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A boy presented at the age of 3.5 months with a developmental delay. He developed infantile spasms with hypsarrhytmia on EEG 1 month later. Additional symptoms were delayed visual development, asymmetrical hearing loss, hypotonia, and choreoathetoid movements. He also had some dysmorphic features and was vulnerable for infections. He was treated successively with vigabatrin, prednisolone, valproic acid, nitrazepam, and lamotrigine without a lasting clinical effect, but showed a treatment response to levetiracetam. Cerebral MRI showed hypoplasia of the corpus callosum and a mild delay in myelination. Further investigations including metabolic screening and glycosylation studies by transferrin isoelectric focusing were all considered to be normal. Whole-exome sequencing identified a de novo mutation in the ALG13 gene (c.320A>G, p.(Asn107Ser)). Mutations in this gene, which is located on the X-chromosome, are associated with congenital disorders of glycosylation type I (CDG-I). Mass spectrometric analysis of transferrin showed minor glycosylation abnormalities. The c.320A>G mutation in ALG13 has until now only been described in girls and was thought to be lethal for boys. All girls with this specific mutation presented with a similar phenotype of developmental delay and severe early onset epilepsy. In two girls glycosylation studies were performed which showed a normal glycosylation pattern. This is the first boy presenting with an epileptic encephalopathy caused by the c.320A>G mutation in the ALG13 gene. Since glycosylation studies are near-normal in patients with this mutation, the diagnosis of ALG13-CDG can be missed if genetic studies are not performed.

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949–959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.

Summary Demyelinating syndromes are an important group of nerve disorders for which no effective therapy exists and which are life threatening in a substantial proportion of the patients. In the present experiments we assessed the ameliorative effect of Org 2766, a degradation resistant ACTH4–9 analogue devoid of corticotrophic and melanotrophic action in experimental allergic neuritis (EAN), an animal model for a human demyelinating disease, the Guillain-Barre syndrome (GBS). In order to mimic the clinical situation, peptide treatment was initiated at the first appearance of neurological symptoms in each animal. The ACTH4–9 analogue treatment substantially suppressed the neurological symptoms in animals with EAN, as assessed by clinical scoring and resulted in a significant improvement of motor performance. Furthermore, histological examination of the sural nerve provided a morphological basis for the beneficial functional effect of peptide treatment: more myelinated fibres were present in EAN animals treated with the ACTH4–9 analogue in comparison with EAN animals treated with saline. Further analysis of the sural nerve indicated a complete preservation of the diameter distribution of myelinated fibres in sural nerves of peptide-treated animals. In contrast, saline-treated EAN animals exhibited a significant loss of small and intermediate size myelinated fibres in the sural nerves. This study provides first evidence for the amelioration of the functional and anatomical deficits in an animal model of the GBS syndrome by an interventive synthetic neurotrophic peptide treatment.

Crush or transection of peripheral nerves of the adult rat is accompanied by changes in protein expression, including the growth associated protein (GAP-43) B-50. Following peripheral nerve crush in rat enhanced B-50 immunoreactivity was observed in regenerating nerve fibres and in newly formed axon terminals. However, before reinnervation was apparent, an unexpected transient increase in B-50 immunoreactivity was observed at denervated motor endplates [J. Neurosci. 8 (1988) 1759]. This study was performed to clarify this observation. Four days following facial nerve crush B-50 immunoreactivity was detected by double immunofluorescence microscopy in Sl00-positive Schwann cells covering the denervated endplates. Using diluted polyclonal and monoclonal B-50 antibodies we found that B-50 immunoreactivity at the denervated motor endplates was strongly increased in comparison to innervated motor endplates in which B-50 immunoreactivity was hardly detectable. However, when a high concentration of B-50 antibodies was applied the normal innervated motor endplates were also B-50 immunoreactive. Muscle fibres did not display B-50 immunoreactivity. Northern blot analysis revealed elevated B-50 mRNA in denervated muscle and in degenerating nerve with respect to the controls. The B-50 mRNA levels in these non-neuronal tissues were very low compared to the intact and injured facial nucleus containing the neuronal cell bodies. Electron microscopy demonstrated that the B-50 protein was localized in the processes of Schwann cells covering axon terminals of intact and vacant motor endplates and in axon varicosities of sympathetic nerves. This study has confirmed that prior to reinnervation B-50 immunoreactivity is increased at denervated motor endplates and shows that B-50 is co-localized with S100 in Schwann cells. Therefore, upregulation of B-50 expression in Schwann cells may explain the early occurrence of B-50 immunoreactivity at the motor endplate.

