B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase.

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.

Polyneuropathies and CNS protein metabolism. III. Changes in protein synthesis rate induced by acrylamide intoxication.

A defect in neuronal protein metabolism has been proposed as one of the primary, molecular events underlying the development of polyneuropathies of the dying back type. Using acrylamide-intoxication as an experimental model to study these polyneuropathies, changes in leucine-incorporation into proteins disability. Proteinsynthesis rates were determined in vivo using flooding concentrations of [1-14C]valine as the precusor. Under conditions of acute and of chronic intoxication, a decrease in synthesis rate was measured preceding the loss of functional ability. Similar changes in protein synthesis rate were observed in peripheral tissues such as heart muscle and liver showing the general toxicity of acrylamide. Methylene bisacrylamide, that was used to discriminate between the neurotoxic action of acrylamide and its systemic effects, interfered with protein synthesis rates in a comparable way. No change in protein synthesis rate was observed under in vitro conditions suggesting that the interference of acrylamide with the synthetic machinery for protein synthesis in vivo is mediated by one or more as yet unknown indirect factors.

Local accumulations of B-50/GAP-43 evoke excessive bleb formation in PC12 cells.

B-50 (GAP-43) is an axonal, plasma membrane-associated protein involved in growth cone morphology and function. We have conducted immunocytochemical, electron microscopic, and time-lapse experiments to visualize morphological consequences of local accumulations of B-50 at the plasma membrane of B-50-transfected PC-B2 cells, a clonal PC12 cell line with very low expression of endogenous B-50. The distribution of the transfected B-50 within these cells was inhomogeneous. At sites where the B-50 concentration was locally increased up to twofold, numerous filopodia were present in growth cone-like, substrate-attached regions. When local B-50 concentrations were even higher (up to 6.2-fold), blebs were formed, often containing vesicular structures, heavily decorated with B-50 immunoreactivity. Double labeling with f-actin binding phalloidin revealed that local B-50 accumulations were accompanied by increased actin filament concentrations. Colocalization of B-50 with actin filaments was prominent in filopodia, but was virtually absent in blebs, suggesting a disconnection of the bleb plasma membrane from the actin cytoskeleton. We conclude that B-50 evokes distinct effects on cell-surface activity in PC12 cells depending on its local concentration.

Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity.

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.

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.

Dexamethasone-induced effects on B-50/GAP-43 expression and neurite outgrowth in PC12 cells.

Undifferentiated PC12 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 PC12 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 PC12 cell line demonstrates the tight coupling of these parameters that might be indicative of a threshold effect of B-50 levels on neurite outgrowth.

Cloning and promoter analysis of the human B-50/GAP-43 gene.

We here report isolation of exon 1 and analysis of the human B-50 promoter. A human genomic lambda EMBL3 library was screened with a homologous PCR probe. Two independent clones were analyzed and partially sequenced: They contained up to 5 kb sequence upstream of the translation start site and approx 13 kb of intron 1 sequence. There was a high degree of homology between the rat and the human gene with 100% homology from -504 to -427, with respect to the translation start codon. However, relatively long GT and GA repeats as seen in the rat gene were absent. Various promoter-reporter constructs, containing 5.0 to 0.12 kb of the upstream region, were transfected into undifferentiated and neuroectodermally differentiated P19-EC. Two promoter activities were found. The minimal fragment with promoter activity still responsive to differentiation was the 0.22 kb construct, similar to rat promoter P2. We conclude that the human B-50 gene is expressed in a similar way to the rat B-50 gene, based on the presence of two transcripts, the high degree of homology between the rat and the human sequence, and the two promoter activities found in P19-EC cells.

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.

Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation.

B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.

Structure of the human gene for the neural phosphoprotein B-50 (GAP-43).

The genomic DNA encoding the exons for the human neural phosphoprotein B-50 (GAP-43) was isolated using rat-based cDNA probes and oligonucleotides. Exons 2 and 3 were isolated from a genomic library, exon 1 was amplified by PCR on total genomic DNA. The gene consists of 3 exons and 2 large introns. The first exon encodes the N-terminal 10 amino acids of B-50 involved in membrane association of the protein. Exon 2 encodes the main part of the protein with the sites for protein kinase C-mediated phosphorylation and calmodulin binding, and includes a 10 amino acid residue insert not found in rodents. Exon 3 encodes the last 29 amino acid residues. The reported sequence extends the known cDNA structure to both the 5' and 3' ends. The 358 bp region upstream of the translational initiation codon, containing the main transcription starts, is purine-rich and does not include TATA or GC boxes. At the 3' end potential polyadenylation signals were found 510 bp and 584 bp downstream of the stopcodon in exon 3. The 5' end of the mRNA is heterogeneous in length, with primer extension products corresponding to a 5' untranslated region of 159 and 343 bases. Northern hybridizations, however, indicate that the majority of B-50 mRNA has a shorter 5' untranslated region, as was reported for the rat (Schrama et al., Soc. Neurosci. Abstr., 18 (1992) 333.4). The structural organization of the human gene is similar to that described for the rat (Grabczyk et al., Eur. J. Neurosci. 2 (1990) 822-827), and both translated and untranslated regions show a high degree of sequence homology to the rat gene.

