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.

B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system.

The growth-associated protein B-50 (GAP-43) is a presynaptic protein. Its expression is largely restricted to the nervous system. B-50 is frequently used as a marker for sprouting, because it is located in growth cones, maximally expressed during nervous system development and re-induced in injured and regenerating neural tissues. The B-50 gene is highly conserved during evolution. The B-50 gene contains two promoters and three exons which specify functional domains of the protein. The first exon encoding the 1-10 sequence, harbors the palmitoylation site for attachment to the axolemma and the minimal domain for interaction with G0 protein. The second exon contains the "GAP module", including the calmodulin binding and the protein kinase C phosphorylation domain which is shared by the family of IQ proteins. Downstream sequences of the second and non-coding sequences in the third exon encode species variability. The third exon also contains a conserved domain for phosphorylation by casein kinase II. Functional interference experiments using antisense oligonucleotides or antibodies, have shown inhibition of neurite outgrowth and neurotransmitter release. Overexpression of B-50 in cells or transgenic mice results in excessive sprouting. The various interactions, specified by the structural domains, are thought to underlie the role of B-50 in synaptic plasticity, participating in membrane extension during neuritogenesis, in neurotransmitter release and long-term potentiation. Apparently, B-50 null-mutant mice do not display gross phenotypic changes of the nervous system, although the B-50 deletion affects neuronal pathfinding and reduces postnatal survival. The experimental evidence suggests that neuronal morphology and communication are critically modulated by, but not absolutely dependent on, (enhanced) B-50 presence.

Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor.

NMDA receptor, Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both alphaCaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant alphaCaMKII and PSD-95. PSD-95(54-256) was able to compete with the binding of both native and recombinant alphaCaMKII to the NR2A C-tail. Accordingly, alphaCaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native alphaCaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of alphaCaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, alphaCaMKII, and PSD-95 in hippocampal synaptic plasticity.

Calcium-dependent turnover of brain polyphosphoinositides in vitro after prelabelling in vivo.

Rat brain phospholipids were labelled in vivo by an intraventricular injection of 32P. The radioactivity was found to accumulate predominantly in limbic structures, particularly hippocampus and diencephalon. A rapid and high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro high specific labelling of the inositol phospholipids and phosphatidic acid was observed. The rate of incorporation into a crude myelin fraction was similar to that into a mitochondrial/synaptosomal fraction although phosphatidyl-myo-inositol 4,5-diphosphate was especially enriched in myelin. Upon incubation in vitro of the brain fraction after 2 h prelabelling in vivo, both phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-diphosphate rapidly lost their radioactivity. Half of the labile fraction of the incorporated 32P was removed within 2 min. None of the other phospholipids changed in the 30 min in vitro incubation period. The metabolism of the polyphosphoinositide proceeded at a lower rate when the temperature was lowered, and was Ca2+-dependent. Further subcellular fractionation revealed that purified synaptosomes and myelin contained highly labelled phosphatidyl-myo-inositol 4-phosphate or phosphatidyl-myo-inositol 4,5-diphosphate. Mitochondria contained highly labelled phosphatidyl-myo-inositol but no phosphatidyl-myo-inositol 4-phosphate or phosphatidyl-myo-inositol 4,5-diphosphate. ACTH1-24 did not inhibit the in vitro dephosphorylation of prelabelled polyphosphoinositide, confirming previous findings that the peptide affects the polyphosphoinositide kinases and not the respective phosphatases.

Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes.

The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with [32P]orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of 32P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with [32P]-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.

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.

AlphaCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.

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.

Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV.

The authors investigated immunocytochemically the innervation of a skin biopsy in a rare case of hereditary sensory and autonomic neuropathy type IV. A few protein gene product 9.5-, growth-associated protein 43-, calcitonin gene-related peptide-, and substance P-immunoreactive nerve fibers were observed in the deeper regions of the dermis. Neuropeptide Y-, nitric oxide-, and vasoactive intestinal polypeptide-immunoreactive fibers were completely absent. Their observations support the hypothesis that the sensory and autonomic defects reported in hereditary sensory and autonomic neuropathy are based on profound developmental alterations of the peripheral nervous system.

Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve.

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution. Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.

B-50/growth-associated protein-43, a marker of neural development in Xenopus laevis.

To study the regulation and function of the growth-associated protein B-50/growth-associated protein-43 (mol. wt 43,000) in Xenopus laevis, B-50/growth-associated protein-43 complementary DNAs were isolated and characterized. The deduced amino acid sequence revealed potential functional domains of Xenopus B-50/growth-associated protein-43 that may be involved in G-protein interaction, membrane-binding, calmodulin-binding and protein kinase C phosphorylation. The expression of B-50/growth-associated protein-43 at the RNA and protein level during development was investigated using the Xenopus complementary DNA and the monoclonal B-50/growth-associated protein-43 antibody NM2. The antibody NM2 recognized the gene product on western blot and in whole-mount immunocytochemistry of Xenopus embryos. Moreover, visualization of the developmentally regulated appearance of B-50/growth-associated protein-43 immunoreactivity showed that this mode of detection may be used to monitor axonogenesis under various experimental conditions. In the adult Xenopus, XB-50/growth-associated protein-43 messenger RNA was shown to be expressed at high levels in brain, spinal cord and eye using northern blotting. The earliest expression detected on northern blot was at developmental stage 13 with poly(A) RNA. By whole-mount immunofluorescence, applying the confocal laser scanning microscope, the protein was first detected in embryos from stage 20, where it was expressed in the developing trigeminal ganglion. Also later in development the expression of the B-50/growth-associated protein-43 gene was restricted to the nervous system in Xenopus, as was previously found for the mouse. In conclusion, we find that XB-50/growth-associated protein-43 is a good marker to study the development of the nervous system in Xenopus laevis.

