CNS Myelin Research Articles

The Nogo-66 Receptor Homolog NgR2 Is a Sialic Acid-Dependent Receptor Selective for Myelin-Associated Glycoprotein

The Nogo-66 receptor (NgR1) is a promiscuous receptor for the myelin inhibitory proteins Nogo/Nogo-66, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). NgR1, an axonal glycoprotein, is the founding member of a protein family composed of the structurally related molecules NgR1, NgR2, and NgR3. Here we show that NgR2 is a novel receptor for MAG and acts selectively to mediate MAG inhibitory responses. MAG binds NgR2 directly and with greater affinity than NgR1. In neurons NgR1 and NgR2 support MAG binding in a sialic acid-dependent Vibrio cholerae neuraminidase-sensitive manner. Forced expression of NgR2 is sufficient to impart MAG inhibition to neonatal sensory neurons. Soluble NgR2 has MAG antagonistic capacity and promotes neuronal growth on MAG and CNS myelin substrate in vitro. Structural studies have revealed that the NgR2 leucine-rich repeat cluster and the NgR2 "unique" domain are necessary for high-affinity MAG binding. Consistent with its role as a neuronal MAG receptor, NgR2 is an axonassociated glycoprotein. In postnatal brain NgR1 and NgR2 are strongly enriched in Triton X-100-insoluble lipid rafts. Neural expression studies of NgR1 and NgR2 have revealed broad and overlapping, yet distinct, distribution in the mature CNS. Taken together, our studies identify NgRs as a family of receptors (or components of receptors) for myelin inhibitors and provide insights into how interactions between MAG and members of the Nogo receptor family function to coordinate myelin inhibitory responses.

Repairing CNS myelin—astrocytes have to do their jobs

Chronic and progressive demyelination is a feature of a wide range of CNS disorders. Demyelination is a hallmark of multiple sclerosis (MS), and is commonly considered the major cause of symptoms in this disease, at least in its earlier phases. Demyelination is widespread also following compression injuries to the brain or spinal cord. This pathology may have a particularly heavy impact on the human spinal cord white matter with its large numbers of heavily myelinated axons. Recent findings indicate that demyelination may contribute to functional deteriorations associated with aging (Davatzikos and Resnick, 2002; Kovari et al., 2004), and to the pathogenesis of psychiatric disorders, notably schizophrenia (Davis et al., 2003; Flynn et al., 2003). The impact of demyelination pathology for the functional disturbances is more difficult to assess in these conditions because of the many other simultaneously ongoing or temporally overlapping pathological processes. In any case, demyelination is a pathological process with implications for diseases causing serious disability and distress, and affecting large numbers of patients. At the same time, restoring action potential conduction in demyelinated axons may lead to rapid recovery of functions. If demyelinated axons maintain functional synaptic target contacts, the return of impulse conduction in these axons may lead to functional improvement more or less immediately. Even if synaptic contacts have been lost, e.g., as a result of absence of activity, there may be rapid return of such contacts through terminal sprouting after action potential propagation has been resumed. Thus, the clinical benefits may be substantial from therapies leading to rapid and efficient remyelination of demyelinated axons. The myelinated axon is a highly elaborate structure, built to ensure faithful saltatory propagation of action potentials

Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo

Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75(NTR). To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p75(NTR)-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p75(NTR) plays a central role in this response. Neither NgR-nor p75(NTR)-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo.

Characterization of the murine splice variantMobp155: Developmental CNS expression pattern and subcellular localization of epitope-tagged protein

Members of the myelin-associated oligodendrocytic basic protein (MOBP) family constitute the third most abundant protein in CNS myelin. Although MOBP localizes to the major dense line (MDL) of CNS myelin, the function of the individual isoforms is unknown. Alternative splicing of pre-Mobp mRNA gives rise to six characterized splice variants in both the mouse and the rat. These splice variants share a common N-terminal encoded in Mobp exon 3 comprising 68 amino acids. The predicted protein isoforms differ in their C-termini. Sequence analysis of intron 3 revealed the presence of a putative initiation codon followed by an open reading frame (ORF) encoding 53 amino acids that extends in frame into Mobp exon 4 yielding a predicted MOBP isoform comprising 155 amino acids, designated MOBP155. This newly characterized isoform possessing a novel N-terminus shares a common C-terminus with MOBP170. Mobp170 message is detectable at low abundance throughout myelinogenesis. In contrast, the novel splice variant encoding MOBP155 is expressed at modest levels late in CNS development, coincident with the expression of the abundant splice variant, Mobp81A. Immunostaining of Cos7 cells transiently expressing an epitope-tagged MOBP155 suggested that most of the product was translocated to mitochondria. Although Mobp155 and Mobp170 encode a common predicted C-terminus they have different expression profiles and their products are targeted to mitochondria and the nucleus, respectively, in transiently transfected Cos7 cells.