Identification of two promoter regions in the rat B-50/GAP-43 gene.

To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5' flanking sequence was isolated. Sequence analysis of 1 kb 5' untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5' transcription start site is located between -465 and -440. Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.

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.

The protein kinase C phosphosite(s) in B-50 (GAP-43) are confined to 15K phosphofragments produced by Staphylococcus aureus V8 protease.

The neuron-specific phosphoprotein B-50 (= GAP-43/F1/pp46/P57) is an endogenous substrate of protein kinase C (PKC) in rat brain. To examine the location of the PKC phosphosites, phosphorylated B-50 was digested by Staphylococcus aureus V8 protease (SAP). The products migrated in SDS-polyacrylamide gel electrophoresis as two phosphoprotein bands of apparent molecular weight of 15 and 28 kDa (indicated as 15 and 28 K). This study reports further characterization of the 15 and 28 K phosphobands. ACTH(1-24), a characteristic inhibitor, inhibited equally effective the [(32)P]phosphate-incorporation into the 15 and 28 K phosphobands formed by SAP from B-50 endogenously phosphorylated in synaptosomal plasma membrane (SPM). Tests using immunoprecipitation or immunoblotting showed that all polyclonal rabbit B-50 antisera recognized the 28 K phosphoband, but only a minor population B-50 antibodies of a recently developed antiserum 8613 reacted with the 15 K phosphoband. The time course of the SAP digestion indicated that B-50 is degraded first to the 28 K band and then to the 15 K band. [(32)P]phosphate incorporated in B-50 was totally recovered in these phosphobands. Isoelectric focusing resolved the SAP products into one 28 K phosphopeptide of isoelectric point (IEP) 4.8 and the 15 K phosphofragment in at least 4 phosphopeptides, with IEP of 6.1, 6.6, 6.9 and 7.0, respectively. SAP digests of extensively phosphorylated B-50 analysed by isoelectric focusing in narrow pH gradients, showed microheterogeneity in the undigested B-50, the 28 and 15 K phosphofragments. The time course of SAP digestion of B-50 in endogenously phosphorylated SPM and in phosphorylated nerve growth cone membranes, demonstrated that in both types of membranes, 28 and 15 K phosphofragments are consecutively formed, with IEP identical to the phosphofragments derived from isolated B-50. Our findings suggest that the PKC phosphosite(s) in the B-50 protein are restricted to neutral 15 K peptides of the B-50 molecule.

N-terminal cysteines essential for Golgi sorting of B-50 (GAP-43) in PC12 cells.

We have examined the subcellular distribution of the growth-associated protein B-50 (GAP-43) in pheochromocytoma (PC12) cells, using confocal microscopy. Proliferating PC12 cells contained very low levels of B-50 in the cytosol. Enhanced expression of B-50 in these cells, evoked by either nerve growth factor (NGF) treatment or transient transfection with rat B-50 cDNA, led to Golgi sorting and membrane targeting of the B-50 protein. Site directed mutagenesis of Cys3Cys4 to Ser3Gly4 in B-50 resulted in a cytosolic distribution. We conclude that Cys3, and Cys4 are essential for accumulation of B-50 both at the plasma membrane and in the Golgi apparatus of PC12 cells.

Pineal protein synthesis highly sensitive to ACTH-like neuropeptides.

Pineal protein synthesis was studied in vitro over a period of 6-8 h after dissection. The level of protein synthetic activity of the pineal gland was greatly dependent on the time of dissection showing a maximum at midnight and a minimum at 10.00 h, 2 h after onset of light. Low concentrations of ACTH1-24 (down to 10(-11) M) could stimulate protein synthesis in vitro. The sensitivity to hormonal stimulation showed a circadian variation similar to that observed in the basal protein synthetic activity. Furthermore, overall synthetic activity appeared to be under neural influence. These neural and hormonal influences seemed to be mediated by beta-receptor stimulation and cyclic AMP. Structure-activity studies of the ACTH-effect on pineal protein synthesis gave results similar to those previously observed for excessive grooming behaviour, synaptic plasma membrane phosphorylation, adenylcyclase-activity and cell-free protein synthesis in brain. It was concluded, that overall pineal protein synthesis is both under neural and hormonal control. The action of ACTH on protein synthesis rate might be mediated by a calcium-dependent release of norepinephrine followed postsynaptically by beta-receptor activation, cAMP production, and stimulation of translation.

ACTH-like neurotropic peptides: possible regulators of rat brain cyclic AMP.