Demyelinating diseases such as multiple sclerosis or the Guillain-Barré syndrome originate from an autoimmune response resulting in the degradation of myelin and impaired neuronal function. Prophylactic administration of the neurotrophic peptide, H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH [an ACTH(4-9) analogue], to rats with experimental allergic neuritis, a model for the Guillain-Barré syndrome, markedly suppresses the clinical symptoms, protects against loss of motor coordination and prevents the degeneration of myelinated axons in the affected peripheral nerve. Therefore, this peptide may provide a new approach to the therapy of peripheral demyelinating polyneuropathies.

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.

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B-50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X-100 (TX-100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B-50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B-50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP-expressing neurons occurs only after peripheral lesion with TX-100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B-50/GAP43-expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP-expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B-50/GAP43 and OMP at long times after peripheral lesion with TX-100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.

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.

Although much progress has been made, current treatments of peripheral nerve damage mostly result in only partial recovery. Local production of neurite outgrowth-promoting molecules, such as neurotrophins and/or cell adhesion molecules, at the site of damage may be used as a new means to promote the regeneration process. We have now explored the ability of an adenoviral vector encoding the reporter gene LacZ (Ad-LacZ) to direct the expression of a foreign gene to Schwann cells of intact and crushed rat sciatic nerves. Infusion of 8 x 10(7) PFU Ad-LacZ in the intact sciatic nerve resulted in the transduction of many Schwann cells with high levels of transgene expression lasting at least up to 12 days following viral vector administration. The efficacy of adenoviral vector delivery to a crushed nerve was investigated using three strategies. Injection of the adenoviral vector at the time of, or immediately after, a crush resulted in the transduction of only a few Schwann cells. Administration of the adenoviral vector the day after the crush resulted in the transduction of a similar number of Schwann cells 5 days after administration, as observed in uncrushed nerves. Regenerating nerve fibers were closely associated with beta-galactosidase-positive Schwann cells, indicating that the capacity of transduced Schwann cells to guide regenerating fibers was not altered. These results imply that the expression of growth-promoting proteins through adenoviral vector-mediated gene transfer may be a realistic option to promote peripheral nerve regeneration.

Publisher Summary Semaphorins comprise a large family of secreted and transmembrane anchored glycoproteins, several members of which have been implicated in repulsive axon guidance. Members of the semaphorin family display specific patterns of expression and are predominantly found within the developing embryo. This chapter discusses the data indicating that in the developing nervous system Sema III plays a critical role in repulsive axon guidance, nerve fasciculation, and the timing of target innervation of a number of neuropilin-1 positive fibers tracts. In addition, the distribution of Sema III binding sites, as assessed by direct binding of a chimeric Sema III-AP probe to sections, neuropilin-1 protein distribution, and sema III RNA expression are compared to elucidate where in the embryo Sema III is likely to function as a guidance cue. Finally, continued adult expression of sema III and neuropilin-1 in distinct structures of the mammalian nervous system is discussed in the context of the role Sema III might play in structural plasticity.

Expression of the growth-associated protein B50 (GAP-43) mRNA in dorsal root ganglia (DRG) of rats was studied by in situ hybridization. In response to treatment with the neurotoxic agent cisplatin, B50 mRNA expression was significantly enhanced following a cumulative cisplatin dose of 14 mg/kg. In the untreated age-matched control animals, only half of the ganglion cells exhibited expression of B50 mRNA (mean hybridization signal, 10 times background), whereas at a cumulative cisplatin dose of 14 mg cisplatin every neuron exhibited well above background expression (mean hybridization signal, 34 times background). Cotreatment with a neuroprotective ACTH4-9 analog known to prevent cisplatin neuropathy in rats did not affect the overall expression of B50 mRNA. However, in the subpopulation of large sensory neurons, B50 mRNA content was significantly higher in the group cotreated with the ACTH4-9 analog as compared with the saline-cotreated group after 14 mg/kg of cisplatin. We conclude that in analogy with the well-known upregulation of B50 mRNA following mechanical nerve lesions, treatment with the neurotoxic drug cisplatin also leads to an increase in B50 mRNA expression. This observation lends strength to the hypothesis that in neuropathies an imbalance between regenerative and degenerative mechanisms exists. The ability of the larger sensory neurons to retain an increased B50 mRNA expression better after cotreatment with the peptide than without may be related to stimulation of regenerative processes by this ACTH4-9 analog.