Human myelination and perinatal white matter disorders

Human myelination progresses caudally to rostrally, beginning in the first trimester in the spinal cord, and continuing into the third decade in the intracortical fibers of the cerebral cortex. The most rapid changes occur between mid-gestation and the end of the second postnatal year. The major pediatric disorder of central white matter, periventricular leukomalacia (PVL), occurs during this critical developmental period, and is the predominant underlying substrate of cerebral palsy and other neurological handicaps in premature infants [ 1 Kinney H.C. Haynes R.L. Folkerth R.D. White matter lesions in the perinatal period. in: Golden J.A. Harding B. Pathology and genetics: acquired and inherited diseases of the developing nervous system. ISN Neuropathology Press, Basel2004: 156-170 Google Scholar , 2 Volpe J.J. Cerebral white matter injury in the premature infant—more common than you think. Pediatrics. 2003; 112: 176-180 Crossref PubMed Scopus (422) Google Scholar , 3 Kinney H.C. Armstrong D.L. Perinatal neuropathology. in: Graham D.I. Lantos P.E. Greenfield's neuropathology. 7th ed. Arnold, London2002: 557-559 Google Scholar ]. This disease affects the immature white matter of the cerebral hemispheres, and peaks at mid-to-late gestation, before cerebral white matter is myelinated. The focus of our laboratory is upon the delineation of the cellular and molecular pathology of PVL [ 4 Haynes R.L. Folkerth R.D. Keefe R.J. Sung I. Swzeda L.I. Rosenberg P.A. et al. Nitrative and oxidative injury to premyelinating oligodendrocytes in periventricular leukomalacia. J. Neuropathol. Exp. Neurol. 2003; 62: 441-450 Crossref PubMed Scopus (391) Google Scholar , 5 Folkerth R.D. Keefe R.J. Haynes R.L. Trachtenberg F.L. Volpe J.J. Kinney C. Interferon-γ expression in periventricular leukomalacia in the human brain. Brain Pathol. 2004; 14: 265-274 Crossref PubMed Scopus (89) Google Scholar ], and upon the regional, cellular, and biochemical sequences of human CNS myelination that are of major relevance to deciphering the pathogenesis of this devastating disorder [ 6 Brody B.A. Kinney H.C. Kloman A.S. Gilles F.H. Sequence of central nervous system myelination in human infancy: I. An autopsy study of myelination. J. Neuropathol. Exp. Neurol. 1987; 46: 283-301 Crossref PubMed Scopus (551) Google Scholar , 7 Kinney H.C. Brody B.A. Kloman A.S. Gilles F.H. Sequence of central nervous system myelination in human infancy: II. Patterns of myelination in autopsied infants. J. Neuropathol. Exp. Neurol. 1988; 47: 217-234 Crossref PubMed Scopus (607) Google Scholar , 8 Kinney H.C. Karthigasan J. Borenshteyn N.I. Flax J.D. Kirschner D.A. Myelination in the developing human brain: biochemical correlates. Neurochem. Res. 1994; 19: 983-996 Crossref PubMed Scopus (100) Google Scholar , 9 Back S.A. Luo N.L. Borenstein N.S. Levine J.M. Volpe J.J. Kinney H.C. Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury. J. Neurosci. 2001; 21: 1302-1312 PubMed Google Scholar , 10 Back S.A. Luo N.L. Borenstein N.S. Volpe J.J. Kinney H.C. Arrested oligodendrocyte lineage progression during human cerebral white matter development: dissociation between the timing of progenitor differentiation and myelinogenesis. J. Neuropathol. Exp. Neurol. 2002; 61: 197-211 PubMed Google Scholar , 11 Folkerth R.D. Haynes R.L. Borenstein N.S. Belliveau R.A. Trachtenberg R. Rosenberg P.A. et al. Developmental lag of superoxide dismutases relative to other antioxidant enzymes in premyelinated human telencephalic white matter. J. Neuropathol. Exp. Neurol. 2004; 63: 999-1009 Google Scholar , 12 Haynes, R.L., Folkerth, R.D., Borenstein, N.S., et al. Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol 2004 (In press). Google Scholar , 13 Billiards S. Haynes R.L. Folkerth R.D. Volpe J.J. Kinney H.C. The developmental profile of microglial density in the cerebral white matter of the human fetus and infant. Abstr.-Soc. Neurosci. 2004; : 609.19 Google Scholar ]. The over-riding hypothesis of our research is that premyelinating oligodendrocytes (pre-OLs) are the targeted cell population in PVL due to preferential vulnerability to free radical, glutamate, and cytokine toxicity in the developmentally immature white matter.

