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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.

STUDY DESIGN: Discussion document. OBJECTIVES/METHODS: To review the Research Strategy of the International Spinal Research Trust (ISRT), which identifies key areas of basic and clinical research that are likely to be beneficial in developing potential treatments for spinal cord injury for funding. This strategy is intended to both guide the programme of research towards areas of priority and stimulate discussion of the different avenues of research. This latest document has been developed to take into account the scientific progress in the 6 years since publication of the previous Research Strategy. RESULTS/DISCUSSION: The latest scientific developments in research designed to repair the spinal cord and restore function following injury and how they might impact on spinal cord injury research are highlighted.

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.

Chronic experimental allergic encephalomyelitis (CEAE) is an established experimental model for multiple sclerosis (MS). The demyelinating lesions in the white matter of the central nervous system observed in CEAE and in MS are accompanied by various neurophysiological alterations. Among the best defined electrophysiological abnormalities are the changes in event-related potentials, in particular evoked potentials involving the spinal cord, i.e. motor and sensory evoked potentials. Less familiar are the changes observed in the electroencephalogram of CEAE-affected animals, which are also encountered in the human equivalent, MS. In the present experiment we evaluated the therapeutic value of a neurotrophic peptide treatment [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH, an ACTH4-9 analogue] and its effect on the delayed flash visual evoked potentials (VEP) and power spectra of the electroencephalogram, during a 17-week follow-up of CEAE. CEAE animals treated with the neurotrophic peptide were protected against the development of neurological symptoms during the course of the demyelinating syndrome. VEPs of animals suffering from CEAE showed a delay of the latencies of the late components which was significantly counteracted by peptide treatment. The peak-to-peak amplitude of the VEP afterdischarge recorded from CEAE animals was significantly increased during the course of CEAE and correlated closely with the progression of the myelinopathy. Furthermore, CEAE animals showed an increase of electroencephalogram (EEG) beta activity of up to 500% as compared with the age-matched control group. This increase in beta power mainly consisted of a prevailing 20-21 Hz peak, a frequency that normally is not dominant in control EEG recordings of the rat during passive wakefulness. All these electrophysiological phenomena were absent in ACTH4-9 analogue-treated animals. The present findings underscore the potential importance of a neurotrophic peptide treatment in the pharmacotherapy of central demyelinating syndromes, and possibly of MS.

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.

Ubiquitin-B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimer's disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquitinate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin-proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin-1 and plexin-A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A-positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A-positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin-C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin-C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.

Chronic experimental allergic encephalomyelitis (CEAE) is an established experimental model for multiple sclerosis (MS). The demyelinating lesions in the white matter of the central nervous system observed in CEAE and in MS are accompanied by various neurophysiological alterations. Among the best defined electrophysiological abnormalities are the changes in event-related potentials, in particular evoked potentials involving the spinal cord, i.e. motor and sensory evoked potentials. Less familiar are the changes observed in the electroencephalogram of CEAE-affected animals, which are also encountered in the human equivalent, MS. In the present experiment we evaluated the therapeutic value of a neurotrophic peptide treatment [H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH, an ACTH4-9 analogue] and its effect on the delayed flash visual evoked potentials (VEP) and power spectra of the electroencephalogram, during a 17-week follow-up of CEAE. CEAE animals treated with the neurotrophic peptide were protected against the development of neurological symptoms during the course of the demyelinating syndrome. VEPs of animals suffering from CEAE showed a delay of the latencies of the late components which was significantly counteracted by peptide treatment. The peak-to-peak amplitude of the VEP afterdischarge recorded from CEAE animals was significantly increased during the course of CEAE and correlated closely with the progression of the myelinopathy. Furthermore, CEAE animals showed an increase of electroencephalogram (EEG) beta activity of up to 500% as compared with the age-matched control group. This increase in beta power mainly consisted of a prevailing 20-21 Hz peak, a frequency that normally is not dominant in control EEG recordings of the rat during passive wakefulness. All these electrophysiological phenomena were absent in ACTH4-9 analogue-treated animals. The present findings underscore the potential importance of a neurotrophic peptide treatment in the pharmacotherapy of central demyelinating syndromes, and possibly of MS.

Sensory axons interrupted in the dorsal roots of adult mammals are normally unable to regenerate into the spinal cord. We have investigated whether the introduction of a neurotrophin gene into the spinal cord might offer an approach to otherwise intractable spinal root injuries. The dorsal roots of the 4th, 5th, and 6th lumbar spinal nerves of adult rats were severed and reanastomosed. Fourteen to nineteen days later, adenoviral vectors containing either the LacZ or NT-3 genes were injected into the ventral horn of the lumbar spinal cord, resulting in strong expression of the transgenes in glial cells and motor neurons between 4 and 40 days after injection. When dorsal root axons were transganglionically labelled with HRP conjugated to cholera toxin subunit B, 16 to 37 days after dorsal root injury, large numbers of labelled axons could be seen to have regenerated into the cord, but only in those animals injected with vector carrying the NT-3 gene. The regenerated axons were found at the injection site, mainly in the grey matter, and had penetrated as deep as lamina V. Gene therapy with adenoviral vectors encoding a neurotrophin has therefore been shown to be capable of enhancing and directing the regeneration of a subpopulation of dorsal root axons (probably myelinated A fibres), into and through the CNS environment.