The neurotrophic peptide Org 2766 accelerates the regeneration of peripheral nerves. Although the mechanism of action of this neuropeptide is not yet understood, functional, pharmacological, and morphological evidence has demonstrated that Org 2766 exerts its beneficial effect during the early stages of nerve regeneration. The induction of some members of the Immediate Early Gene (IEG) family such as c-jun and c-fos is one of the first molecular events following peripheral nerve damage. The Fos and Jun proteins act as a transcription factor and may stimulate the expression of a number of genes implicated in nerve regeneration. We examined whether Org 2766 stimulates nerve regeneration by enhancing or prolonging the expression of c-fos mRNA. Following a crush lesion of the sciatic nerve, the expression of c-fos mRNA was induced in the spinal cord and in the damaged nerve at 30 min following injury in untreated animals as demonstrated with Northern blot. No effect of the crush lesion was observed in dorsal root ganglia (DRG). The induction of c-fos mRNA in the damaged nerve was more robust as compared to the relatively small induction observed in the spinal cord. With in situ hybridization an increase in c-fos mRNA expression both in the dorsal and in the ventral horn of the spinal cord was demonstrated at 30 min post-lesion. In the distal sciatic nerve portion the expression of c-fos mRNA was predominantly localized around Schwann cell nuclei at 30 min after nerve crush. The effect of Org 2766 treatment on the expression of c-fos mRNA was investigated using semiquantitative dot blots. Dot blots containing RN A samples of DRG, spinal cord or sciatic nerve of control animals, saline treated and Org 2766 treated animals isolated 15 min, 30 min, l hr, 1 day and 2 days following nerve crush were scanned densitometrically. No significant effect of Org 2766 on the expression of c-fos mRNA was detected. We conclude that Org 2766 does not stimulate peripheral nerve regeneration by affecting the expression of the c-fos mRNA following sciatic nerve crush. The putative effect of Org 2766 on other immediate early transcription factors of the fos and jun family is currently under investigation.

Publisher Summary This chapter reviews the evidence concerning the putative efficacy of neurotrophic peptides related to adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormones (MSH) in animal and human disorders of the peripheral nervous system. In two different experimental models of diabetic neuropathy, the efficacy of chronic treatment with the ACTH-analog was determined. The first model involved the single administration of the cytostatic drug streptozotocin, which is known to kill the β-cells in the islets of Langerhans and thus to increase blood glucose. The second model employed the BB/Wor rat, a strain of rats that genetically develop an autoimmune disease resulting in hyperglycaemia. These two model demonstrates the efficacy of the of neurotrophic peptides was apparent. Based on these data, a first clinical double blind study on the efficacy of chronic treatment with the ACTH-analog in diabetic neuropathy was performed involving 62 patients suffering from Type 1 diabetes mellitus randomly divided over a placebo and a peptide treatment group. During the treatment period of 12 months, a number of parameters for peripheral and autonomic neuropathy were assessed. The peptide treatment was effective in lowering the vibration threshold in patients with diabetic neuropathy.As their neurotrophic or protective efficacy is manifest irrespective of the nature of the threatening conditions, eventual application may involve many disorders that have the dysfunction of neurons in common.

Neurons in the central nervous system regenerate poorly or not at all. In contrast neurons of the peripheral nervous system have the ability to regrow their nerve fibers over considerable distances. Previously it has been suggested that the absence of the reinduction of the expression of growth associated proteins such as B-50 (GAP43) may be an important factor in the differential response of CNS and PNS neurons to injury. We studied B-50(GAP43) mRNA expression following lesioning of a class of CNS neurons, the olfactory bulb mitral cells. Expression of B-50 mRNA in approximately 40% of the mitral cells was upregulated in response to transection of their axons in the lateral olfactory tract (LOT). Enhanced expression persisted for 10 days postlesion but had virtually declined to control levels by 4 weeks after the lesion. A large proportion of the mitral cells gradually degenerated subsequent to LOT transection. Thus a subpopulation of mitral cells maintains their ability to upregulate B-50, a protein characteristic of growing axons, but enhanced B-50 expression is not accompanied by regeneration of the severed LOT.