Pelizaeus–Merzbacher disease: pathogenic mechanisms and insights into the roles of proteolipid protein 1 in the nervous system

After over 50 years of study, a clear understanding of the biological roles of proteolipid protein 1 (PLP1) is lacking [1]. The PLP1 gene encodes two major products, PLP1 itself, and DM20, encoded by an alternatively spliced transcript, that lacks 35 residues from the cytoplasmic domain of PLP1. PLP1 comprises about half the protein mass of central nervous system myelin. PLP1 is also present in the peripheral nervous system, however it constitutes only a small fraction of myelin protein. PLP1 is a highly hydrophobic transmembrane protein. In addition to a high content of hydrophobic amino acids, PLP1 is anchored to the lipid bilayer by fatty acylation of several cysteine residues. DM20 is present at about 10% of the level of PLP1 in the CNS while in the PNS it is present at approximately equivalent amounts [2]. Because of its abundance in CNS myelin, PLP1 has generally been thought of as an important structural protein. Mutations of the PLP1 gene cause a broad range of clinical phenotypes that affect the central nervous system and, in some instances, the peripheral nervous system as well [3,4]. Analysis of the consequences of these mutations offers glimpses into the functions of PLP1, the most abundant protein in CNS myelin, as well as into the cellular effects of abnormalities in PLP1. Null mutations are the most informative for inferring the normal biological functions of a gene product. Belying its abundance in normal CNS myelin, complete lack of PLP1 and DM20 results in a surprisingly mild phenotype in both mice and in humans. Griffiths et al. [5] found that Plp1 knockout mice behave normally until about 15 months of age, when they develop progressive extremity weakness. Early myelination

Immunity to the Extracellular Domain of Nogo-A Modulates Experimental Autoimmune Encephalomyelitis

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE). Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.

Nogo-66 Receptor Prevents Raphespinal and Rubrospinal Axon Regeneration and Limits Functional Recovery from Spinal Cord Injury

Axon regeneration after injury to the adult mammalian CNS is limited in part by three inhibitory proteins in CNS myelin: Nogo-A, MAG, and OMgp. All three of these proteins bind to a Nogo-66 receptor (NgR) to inhibit axonal outgrowth in vitro. To explore the necessity of NgR for responses to myelin inhibitors and for restriction of axonal growth in the adult CNS, we generated ngr(-/-) mice. Mice lacking NgR are viable but display hypoactivity and motor impairment. DRG neurons lacking NgR do not bind Nogo-66, and their growth cones are not collapsed by Nogo-66. Recovery of motor function after dorsal hemisection or complete transection of the spinal cord is improved in the ngr(-/-) mice. While corticospinal fibers do not regenerate in mice lacking NgR, regeneration of some raphespinal and rubrospinal fibers does occur. Thus, NgR is partially responsible for limiting the regeneration of certain fiber systems in the adult CNS.

Exposure of rat optic nerves to nitric oxide causes protein S-nitrosation and myelin decompaction.