The present study was undertaken to determine the effects of neurotrophin-3 (NT3) and spontaneous bioelectric activity (SBA) on dendritic elongation and branching in long-term isolated organotypic explants of rat neocortex. Viral vector-directed expression of NT3 was used as an effective means to ensure a continuous, local production of the neurotrophic factor. Quantitative light microscopic measurement of dendritic branching patterns was carried out on Golgi-stained materials. Explants were exposed to an adenoviral vector encoding the genetic sequence for neurotrophin-3 (Ad-NT3), or to exogenous additions of the neuropeptide NT3. In order to test for activity-dependent growth effects under control and experimental conditions, explants were exposed to glutamatergic blockade using a cocktail of APV and DNQX. Both Ad-NT3 and NT3 peptide potently promoted apical and basal dendritic growth (elongation and branching) in pyramidal neurons. This growth was observed to be significant in layers II-IV and V. These growth effects were also not activity dependent, inasmuch as they were elicited from explants in which spontaneous bioelectric activity had been suppressed. Non-pyramidal neurons, throughout the neocortical slice, showed no significant dendritic responses to the prolonged presence of NT3. These findings show that pyramidal dendritic growth in long-term neocortical explants responds to at least one neurotrophic growth factor, NT3, and is independent of intrinsic bioelectric activity. The use of viral vectors in delivering a continuous high level of neurotrophic factor within developing neural tissues demonstrates its potential application to in vivo tissues during development, or in the stimulation of neuritogenesis and neuroregeneration following injuries.

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.

Alterations in neuronal connectivity of the mature central nervous system (CNS) appear to depend on a delicate balance between growth-promoting and growth-inhibiting molecules. To begin to address a potential role of the secreted chemorepulsive protein semaphorin(D)III/collapsin-1 (semaIII/coll-1) in structural plasticity during adulthood, we used high-resolution nonradioactive in situ hybridization to identify neural structures that express semaIII/coll-1 mRNA in the mature rat and human brain. SemaIII/coll-1 was expressed in distinct but anatomically and functionally linked structures of the adult nervous system. The olfactory-hippocampal pathway displayed semaIII/coll-1 expression in a continuum of neuronal structures, including mitral and tufted cells of the olfactory bulb, olfactory tubercle, and piriform cortex; and distinct nuclei of the amygdaloid complex, the superficial layers of the entorhinal cortex, and the subiculum of the hippocampal formation. In addition, prominent labeling was found in neuronal components of the motor system, particularly in cerebellar Purkinje cells and in subpopulations of cranial and spinal motoneurons. Retrograde tracing combined with in situ hybridization also revealed that the staining of semaIII/coll-1 within the entorhinal cortex was present in the stellate neurons that project via the perforant path to the molecular layer of the dentate gyrus. Like in the rat, the human brain displayed discrete expression of semaIII/coll-1. Among the structures examined, the most prominent staining was observed in the cellular islands of the superficial layers of the human entorhinal cortex. The constitutive expression of the chemorepellent semaIII/coll-1 in discrete populations of neurons in the mature rat and human CNS raises the possibility that, in addition to its function as repulsive axon guidance cue during development, semaIII/coll-1 might be involved in restricting structural changes that occur in the wiring of the intact CNS.

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.

The neurotrophins are a family of proteins that promote neuronal survival and neurite outgrowth during development and can also enhance the regeneration of injured adult neurons. The local and continuous delivery of these proteins at the site of injury is problematic, since this requires repeated intraparenchymal injections or the use of invasive canula-micropump devices. In the present study we report the generation and characterization of an adenoviral vector for a member of the neurotrophins, neurotrophin-3 (Ad-NT-3). Using Ad-NT-3, we examined the expression and biological activity of NT-3 in dorsal root ganglia (DRG) explant cultures. Gene transfer with Ad-NT-3 results in the synthesis of genuine NT-3 and in a dosage-dependent neurite outgrowth response in DRG explants. Transduction of DRG explants with a viral vector dosage of 5 x 10(5) to 5 x 10(6) plaque-forming units induced the formation of a dense halo of neurites comparable to outgrowth observed following the addition of 100 ng/mL exogenous NT-3. In addition, a single infection with Ad-NT-3 produced biologically active NT-3 for at least 20 days in culture, as evidenced by continued neurite extension. This indicates that adenoviral vector-mediated expression of NT-3 results in high-level production of biologically active NT-3 and could therefore be used as a strategy to promote the regeneration of injured peripheral and central nerve projections.

