Expression in ERα-Transfected PC12 Cell Lines

The potential of gonadal steroids like estrogen (E) to promote neurite sprouting is of interest in development and aging, as well as after neural trauma. The specific roles of the two main estrogen receptors, ERα and ERβ, in neuronal sprouting are not yet well understood. We examined the hypothesis that E can enhance nerve growth factor (NGF)-stimulated neurite sprouting in an ERα-dependent manner. PC12 cells that were stably transfected with the full-length rat ERα gene (PCER) and a control line of cells transfected with vector DNA alone (PCCON) were compared. Both cell lines vigorously differentiate neurites when treated with NGF. We determined that both lines show basal expression of ERβ mRNA, but only the PCER cells express ERα mRNA. Estrogen treatment markedly enhanced NGF-stimulated neurite outgrowth from PCER but not from PCCON cells. Significantly larger proportions of PCER cells (34 and 53% at 24 and 48 h, respectively) had neurites than did the PCCON cells (17 and 26% at 24 and 48 h) after E plus NGF treatment. We also examined the effects of E and NGF treatment of PCER and PCCON cells on peripherin, α-tubulin, and tau mRNA expression. In undifferentiated PCER cells, E treatment increased peripherin, reduced α-tubulin, and did not alter tau mRNA levels. No changes in these mRNAs were observed in the controls (undifferentiated PCCON cells) after E treatment. NGF treatment markedly stimulated expression of peripherin, α-tubulin, and tau mRNAs in both PCER and PCCON cells. From these observations we conclude that E synergizes with NGF and stimulates neurite sprouting and also modulates expression of several cytoskeletal mRNAs through ERα.     

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes

Recently, we have shown that stimulation of [³H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K⁺, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 μM to 5 mM. 4-AP (100 μM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K⁺ for 15 seconds. The effects of both 4-AP or K⁺ depolarization on B-50 phosphorylation were abolished at low extracellular Ca²⁺ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K⁺ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca²⁺-dependent transmitter release evoked by 4-AP. This research was supported by CLEO-TNO grant A66 of the Dutch Epilepsy Foundation.     

Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain

GAP-43 (B-50,F1,pp46) is a neuron-specific phosphoprotein that has been implicated in the development and modulation of synaptic relationships. Although most neurons cease expressing high levels of GAP-43 after the completion of synaptogenesis (Jacobson et al., 1986), certain brain regions continue to have considerable amounts of the protein throughout life (Oestreicher et al., 1986); in at least one such area, the phosphorylation of the protein has been linked with the events that underlie synaptic potentiation (Lovinger et al., 1985). In this study, we used the indirect immunoperoxidase method to map the distribution of GAP-43/B-50 in the brains of 8 adult rats with 2 different antibodies: a monospecific, polyclonal antibody prepared in sheep against the purified protein and an affinity-purified IgG prepared in rabbits. Specific immunoreactivity was found primarily in the neuropil and followed a generally increasing caudal-to-rostral gradient along the neuraxis. Densest staining occurred in layer I of the cortex, the CA1 field of the hippocampus, and in a continuum of subcortical structures that included the caudate-putamen, olfactory tubercle, nucleus accumbens, bed nucleus of the stria terminalis, amygdala, and medial preoptic area-hypothalamus. In the brain stem, staining was seen in the central gray and in ascending visceral relay nuclei, but was essentially absent in areas related to ascending somatosensory information (e.g., the cochlear nuclei or vestibular complex) and motor control (e.g., nucleus ruber or the motor nuclei of the cranial nerves). Staining in dorsal thalamus was likewise modest in most somatosensory and somatomotor relay nuclei, but dark in certain other structures (e.g., mediodorsal nucleus, lateral complex). This distributional pattern raises the question of whether synapses in all areas containing high levels of GAP-43/B-50 are capable of undergoing functional plasticity, or whether the protein may function in some of these areas in some other capacity (e.g., general signal transduction).     

Inhibition of PC12 Cell Attachment and Neurite Outgrowth by Detergent Solubilized CNS Myelin Proteins

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.     

Selective alteration in B-50/GAP-43 phosphorylation in brain areas of animals characterized by cognitive impairment

When methylazoxymethanol acetate is administered to pregnant rats at gestational day 19, the offspring are greatly impaired in the learning of a two-way active avoidance task and these behavioral changes are paralleled by a change in the phosphorylation of the protein B-50/GAP-43 in hippocampus but not in cortex. The expression of the protein is not altered, indicating that the phosphorylation of B-50 is a sensitive marker of alterations in synaptic plasticity associated with impairments of learning abilities in rats.     

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: content and phosphorylation of B-50.

The content and phosphorylation of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B-50 immunolocalization in the same cultures. In control cultures, the concentration of B-50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B-50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B-50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B-50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B-50 and NSE after 14 DIV. The basal phosphorylation of B-50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B-50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B-50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B-50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.     

Decreased GAP-43/B-50 phosphorylation in striatal synaptic plasma membranes after circling motor behavior during development

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (-29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (-29.3%; p<0. 001) in the incorporation of 32P-phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [(r=0.9443, p=0.0046, n=6, relative to control group) and (r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.     

Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat

Neurons that can regenerate their axons following axotomy increase their synthesis and axonal transport of a growth-associated protein, called GAP-43, which has been shown to be identical to the synaptic phosphoprotein B-50. The function of B-50/GAP-43 to the process of regeneration is unknown. We used a polyclonal, affinity-purified antibody against B-50 to study the axonal transport and localization of B-50/GAP-43-like immunoreactivity (B50LI) in the regenerating sciatic and facial nerves of adult rats. Quantitative data were obtained by densitometry of the B-50 band in immunoblots of nerve segments, which had been run on SDS-polyacrylamide gels. In the regenerating sciatic nerve, anterograde accumulation at a collection ligature was 3.0 times higher than retrograde accumulation. The mobile fraction of B50LI was only 0.28 of total B50LI and traveled with a mean anterograde velocity of 5.3 mm/hr. B50LI distribution in the newly regenerated portion of the nerve revealed maximal B50LI levels midway between the position of the crush and the fastest-growing axons. Immunocytochemistry of this portion of the nerve demonstrated B50LI to be associated with regenerating axons but also to a large extent with extra-axonal structures outlining the Schwann cell bands of Büngner. This zone of B50LI-positive Schwann cell bands was found to extend more distally in nerves in which regeneration had processed longer, e.g., up to 5 mm distal to the crush after 3 d and 8 mm after 4 d. Further distal to this zone, many fine regenerating axonal profiles could be detected with B-50 antibody, but were neurofilament negative. These findings raise the possibility of an extra-axonal function of B-50/GAP-43, as this protein might be secreted from regenerating axons and might play a role in axon-Schwann cell interactions during axonal maturation.     

Delayed and bilateral changes of GAP-43/B-50 phosphorylation after circling training during a critical period in rat striatum

During the critical period of activity-dependent plasticity in rat striatum (30-37 days after birth) physiological circling behavior induces delayed modifications in GAP-43/B-50 phosphorylation by PKC. Postexercise, ipsi- and contralateral striatum to the circling direction show a similar temporal pattern of GAP-43/B-50 phosphorylation, with an initial decrease followed by a subsequent increase. However, there is a lag between initiation of the phosphorylation response in this asymmetrical task which does not occur when animals are subjected to exercise under conditions of symmetrical motor activity.