This study investigates the effect of nitric oxide (NO) on both the chemical modifications of CNS proteins and the architecture of the myelinated internode. Incubation of rat optic nerves for 2 h with 1 mM concentration of the NO-donors S-nitroso-N-acetyl-penicillamine (SNAP), ethyl-2-[hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), and 4-phenyl-3-furoxan carbonitrile (PFC) led to decompaction of myelin at the level of the intraperiod line (IPL). In contrast, incubation with 1 mM sodium nitroprusside, which slowly releases NO, sodium nitrite, and N-nitrosopyrrolidine failed to cause myelin disassembly. This suggests that free NO and/or some of its direct oxidation products (e.g., N2O3) are the active molecular species. NO-induced alterations in myelin architecture could not be assigned to protein or lipid degradation, lipid peroxidation, ATP depletion, calcium uptake, protein nitration, protein carbonylation, and nerve depolarization. NO-treatment, however, resulted in the S-nitrosation of a number of proteins. In myelin, one of the major S-nitrosated substrates was identified as proteolipid protein (PLP), an abundant cysteine-rich protein that is responsible for IPL stabilization. Peripheral nervous system myelin, whose stability depends on proteins other than PLP, was not decompacted upon incubation of sciatic nerves with SNAP. It is proposed that NO-mediated nitrosation of sulfhydryl groups is likely to interfere with the normal function of PLP and other important CNS myelin proteins leading to the structural demise of this membrane. These findings are relevant to multiple sclerosis and other inflammatory demyelinating disorders where both excessive NO production and myelin instability are known to occur.

Effect of Oligodendroglial Microdomain Components on NGF Signaling

Recent results indicate that the integrity of the myelin sheath is compromised with age [1], and that myelin destruction could be a precipitating event in age-related disorders [2]. Furthermore, oligodendrocytes (OL), which furnish the CNS myelin, do not support remyelination in older animals to the same extent as in younger animals. An increased process formation reflects an OL response to NGF, however, differentially depending on age of the donor [3]. We were interested to know as to whether NGF signaling is modulated by microdomain components such as caveolin and cholesterol, which might undergo age-related changes. Immunocytochemistry of cultured pig OL revealed the co-expression of caveolin-1 and the 140 kDa NGF receptor TrkA. Caveolin-containing microdomains were isolated via previously published buoyant density centrifugation methods (±Triton X-100) and by using MACS technology. Western blotting showed a co-labeling of the caveolar protein, flotillin-1, in addition to TrkA, which is enriched in the Triton X-100 insoluble fraction, p75 NTR, and p21 Ras. Preliminary results indicate that oligodendroglial caveolin and cholesterol are up-regulated from 8 DIV to 16 DIV, a period of extensive oligodendroglial process regeneration. Cells exposed to PEG-cholesterol increased their process formation; PEG-cholesterol plus NGF accelerated the NGF response; under both conditions, an in-gel-kinase assay demonstrated an increased MAPK activity, a step of the downstream TrkA signaling. On the other hand, exposure to cyclodextrin (1.2 mM), which disrupts caveolar microdomains by removing cholesterol from the plasma membrane, resulted in a less effective NGF response. The results at present indicate that NGF signaling is modulated by cholesterol; the effect of caveolin-1 siRNA is under investigation.

Promotion of axon regeneration by myelin-associated glycoprotein and Nogo through divergent signals downstream of Gi/G.

Several myelin-derived proteins have been identified as components of the CNS myelin that prevents axonal regeneration in the adult vertebrate CNS. Activation of RhoA has been shown to be an essential part of the signaling mechanism of these proteins. Here we report an additional signal, which determines whether these proteins promote or inhibit axon outgrowth. Myelin-associated glycoprotein (MAG) and Nogo trigger the intracellular elevation of Ca2+ as well as the activation of PKC, presumably mediated by G(i)/G. Neurite outgrowth inhibition and growth cone collapse by MAG or Nogo can be converted to neurite extension and growth cone spreading by inhibiting conventional PKC, but not by inhibiting inositol 1,4,5-triphosphate (IP3). Conversely, neurite growth of immature neurons promoted by MAG is abolished by inhibiting IP3. Activation of RhoA is independent of PKC. Thus, a balance between PKC and IP3 is important for bidirectional regulation of axon regeneration by the myelin-derived proteins.