Distribution of glutamate transporters in the human placenta.

Glutamate metabolism is known to be important for growth and development of the human fetus. The glutamate transporters EAAT1, EAAT2 and EAAT3 are key components of the glutamate-glutamine cycle and responsible for active transport of glutamate over the cell membrane. The placenta is thought to regulate glutamate transport during fetal development. Glutamate transporters have been found in placentae of rats, but their distribution in the human placenta is unknown. Therefore, the distribution of glutamate transporters EAAT1, EAAT2 and EAAT3 were analysed in the human placenta during normal pregnancies ending between 8 and 40 weeks of gestation and in placentae of intrauterine growth restricted infants with gestational ages between 28 and 35 weeks of pregnancy. Using immunohistochemistry, EAAT1 expression was found in the syncytiotrophoblast layer, while EAAT2 was detected in the syncytiotrophoblast layer and in endothelial cells of about 5 per cent of all fetal blood vessels. EAAT3 was observed in the endothelium of the fetal blood vessels in all placentae examined. However, expression was also found in the syncytio- and the cytotrophoblast layer of the fetal villi at 8 weeks of gestational age. The expression patterns of EAAT1, EAAT2 and EAAT3 suggest involvement in active transport of glutamate between the fetal and maternal blood circulation. No differences were found in the distribution of the glutamate transporters between control and IUGR placentae. Our data show specific localization of EAAT1, EAAT2 and EAAT3 in the human placenta during development.

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 microM to 5 mM. 4-AP (100 microM) 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.

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.

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.

Expression of the pro-opiomelanocortin gene in dorsal root ganglia, spinal cord and sciatic nerve after sciatic nerve crush in the rat.

Neuropeptides related to alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate nerve outgrowth following peripheral nerve injury and may play an important physiological role in peripheral nerve regeneration. The mechanism of action underlying the neurotrophic effect of pharmacologically administered alpha-MSH is unknown. Here we investigate the hypothesis that reexpression of the proopiomelanocortin (POMC) gene, the prohormone of alpha-MSH/adrenocorticotropic hormone (ACTH)-like peptides, is part of the endogenous repertoire of peripheral nerve responses following injury. The effect of sciatic nerve crush on the expression of POMC mRNA between 0.5 h and 14 days after crush was investigated using polymerase chain reaction (PCR) and Northern blot analysis. The presence of a POMC transcript in dorsal root ganglia (DRG), spinal cord and in the sciatic nerve at the crush site could be demonstrated in both control and lesioned animals by PCR using primers located in exon 1 and 3 of the POMC gene. Minute quantities of two POMC transcripts (1200 nt and 800 nt) could be detected by Northern blot analysis of total RNA prepared from DRG, spinal cord and the sciatic nerve of control animals and of animals subjected to nerve crush. POMC mRNA expression was, however, not increased following nerve crush. Probes specific for exons 1 and 2 or specific for exon 3 of the POMC gene were employed to demonstrate that the 800 nt transcript represents the truncated POMC mRNA previously shown to be present in extra-pituitary tissue. The larger 1200 nt transcript comigrates with the full length POMC mRNA expressed in the pituitary gland. The present results demonstrate the expression of small amounts of POMC mRNA in all compartments of the sciatic nerve. The absence of an induction of POMC expression in response to nerve crush suggests that the stimulating effect of exogenously applied alpha-MSH does not mimic a POMC derived neurotrophic peptide induced in the nerve following nerve injury.

Effects of enriched housing on functional recovery after spinal cord contusive injury in the adult rat.

To date, most research performed in the area of spinal cord injury focuses on treatments designed to either prevent spreading lesion (secondary injury) or to enhance outgrowth of long descending and ascending fiber tracts around or through the lesion. In the last decade, however, several authors have shown that it is possible to enhance locomotor function after spinal cord injury in both animals and patients using specific training paradigms. As a first step towards combining such training paradigms with pharmacotherapy, we evaluated recovery of function in adult rats sustaining a spinal cord contusion injury (MASCIS device, 12.5 mm at T8), either housed in an enriched environment or in standard cages (n = 15 in both groups). The animals in the enriched environment were stimulated to increase their locomotor activity by placing water and food on opposite sides of the cage. As extra stimuli, a running wheel and several other objects were added to the cage. We show that exposure to the enriched environment improves gross and fine locomotor recovery as measured by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, the BBB subscale, the Gridwalk, and the Thoracolumbar height test. However, no group differences were found on our electrophysiological parameters nor on the amount of spared white matter. These data justify further studies on enriched housing and more controlled exercise training, with their use as potential additive to pharmacological intervention.