We have detected mRNA for B-50 (GAP-43, pp46, F1, neuromodulin), which was originally believed to be a neuron-specific protein, in non-neuronal cells in the rat sciatic nerve. In control rats, the level of B-50 mRNA in sciatic nerve tissue was much lower than in dorsal root ganglia. Following nerve crush or transection, the expression of B-50 mRNA in the distal nerve stump increased dramatically between 1 and 2 days post-injury. The B-50 mRNA levels in the distal stump of crushed nerves remained elevated for up tp 4 weeks and subsequently returned to control levels after 7 weeks. In contrast, after nerve transection B-50 mRNA levels in the distal nerve portion continued to increase up to 7 weeks post-lesion. No changes in the levels of the B-50 transcript were observed in the proximal portion of either crush-lesioned or transected sciatic nerves. In situ hybridization demonstrated B-50 mRNA associated with Schwann cells in the distal nerve stump. The observation that Schwann cells are capable of producing B-50 mRNA was confirmed by Northern blot analysis of total RNA isolated from primary Schwann cell cultures. Taken together, these data show the expression of B-50 mRNA by Schwann cells and the up-regulation of B-50 mRNA in reactive Schwann cells upon loss of axonal contact.

Because the growth-associated protein B-50 (GAP-43) has been implicated in neurite outgrowth as well as in synaptic plasticity, we studied its light and electron microscopical distribution in the mouse olfactory bulb, an area of the nervous system which exhibits a high degree of synaptic plasticity. Immunofluorescent staining with monospecific affinity-purified anti-B-50 antibodies revealed that B-50 is most abundantly expressed in the olfactory nerve fibre layer and the granule cell layer neuropil, while little staining was observed in the external plexiform layer and in cell bodies. B-50 is absent from dendrites and myelinated axons as indicated by double labelling with monoclonal antibodies against microtubule-associated protein 2 and the large neurofilament protein, respectively. Using post-embedding immunogold labelling on ultrathin Lowicryl sections, B-50 was found to be highly concentrated in presumed growth cones in the olfactory nerve fibre layer and in thin unmyelinated axons and presynaptic terminals in the granule cell layer neuropil. Near background immunolabelling was seen in perikarya, dendrites and myelinated axons. In view of the implication of B-50 in plasticity-related phenomena, its abundance in the thin unmyelinated preterminal axons suggests that these are potential sites of extrasynaptic plasticity.

Publisher Summary The olfactory neuroepithelium exhibits neurogenesis throughout adult life, and in response to lesions, a phenomenon that distinguishes this neural tissue from the rest of the mammalian brain. This chapter reviews studies on the expression of B50/GAP43 during ontogeny of the olfactory system and following lesioning. This analysis includes the characterization of the expression of olfactory marker protein (OMP), a protein expressed in mature olfactory neurons, as well as protein kinase-C and calmodulin. The latter two molecules are of particular interest to the function of B50/GAP43 because the degree of phosphorylation of B50/GAP43 appears to determine B50/GAP43's ability to bind calmodulin. In the mature olfactory epithelium, B50/ GAP-43 expression is restricted to a subset of cells located in the basal region. Because the expression of B50/GAP43 is high in developing and regenerating nerve cells, the B50/GAP43 positive cells are new neurons derived from the stem cells in the basal region of the epithelium. B50/GAP43 is absent from the stem cells themselves and also from the mature OMP-expressing neurons.