A Structurally Available Encephalitogenic Epitope of Myelin Oligodendrocyte Glycoprotein Specifically Induces a Diversified Pathogenic Autoimmune Response

Multiple sclerosis is an inflammatory disease of the CNS that involves immune reactivity against myelin oligodendrocyte glycoprotein (MOG), a type I transmembrane protein located at the outer surface of CNS myelin. The epitope MOG92-106 is a DR4-restricted Th cell epitope and a target for demyelinating autoantibodies. In this study, we show that the immune response elicited by immunization with this epitope is qualitatively different from immune responses induced by the well-defined epitopes myelin basic protein (MBP) 84-96 and proteolipid protein (PLP) 139-151. Mice with MOG92-106-, but not with MBP84-96- or PLP139-151-induced experimental autoimmune encephalomyelitis developed extensive B cell reactivity against secondary myelin Ags. These secondary Abs were directed against a set of encephalitogenic peptide Ags derived from MBP and PLP as well as a broad range of epitopes spanning the complete MBP sequence. The observed diversification of the B cell reactivity represents a simultaneous spread toward a broad range of antigenic epitopes and differs markedly from T cell epitope spreading that follows a sequential cascade. The Abs were of the isotypes IgG1 and IgG2b, indicating that endogenously recruited B cells receive help from activated T cells. In sharp contrast, B cell reactivity in MBP84-96- and PLP139-151-induced experimental autoimmune encephalomyelitis was directed against the disease-inducing Ag only. These data provide direct evidence that the nature of the endogenously acquired immune reactivity during organ-specific autoimmunity critically depends on the disease-inducing Ag. They further demonstrate that the epitope MOG92-106 has the specific capacity to induce a widespread autoimmune response.

CD44 overexpression by oligodendrocytes: a novel mouse model of inflammation-independent demyelination and dysmyelination.

The CD44 transmembrane glycoprotein family has been implicated in cell-cell adhesion and cell signaling in response to components of the extracellular matrix but its role in the nervous system is not understood. CD44 proteins are elevated in Schwann cells and oligodendrocytes following nervous system insults, in inflammatory demyelinating lesions, and in tumors. Here, we tested the hypothesis that elevated CD44 expression influences Schwann cell and oligodendrocyte functions by generating transgenic mice that express CD44 under the control of the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) promoter. These mice failed to develop peripheral nerve or CNS tumors. However, they did develop severe tremors that were associated with CNS dysmyelination and progressive demyelination. Loss of CNS myelin was not due to alterations in early oligodendrocyte precursor differentiation, proliferation, or survival. Myelination in the PNS appeared normal. In no instance was there any evidence of an inflammatory response that could account for the loss of CNS myelin. These findings suggest that CNPase-CD44 mice are a novel model for noninflammatory progressive demyelinating disease and support a potential role for CD44 proteins expressed by glial cells in promoting demyelination.

Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity

Exposing rat dorsal root ganglion (DRG) neurons to dibutyryl cAMP (db-cAMP) enables central branches to regenerate in the spinal cord by nullifying the ability of CNS myelin to inhibit elongation. A conditioning lesion (CL) promotes similar regeneration of central branches in the spinal cord by increasing neuronal cAMP levels. It is a matter of speculation whether any of the other effects of a CL are triggered by elevated cAMP. We found that like a CL, intraganglionic injection of db-cAMP increases the expression of growth-associated tubulin isotypes. However, unlike a CL, db-cAMP does not increase the velocity at which tubulin is delivered to the tips of growing axons by slow component b (SCb). db-cAMP also fails to increase intrinsic axon growth capacity enough to raise the rate of regeneration of peripheral branches in the sciatic nerve or enable central branches to elongate long distances in an environment free of all CNS inhibitors of elongation (i.e., a peripheral nerve graft transplanted into the spinal cord at the site of dorsal column transection). Thus, the increase in cAMP induced by a CL induces some, but not all, of the changes that may be necessary to increase intrinsic axon growth capacity.

Axonal cell-adhesion molecule L1 in CNS myelination

Of the axonal signals influencing myelination, adhesion molecules expressed at the axonal surface are strong candidates to mediate interactions between myelinating cells and axons. The recognition cell-adhesion molecule L1, a member of the immunoglobulin superfamily has been shown to play important roles in neuronal migration and survival, and in PNS myelination. We have investigated the role of axonally expressed L1 in CNS myelination. In co-cultures of myelinating oligodendrocytes and neurons derived from murine brain, we demonstrate that, before myelination, L1 immunoreactivity is confined to neurites. After myelination commences, L1 expression is downregulated on myelinated axons and adjacent, but not yet myelinated, internodes.Interfering with L1 before the onset of myelination, by adding either anti-L1 antibody or L1-Fc fusion proteins to the culture medium, inhibits myelination. In addition, in purified cultures of oligodendrocytes, L1-Fc fusion protein prevents lysophosphatidic acid-induced activation of the mitogen-activated kinase (MAP)-kinase pathway. Together, our data indicate that L1 is involved in the initiation of CNS myelination, and that this effect might involve the dephosphorylation of oligodendroglial phosphoproteins.