The influence of behaviorally active, N-terminal fragments of ACTH on the accumulation of cAMP in rat brain investigated in broken cell preparations of subcortical tissue, in slices of neostriatum and in vivo. ACTH1--24 has a biphasic effect on the activity of adenylate cyclase in broken cell preparations of rat brain subcortical tissue: concentrations below 25 micrometer stimulated, whereas concentrations of 0.1 mM and higher inhibited adenylate cyclase activity. The magnitude of the stimulation was dependent on the concentrations of ATP and Mg2+ in the incubation medium. Structure activity studies revealed that at a concentration of 10(-4) M ACTH1--16-NH2 and ACTH4--7 also inhibited the activity of adenylate cyclase, whereas ACTH11--24, ACTH1--10, ACTH4--10, [D-Phe7]ACTH1--10 and [D-Phe7]ACTH4--10 were inactive in this respect. Addition of 0.8 mM EGTA but not of 0.25 mM Ca2+ prevented the inhibition by 10(-4) M ACTH1--24. GMP-N-P (10(-5) M), naltrexone (10(-3) M) and ergometrine (10(-3) M) did not influence the inhibitory effect. ACTH1--24 enhanced the accumulation of cAMP in slices from rat brain neostriatum in a dose-dependent manner. This effect was already maximal 7.5 min after the addition of the peptide and was potentiated by isobutylmethylxanthine, a potent inhibitor or phosphodiesterase. Intraventricular injection of 1 microgram ACTH1--16-NH2 in rats significantly elevated (+ 27%) the concentration of cAMP in the septal region 60 min after the injection of the peptide. The results are discussed in terms of a possible involvement of cAMP as a second messenger in the central nervous system for N-terminal fragments of ACTH.

Early effect of an ACTH4-9 analog (Org.2766) on regenerative sprouting demonstrated by the use of neurofilament-binding antibodies isolated from a serum raised by alpha-MSH immunization.

Antibodies binding to the 150 kDa neurofilament protein NF150 have been purified from a serum raised by immunizing a rabbit with alpha-MSH. The NF150-binding antibodies were purified by affinity chromatography on a column of cytoskeletal proteins coupled to CNBr-activated Sepharose-4B. Immunocytochemical application of these antibodies, followed by a FITC-coupled second antibody, labels axons in intact and regenerating nerves and provides a means of identifying and counting small regenerating sprouts from 48 h after nerve crush. We have been able to demonstrate that treatment with the neurotrophic melanocortin analog, Org.2766, increases the number of regenerating axons present in the nerve as early as 72 h after nerve crush.

Changes in synaptic membrane phosphorylation after tetanic stimulation in the dentate area of the rat hippocampal slice.

Slices of rat brain hippocampus were stimulated electrically in the perforant pathway. After electrical stimulation, known to produce long-lasting post-tetanic potentiation, endogenous phosphorylation of membrane proteins was measured in a crude mitochondrial fraction, prepared from stimulated and unstimulated slices. Tetanic stimulation specifically enhanced the incorporation of [32P]phosphate into a protein band with apparent molecular weight of 50K. When the same number of pulses were given, but at a much slower rate (1 pulse per 4 sec instead of 15 pulses per sec) no posttetanic stimulation and concomitantly, no enhanced incorporation of [32P]phosphate were observed into the 50K band. When the stimulation of the slices was performed in Ca2+-free medium, again no potentiation and no enhanced incorporation into the 50K protein band were observed. It is suggested that electrical stimulation enhances the activity of the protein kinase that phosphorylates the 50K protein.

Enkephalins affect hippocampal membrane phosphorylation.

Slices of rat brain hippocampus were incubated with methionine-enkephalin, leucine-enkephalin, [Des-Tyr1] methionine-enkephalin or etorphin. After incubation the endogenous phosphorylation of proteins was measured using crude mitochondrial fractions prepared from the incubated slices. Methionine- and leucine-enkephalin specifically enhanced the radiophosphate incorporation into one protein band with an apparent molecular weight of 50K. The effect could also be detected in a fraction containing purified synaptosomal plasma membranes. The effect was time- and dose-dependent and could be mimicked by etorphin and blocked by naloxone. If methionine-enkephalin was added directly to a membrane fraction a marked inhibition of incorporation of phosphate into the 50K band was observed. It is suggested that opiate-receptor activation underlies the effects of enkephalin on hippocampal phosphoprotein metabolism.

Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: structure-activity study.

The recovery of sensorimotor function in rats was studied using a foot-flick response test after crushing the sciatic nerve. Every other day, the animals received a subcutaneous injection of ACTH1-24 or vehicle, immediately after the 'foot-flick' test. Rats treated with ACTH1-24 showed a faster recovery of sensorimotor function as compared to vehicle-treated rats. This beneficial effect was dose-dependent. In contrast, no effect on toe-spreading could be detected. To investigate what part of the peptide molecule would contain the active site and to exclude steroid mediation, smaller sequences of ACTH1-24 devoid of corticotrophic activity were tested. Treatment with ACTH1-16NH2, ACTH4-10 or [Met(O2)4,D-Lys8,Phe9]ACTH4-9 (Org. 2766) mimicked the effect of ACTH1-24, whereas treatment with ACTH11-24 did not effect the return of sensorimotor function. The stimulatory effects of corticotrophin1-24 and congeners on recovery of sensorimotor function are discussed in terms of a direct effect of these peptides on nervous tissue, probably through enhanced brain and spinal cord protein synthesis.