A new semaphorin is defined as a fully sequenced gene, cDNA, or protein which by sequence homology falls into the semaphorin family of proteins. We have established a Semaphorin Nomenclature web site (http://www.semaphorin-nomenclature.bs.jhmi.edu) that provides details for assigning appropriate names and numbers to new semaphorins, apparent orthologs, gene loci, splice variants, or EST sequences.We recommend that this system of nomenclature be used in all future publications.Semaphorin Nomenclature Committee33441The following scientists have endorsed the use of this nomenclature system: J.A. Bamberg, S. Baumgartner, H. Betz, J. Bolz, A. Chedotal, C.R.L. Christensen, P.M. Comoglio, J.G. Culotti, P. Doherty, H. Drabkin, A. Ensser, M.C. Fishman, B. Fleckenstein, G.J. Freeman, H. Fujisawa, A. Ghosh, D.D. Ginty, C.S. Goodman, S. Guthrie, S. Inagake, R. Keynes, T. Kimura, M. Klagsbrun, A.L. Kolodkin, J.Y. Kuwada, Y. Luo, J.D. Minna, S.L. Naylor, T.P. O’Connor, D.D.M. O’Leary, A. Pini, M.-m. Poo, A.W. Puschel, J.A. Raper, J. Roche, C.J. Shatz, W.D. Snider, E. Soriano, M.K. Spriggs, S.M. Stritmatter, S. Sullivan, L. Tamagnone, M. Tessier-Lavigne, T. Tohyama, J. Verhaagen, F.S. Walsh, and T. Yagi.2Three-letter species designations are based on the first letter of the genus and the first two letters of the species. Additional details about gene nomenclature conventions can be obtained at the Human Gene Nomenclature web site (http://www.gene.ucl.ac.uk/nomenclature/) and the links therein.3The appropriate citation for this letter is: (Semaphorin Nomenclature Committee, 1999).4The committee consists of: please see the list of authors.

The olfactory neuroepithelium exhibits neurogenesis throughout adult life, and in response to lesions, a phenomenon that distinguishes this neural tissue from the rest of the mammalian brain. The newly formed primary olfactory neurons elaborate axons into the olfactory bulb. Thus, denervation and subsequent re-innervation of olfactory bulb neurons may occur throughout life. In this study the authors demonstrate the distribution of the growth-associated phosphoprotein B-50/GAP43 and its mRNA in the olfactory neuroepithelium and olfactory bulb during development and aging. In neonatal rats B-50/GAP43 mRNA was expressed in primary olfactory neurons throughout the olfactory epithelium and in their target neurons in the olfactory bulb, the mitral, juxtaglomerular and tufted cells. In contrast, in adult (7.5 weeks) and aging animals (6 - 18 months of age) B-50/GAP43 mRNA expression was progressively restricted to neurons in the basal region of the neuroepithelium and to some of their target mitral and juxtaglomerular cells in the olfactory bulb. The continuing expression of B-50/GAP43 mRNA in mitral- and juxtaglomerular cells in mature animals is thought to be related to their capacity to respond to continuously changing input from the primary olfactory neurons present in the olfactory neuroepithelium.

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.

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 olligonucleotides 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 effects 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.

Peptides related to alpha-MSH (collectively termed melanocortins) stimulate nerve growth following injury and may play a physiological role in the repair process. Melanocortins are not normally present in mature peripheral nerves but MSH-like bioactivity has been observed in extracts of injured nerves. alpha-MSH could derive from reexpression of the POMC prohormone in injured nerves or from proteolysis of the intermediate-size neurofilament protein that bears antigenic similarities to melanocortins. Using a radioimmunoassay that will distinguish between alpha-MSH and neurofilament-derived fragments, we have shown that alpha-MSH is not present (detection limit 74 pg alpha-MSH/mg protein) in damaged rat sciatic nerves.

The effects of ACTH 4–10 , a peptide fragment of corticotropin, on rat dorsal root ganglia (DRG), spinal cord and sciatic nerve were studied following a crush lesion of the sciatic nerve. The in vitro total protein synthesis rate of DRG L4, L5 and L6, measured one and three days after ipsilateral nerve crush, were not altered by various ACTH 4–10 treatment regimes. Likewise, neither ACTH 4–10 treatment of sham-operated rats nor in vitro exposure of control ganglia to peptide, resulted in changes in synthesis rate. Four days after crush lesion, the amounts of actin and tubulin in the ventral horn L2–L5 region of the spinal cord and of actin in DRG L5 were estimated following 2-dimensional separation. No significant effect of ACTH treatment was found. Degeneration-associated changes in the protein profiles of segments of sciatic nerve were not altered by ACTH 4–10 treatment. The data are discussed in relation to the possible site of action of neurotrophic ACTH-like peptides.

Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5–5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.

B50/GAP43 is a neuron-specific phosphoprotein whose expression is associated with neural development and synaptic plasticity. Its postnatal ontogeny was investigated in the primary olfactory pathway of the rat using immunohistochemical methods. The unique ability of the olfactory neuroepithelium to generate new neurons from a population of precursor cells present in the basal cell layer of this tissue makes it a valuable model in the study of neural development. In newborn rats B50/GAP43 is present throughout the entire population of olfactory receptor neurons. These cells are stained throughout, from the ciliated dendritic knob to their axon terminals in the bulb. This appears to be the first example of unambiguous B50/GAP43 expression in dendritic processes. With increasing age the distribution of this protein becomes progressively restricted to a subpopulation of olfactory neurons. Comparison of the expression of B50/GAP43 and the olfactory marker protein (OMP), a polypeptide only present in mature olfactory neurons, revealed that during postnatal development of the olfactory system these 2 proteins are expressed in a nearly reciprocal fashion. In adult animals (3.5 months-6 months of age), B50/GAP43-positive cells are exclusively present adjacent to the basal cell layer of the neuroepithelium. Basal cells appear to be unstained. The region of the epithelium containing the B50/GAP43-positive cells is virtually devoid of OMP-positive neurons. A significant fraction of these B50/GAP43- containing cells bear dendritic and neuritic processes. However, these cells do not express olfactory cilia. It is probable that the olfactory neurons expressing the growth-associated B50/GAP43 protein may correspond to a particular subset of olfactory neurons at an intermediate state of maturation.

As reported previously the prominent protein kinase C substrate protein B-50 is present in growth cones isolated from fetal rat brain and in outgrowing hippocampal neurites. These findings suggest that B-50 plays a role in axonal growth during development of the nervous system. In the present paper the fate of B-50 is investigated in regenerating rat sciatic nerve. Using affinity-purified anti-B-50 antibodies B-50 levels have been compared in crushed and contralateral intact nerves by means of immunoblotting and radioimmunoassay. B-50 levels in the crushed nerve increased 5.3-fold as compared to non-crushed controls. Furthermore, the cellular localization of B-50 has been assessed by immunohistochemistry. Virtually no B-50 immunoreactivity was seen in control nerves, but bright immunofluorescence appeared in regenerating sprouts. Our data are in line with current evidence from several laboratories that B-50 is a member of a small family of growth-associated proteins and support the hypothesis that B-50 is involved in axonal growth.

Publisher Summary This chapter discusses the neurotrophic action of melanocortins. Upon binding to specific receptors on the outside of the cell membrane, peptide hormones initiate a specific physiological response in their target cells. For adrenocorticotropin hormone (ACTH) and the adrenal cortex cell, this response involves the enhanced synthesis and release of corticosteroid hormone. The chapter discusses the trophic influence of melanocortins in repair of neural damage; the possible role of these peptides in brain and body development and growth is surveyed. The neurotrophic effect of these peptides are related to a naturally occurring signal early in the regeneration process, and such peptides or their active analogs thus may prove to be a new lead in the research for effective pharmacotherapy of nerve damage. The production of a neurotrophic peptide in the injured nerve is by no means the only or the predominant event in the repair process.

The neuron-specific phosphoprotein B-50 was originally identified as a phosphoprotein in synaptic plasma membranes isolated from adult brain tissue. In this paper we study the reinnervation of the soleus muscle, a target muscle of sciatic nerve axons, using affinity-purified anti-B- 50 antibodies. Light-microscopical evaluation of the reinnervation process revealed that the period of muscle fiber reinnervation corresponds closely with the time in which high B-50 immunoreactivity was observed in the nerve fibers that invade the muscle and in the newly formed neuromuscular junctions. Upon completion of reinnervation, B-50 immunoreactivity decreased. In the newly innervating terminals, B- 50 was associated with presynaptic vesicular structures and with the presynaptic plasma membrane. In intact mature neuromuscular junctions, virtually no B-50 immunoreactivity could be detected with either light- or electron-microscopic procedures. These observations corroborate the association of high levels of B-50/GAP43 during axon outgrowth and support the concept that B-50 may be a key molecule in the reconstruction of axonal structures. We also observed an unexpected transient increase in B-50 immunoreactivity in the degenerating neuromuscular junctions. This observation cannot be explained in terms of increased neuronal synthesis of B-50, since the degenerating axon processes have been completely disconnected from their cell bodies. Thus, our evidence implies that a rise of B-50 immunoreactivity can be associated with stages of neuronal degeneration as well as with those of neuronal differentiation and axon outgrowth.