Repairing the nervous system

The winter meeting of the Anatomical Society, on the subject of ‘Repairing the nervous system’, was held at the Royal Holloway College from 7th to 9th January 2003. The organisers focused on reviewing the current status of ideas about the cellular mechanisms involved in both stimulating and preventing CNS repair. We need to try to understand the complexity of environment through which regenerating axons have to grow, and how this abnormal situation compares with the normal process of navigation in development. Critically, we need to understand how successfully cells respond to these challenges by changing gene expression to generate new or recapitulate old programs for axonal growth and navigation. A major issue is how the environment of the CNS limits repair and how we can try to devise strategies to overcome these limitations and enhance the repair process. This may involve the use of strategies to reverse negative influences of glial cells on axonal regeneration, or the transplantation of cells to promote the regeneration process. This was an excellent meeting, with many stimulating presentations which reviewed cellular and molecular approaches to CNS repair. Some of these talks are presented in this volume and hopefully give an overview of some of the major issues and approaches being used to stimulate CNS repair. John Nicholls reviewed the work on spinal cord regeneration, which has used an elegant in vitro model system from the marsupial and now complemented these cellular approaches with an investigation into differential gene expression during regeneration. Susanna Blackshaw highlighted the beauty of the leech CNS as a model for studying neuronal regeneration in identified neurones. Her recent work shows how this relatively simple model organism can be used to generate a wealth of information on the changes in neuronal gene expression during the regeneration process, and how this data will inform studies of CNS repair in both invertebrate and vertebrates. Jeremy Taylor reviewed the advantages of using immature animals for studying CNS repair, and how the major limitation for successful repair at these stages is trophic support. To overcome this obstacle, glial cells in the peripheral and central nervous system are being studied, and are shown to provide novel factors which act with well studied neurotrophins to stimulate regeneration. James Fawcett discussed the roles of chondroitin sulphate proteoglycans in limiting both CNS regeneration at glial scars and also the more widespread phenomenon of neuronal plasticity. This exciting field has revealed unexpected controls on neuronal plasticity and has shown how these inhibitory mechanisms could be harnessed to promote CNS repair. Marie Filbin demonstrated the remarkable change in neuronal response to CNS myelin that occurs during maturation of the CNS. Again, an understanding of the cellular mechanisms that control these responses has opened a window for overcoming these limiting factors. The negative inhibitory response that myelin proteins have on regenerating axons, can be reversed to a positive growth enhancing response. Susan Barnett has studied the use of the special glial cells, the olfactory ensheathing cells that allow continual renewal of the olfactory system throughout life, in transplants to the CNS. Her careful studies on the preparation, purification and behaviour of these cells when grafted into the CNS highlight the importance of understanding the cell biology underpinning the use of transplanted cells for CNS repair.

AMPA receptors are the major mediators of excitotoxic death in mature oligodendrocytes

Myelination of axons is important for central nervous system function, but oligodendrocytes, which constitute CNS myelin, are vulnerable to excitotoxic injury and death. Although mature oligodendrocytes express functional α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) and kainate-type glutamate receptors, the relative roles of these subtypes in excitotoxicity are not well understood. Using recently developed selective antagonists for subtypes of ionotropic non-NMDA receptors, we addressed this issue. By examining the pharmacological, biochemical, and morphologic features of kainite-induced excitotoxic death, we also determined whether it occurs by apoptosis, necrosis, or both. We conclude that when mature oligodendrocytes die after exposure to kainate: (1) AMPA receptors are the most important mediators, (2) kainate receptors play a smaller role, and (3) death occurs predominantly by necrosis, not apoptosis.