Proton magnetic resonance imaging enables non-invasive monitoring of lesion formation in multiple sclerosis and has an important role in assessing the potential effects of therapy. T2-weighted and short tau inversion recovery magnetic resonance imaging were used to assess the effect of a neurotrophic adrenocorticotrophic hormone4-9 analogue [H-Met(O2)-Glu-His-Phe--Lys-Phe-OH] on the volume of lesions in the brains of rats suffering from chronic experimental allergic encephalomyelitis, an animal equivalent of multiple sclerosis. Lesion volume was monitored during a five-month period. Magnetic resonance imaging indicated that treatment with the adrenocorticotrophic hormone4-9 analogue significantly reduced the lesion volume by 84 and 85% 10 and 20 weeks after lesion induction, respectively. Furthermore, peptide treatment significantly reduced chronic experimental allergic encephalomyelitis-related neurological symptoms during the chronic phase of the disease (week 3 until week 20 after lesion induction). Both functional and morphological recovery were considerably advanced by peptide treatment. Twenty weeks after lesion induction rats with chronic experimental allergic encephalomyelitis were killed for histological analysis, to correlate magnetic resonance imaging findings with morphological changes. The regions of abnormally high signal intensities on T2-weighted magnetic resonance images coincided with areas of demyelination and concomitant widespread inflammatory infiltration, oedema formation and enlarged ventricles. The improved neurological status and the 84% reduction in the lesion volume in the cerebrum of rats chronic experimental allergic encephalomyelitis point to the potential value of trophic peptides in the development of strategies for limiting the damage caused by central demyelinating lesions in syndromes such as multiple sclerosis.

Semaphorin III/collapsin-1 (semaIII/coll-1) is a chemorepellent that exhibits a repulsive effect on growth cones of dorsal root ganglion neurons. To identify structures that express semaIII/coll-1 in developing mammals, we cloned the rat homologue and performed in situ hybridization on embryonic, neonatal, and adult rats. The relationship between semaIII/coll-1 mRNA distribution and developing nerve tracts was studied by combining in situ hybridization with immunohistochemistry for markers of growing nerve fibers. At embryonic day 11, semaIII/coll-1 expression was restricted to the olfactory pit, the basal and rostral surface of the telencephalic vesicle, the anlage of the eye, the epithelium of Rathke's pouch, and the somites. At later developmental stages, semaIII/coll-1 mRNA was found to be widely distributed in neuronal as well as in mesenchymal and epithelial structures outside the nervous system. Strong expression was found in the olfactory bulb, retina, lens, piriform cortex, amygdalostriatal area, pons, cerebellar anlage, motor nuclei of cranial nerves, and ventral spinal cord. After birth, mesenchymal staining decreased rapidly and expression became progressively restricted to specific sets of neurons in the central nervous system (CNS). In the mature CNS, semaIII/coll-1 mRNA remains detectable in mitral cells, neurons of the accessory bulb and cerebral cortex, cerebellar Purkinje cells, as well as a subset of cranial and spinal motoneurons. The temporal and spatial expression pattern of semaIII/coll-1 mRNA and its relationship to emerging nerve tracts suggests that semaIII/coll-1 is involved in guiding growing axons towards their targets by forming a molecular boundary that instructs axons to engage in the formation of specific nerve tracts.

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.

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 possible involvement of alpha-MSH-like peptides in the regenerative response of peripheral nerves was investigated with a competitive antagonist of alpha-MSH, the synthetic hexapeptide [D-Trp7,Ala8,D-Phe10)alpha-MSH(6-11)-amide. Subcutaneous administration of the alpha-MSH antagonist during the first 10 days following sciatic nerve crush significantly decreased functional recovery as measured by the foot flick withdrawal test and the walking pattern analysis. Hypophysectomy delayed both the initial sprouting response and the outgrowth rate after major caudal nerve crush. When hypophysectomized rats were treated with the alpha-MSH antagonist, a further delay in initial sprouting was observed, whereas the outgrowth rate of nerve fibers was not affected. These results suggest that 1) endogenous alpha-MSH-like peptides stimulate nerve outgrowth following peripheral nerve injury and 2) alpha-MSH-like peptides derived from a source other than the pituitary may contribute to the physiological stimulus leading to sprouting.

Efficient methods to introduce and express therapeutic genes in the central and peripheral nervous system will find applications in the treatment of neurodegenerative diseases caused by single gene mutations or degeneration with a mechanical, metabolic, or immunological origin. The goal of our research is to develop experimental gene therapy, based on in vivo gene delivery with viral vectors, to promote neuroregeneration in the peripheral and central nervous system. This paper provides an overview of work to determine the capacity of herpes simplex and adenoviral vectors, encoding members of the neurotrophin gene family or the intraneuronal growth-associated protein B-50/GAP-43, to stimulate neurite outgrowth. Initial results demonstrate that viral vector-mediated transfer of genes encoding these neurite growth promoting molecules could be an effective strategy to enhance the growth potential of injured neurons. A number of biological and technical hurdles that have to be settled in order to move closer to future clinical applications will be discussed briefly.

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.