The ability of α-MSH to facilitate the recovery of sensorimotor nerve function following crush lesion is restricted to a critical period following such a lesion. This period coincided with the initiation of sprouting and the disappearance of the 150 kD neurofilament protein from the degenerating distal stump of the nerve. Degenerating nerve contains a factor that is active in a bioassay system for MSH. This factor could not be detected in control nerves. The hypothesis is forwarded that a neurotrophic factor known to be present in degenerating nerve stumps is an α-MSH-like peptide formed by the breakdown of the 150 kD neurofilament protein.

Adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate the initial sprouting response in the crushed rat sciatic nerve. In this report a detailed analysis of the neurotrophic action of Org.2766 [a degradation resistant ACTH(4-9) analog] and alpha-MSH is described. Org.2766 treatment results in enhanced numbers of outgrowing sprouts in the damaged nerve. The growth velocity of the sprouts is not affected. The peptide effect is dose-dependent. A single peptide injection administered immediately following the crush stimulates the formation of sprouts significantly. Continued high blood levels of Org.2766 are probably not critical for the neurotrophic effect of these peptides, since a more moderate dosing protocol (injections given every 48 hr) was more effective than more frequent injections (injections given every 12 hr). The present results further the understanding of the mode of action of ACTH/alpha-MSH-like peptides and underscore the necessity to test a wide range of doses and injection protocols to avoid false negative results in clinical work being planned to start in the near future.

Severing of a nerve is accompanied by an assortment of morphological and biochemical changes in the neuronal cell bodies, the axonal processes and in the neuronal interstitium. In the peripheral nervous system (PNS) axons normally respond to damage by axonal regeneration, while in the central nervous system (CNS) axons generally regenerate poorly or not at all. The successful regeneration of axons appears to be dependent on the establishment of a neuronal micro-environment which is favorable for axonal regrowth. The importance of trophic factors and extracellular surface components in sustained axonal regrowth has been demonstrated widely (Berg 1984). The difference in the regenerative capacities of CNS and PNS neurons seems largely based on the failure of CNS tissue to provide the damaged axon with a milieu appropriate for axonal regeneration. This has, for instance, been shown in experiments in which damaged CNS axons were exposed to grafts of sciatic nerve tissue. One month after implantation of these PNS grafts, a large number of CNS axons had grown into the graft for distances of 3 cm or more. In the absence of the PNS graft the severed CNS axons would only have exhibited some abortive sprouting (Aguayo et al. 1982). Thus, CNS axons are not necessarily unable to regenerate, but it seems that the CNS environment is not permissive for axonal sprout formation.

Immunoblot techniques and immunohistochemistry have been used to investigate epitopes shared by alpha-melanocyte-stimulating hormone (alpha-MSH) and the 150-kD neurofilament protein (NF150). Three anti-alpha-MSH antisera (namely 31H2T, 4394, and M5) from a total of 12 sera were found to react with NF150. The three crossreacting sera are different in their binding properties to peptide fragments related to alpha-MSH, suggesting that at least two distinct epitopes are shared by NF150 and alpha-MSH. The only known sequence common to NF150 and alpha-MSH, the N-terminal Ac-S-Y residues, appears to be essential for binding of sera 31H2T and 4394. However, the binding of antiserum M5 involves other, currently unknown, similarities between NF150 and alpha-MSH. This is shown by the binding of M5 to peptides such as ACTH(4-10), which do not contain the N-terminal Ac-S-Y sequence. Binding of M5 to tobacco mosaic virus coat protein (TMV-coat protein), which is homologous with alpha-MSH and NF150 in its Ac-S-Y residues, was negligible. The peptide structures that are recognized by M5 have previously been shown to exert neurotrophic activity. The data are discussed in the light of the hypothesis that similarity between NF150 and alpha-MSH, as illustrated by binding to M5, may be significant in the neurotrophic activity of MSH-related peptides.