Gradual loss of myelin and formation of an astrocytic scar during Wallerian degeneration in the human spinal cord

Axons undergo Wallerian degeneration distal to a point of injury. Experimental investigations have documented many of the cellular and molecular events that underlie this behaviour. Since relatively little is known about such events in human CNS pathologies and current experimental intervention strategies indicate the possibility of significant axon regeneration along the original degenerated fibre tract, we performed an immunohistochemical investigation of the dynamics of Wallerian degeneration in post mortem spinal cords of patients who died 2 days to 30 years after either cerebral infarction or traumatic spinal cord injury. Neurofilament (NF) staining demonstrated a spatio-temporal pattern of axonal loss within degenerating descending nerve fibre tracts that could be detected close to the lesion as early as 12 days after injury and progressed to an almost complete loss of NF immunoreactivity at survival times of 1 year and longer. Immunohistochemistry for glial fibrillary acidic protein revealed a late astrocytic reaction starting at 4 months after injury in the degenerating tracts, leading to the long-term deposition of a dense astrocytic scar. These events were accompanied by the gradual reduction of myelin basic protein in affected nerve fibre tracts, leading to almost complete loss by 3 years after injury. Since the extracellular matrix molecule chondroitin sulphate proteoglycan (CSPG) is known to be strongly inhibitory for axonal regeneration and to be a major component of gliotic scar tissues, we investigated the possible deposition of CSPG within the degenerating nerve fibre tracts. Apart from a local up-regulation close to the lesion site, our results show no enhanced CSPG expression within degenerated tracts at any survival time. This suggests that despite the apparent lack of CSPG in Wallerian degeneration, the slow reduction of CNS myelin and the long-term deposition of a dense astrocytic scar may present an environment that is non-supportive for axon regrowth.

Solid-phase synthesis of a biotin-labelled thiopalmitoylated myelin proteolipid protein epitope and application in the study of uptake of antigen by macrophages

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of palmitic acid to cysteine residues viaa thioester linkage. It was previously shown that thiopalmitoylation of encephalitogenic PLP T-cell epitopes enhanced immune responses as well as the development and chronicity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). To understand how the thiopalmitoylated peptides (S-palm peptide) can play a role in the development of autoreactivity in EAE and MS, the mechanism by which they are taken up by antigen presenting cells (APC) and presented to the immune system must be determined. This paper describes the solid-phase synthesis and purification of biotin-labelled thiopalmitoylated PLP(139–151) for use in studying the uptake of S-palm peptides by macrophages using flow cytometry analysis. Our aim was to obtain the labelled lipopeptide after on-resin biotinylation and palmitoylation, but, due to the reactivity of biotin towards acylation, we had to modify the conditions of thiopalmitoylation we had previously described, i.e. palmitic acid activated ester instead of palmitoyl chloride. Using flow cytometry analysis, we were able to show that the entry of S-palm peptide in the macrophages is very rapid compared to the non-palmitoylated peptide, and that the lipopeptide is taken up more efficiently into the macrophages.

Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum.

We have previously shown that progesterone (PROG) is synthesized by Schwann cells and promotes myelin formation in the peripheral nervous system (PNS). We now report that this neurosteroid also stimulates myelination in organotypic slice cultures of 7-day-old (P7) rat and mouse cerebellum. Myelination was evaluated by immunofluorescence analysis of the myelin basic protein (MBP). After 7 days in culture (7DIV), we found that adding PROG (2(-5) x 10(-5) M) to the culture medium caused a fourfold increase in MBP expression when compared to control slices. The effect of PROG on MBP expression involves the classical intracellular PROG receptor (PR): the selective PR agonist R5020 significantly increased MBP expression and the PR antagonist mifepristone (RU486) completely abolished the effect of PROG on this MBP expression. Moreover, treatment of P7-cerebellar slice cultures from PR knockout (PRKO) mice with PROG had no significant effect on MBP expression. PROG was metabolized in the cerebellar slices to 5alpha-dihydroprogesterone (5alpha-DHP) and to the GABAA receptor-active metabolite 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP, allopregnanolone). The 5alpha-reductase inhibitor L685-273 partially inhibited the effect of PROG, and 3alpha,5alpha-THP (2(-5) x 10(-5) M) significantly stimulated the MBP expression, although to a lesser extent than PROG. The increase in MBP expression by 3alpha,5alpha-THP involved GABAA receptors, as it could be inhibited by the selective GABAA receptor antagonist bicuculline. These findings suggest that progestins stimulate MBP expression and consequently suggest an increase in CNS myelination via two signalling systems, the intracellular PR and membrane GABAA receptors, and they confirm a new role of GABAA receptors in myelination.