Levels Of Myelin-associated Glycoprotein Research Articles

Injection of Cerium Oxide Nanoparticles to Treat Spinal Cord Injury in Rats.

This study investigated the effects of local injection of cerium oxide nanoparticles (CeONPs) in a rat spinal cord injury (SCI) model. Thirty-six adult male Wistar rats were divided into 4 groups: controls (healthy animals), sham (laminectomy), SCI (laminectomy+SCI induction), and treatment (laminectomy+SCI induction+intrathecal injection of CeONPs immediately after injury). SCI was induced using an aneurysm clip at the T12-T13 vertebral region. Motor performance and pain threshold tests were performed weekly; H&E staining and measurement of cavity sizes were performed 6 weeks after injury. The expression of granulocyte colony-stimulating factor (GCSF), P44/42 MAPK, P-P44/42 MAPK, Tau, myelin-associated glycoprotein(MAG) was evaluated after 6 weeks by Western blot. The Basso, Beattie, and Bresnahan locomotor scoring scales improved in animals receiving CeONPs compared with SCI animals. The cavity sizes were less in the treatment group. GCSF expression was similar in the animals receiving CeONPs compared with the SCI group but the expression of ERK1/ERK2 and phospho-ERK was lower than in the SCI group. Expression levels of Tau and MAG were significantly increased in treated animals compared to the SCI group. These data indicate that the use of CeONPs may improve motor functional recovery in SCI.

Comparison between cerebrospinal fluid and serum levels of myelin-associated glycoprotein, total antioxidant capacity, and 8-hydroxy-2′-deoxyguanosine in patients with multiple sclerosis

BackgroundMultiple sclerosis (MS) is a chronic inflammatory disease characterized by demyelinated lesions in the brain, the spinal cord, and the optic nerve. It is one of the most common neurological disorders. In this study, serum and cerebrospinal fluid (CSF) levels of total antioxidant capacity (TAC), myelin-associated glycoprotein (MAG), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were investigated to determine their effects on MS. Materials and methodIn this study, 25 serum and cerebrospinal samples from MS patients as a case group and 40 serum and CSF samples from healthy participants as a control group were collected and analyzed. Concentrations of TAC, MAG, and 8-OhdG were determined in the samples using a dedicated kit and relayed using the ELISA device. ResultsThe mean serum antibody levels of MAG and TAC were higher in the case group than the control group, although the difference in the MAG level was not significant (P > 0.05). However, the mean serum level of −8 OHdG was lower in the case group than the control group. Moreover, the mean levels of the evaluated biomarkers in the CSF samples were higher in the case group than in the control group. Still, the difference was only significant in terms of TAC levels (P < 0.05). Receiver operating characteristics curve analysis showed that the area under the curve was 0.71 and 0.69 for 8-OhdG and TAC serum levels, respectively, and 0.73 for both TAC and CSF levels, which was not significantly different from that in other biomarkers. ConclusionElevated TAC levels in serum and CSF samples and 8-OhdG in serum samples may be associated with MS pathogenesis. However, further investigation is needed to consider these cases as a follow-up to the therapeutic goals or treatment process.

Combination of Quercetin, Hirudin and Cinnamaldehyde Promotes Schwann Cell Differentiation and Myelination against High Glucose by Inhibiting ERK Signaling Pathway.

To investigate the therapeutic and synergistic effects of QHC (combination of quercetin (Q), hirudin (H) and cinnamaldehyd (C)) on Schwann cell differentiation and myelination against high glucose (HG) induced injury. Primary-culture Schwann cells exposed to HG (50 mmol/L) for 72 h and Schwann cell-dorsal root ganglion (DRG) neuron cocultures exposed to HG (50 mmol/L) for 7 days were employed as in vitro model of diabetic neuropathy. The cells were randomly divided into 10 groups: control (CON, 25 mmol/L glucose), HG (50 mmol/L glucose), HG plus 10 μmol/L quercetin (Q), HG plus 0.04 IU/mL hirudin (H), HG plus 100 nmol/L cinnamaldehyd (C), HG plus 10 μmol/L quercetin and 0.04 IU/mL hirudin (QH), HG plus 10 μmol/L quercetin and 50 nmol/L cinnamaldehyd (QC), HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (HC), HG plus 10 μmol/L quercetin, 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (QHC) or 10 μmol/L U0126. Cell differentiation was evaluated by periaxin immunofluorescence staining. The protein expression levels of myelin protein zero (P0), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), extracellular signal-regulated kinase (ERK), p-ERK, p-c-Jun, c-Jun, notch intracellular domain (NICD) and the mRNA expression levels of P0, MBP, MAG, Krox-20, Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis. The secretion of ciliary neurotrophic factor (CNTF) was determined by enzyme-linked immunosorbent assay (ELISA). The number and length of the myelin segments were evaluated by MBP immunofluorescence staining. The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining. Co-treatment with Q, C, H and their combination promoted Schwann cell differentiation, increased CNTF secretion, up-regulated the protein and mRNA expressions of myelin, and increased the number and length of the myelin segments (P<0.01 or P<0.05). In particular, the combination therapy of Q, H and C was superior to the respective monotherapy (P<0.01). Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers (P<0.01). QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway, providing scientific evidence for better understanding of combination of Q, H and C in clinical applications.

Association between abnormal serum myelin-specific protein levels and white matter integrity in first-episode and drug-naïve patients with major depressive disorder

BackgroundAlthough the structural abnormalities of white matter (WM) have been described in patients with major depressive disorder (MDD), the neuropathological changes remain unclear. The current study aimed to investigate the myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG) levels and their correlations with WM integrity in first-episode, drug-naïve MDD patients. MethodsWe obtained diffusion tensor images of 102 first-episode, drug-naïve MDD patients and 81 age- and sex-matched controls. Serum MOG and MAG levels of all participants were measured and compared between the two groups. The correlations between WM integrity and MOG and MAG levels were examined. ResultsMOG and MAG serum levels were significantly higher in MDD patients than in controls. Patients with MDD also showed decreased fractional anisotropy (FA) and axial diffusivity in the WM of the bilateral thalamus, right hippocampus, right temporal lobe, and left pulvinar. At the whole-brain level, no regions showed any correlations of diffusivity parameters with MOG or MAG levels in healthy subjects. However, we observed two-way correlations between the MOG and MAG levels and the FA and mean diffusivity values in the WM of the left middle frontal lobe, right inferior parietal lobe, and right supplementary motor area in MDD patients. LimitationsFurther investigation with a larger sample size and longitudinal studies are required to better understand the neuropathology of WM integrity in MDD. ConclusionsOur findings represent the first evidence of a relationship between abnormal serum myelin-specific protein levels and impaired WM integrity, which may help to better understand the neurobiological mechanisms of MDD.

Erythropoietin attenuates axonal injury after middle cerebral artery occlusion in mice

Objective: Erythropoietin (EPO) confers potent neuroprotection against ischemic injury through a variety of mechanisms. However, the protective effect of EPO on axons after cerebral ischemia in adult mice is rarely covered. The purpose of this study was to investigate the potential neuroprotective effects of EPO on axons in mice after cerebral ischemia.Methods: A total of 30 adult male C57 BL/6 mice were treated with EPO (5000 IU/kg) or vehicle after transient middle cerebral artery occlusion (MCAO). The mortality rate of each experimental group was calculated. Neurological function was assessed by Rota-rod test. Frozen sections from each mouse brain at 14 days after reperfusion were used to evaluate the fluorescent intensity of myelin basic protein (MBP) and neurofilament 200 (NF-200). Immunofluorescence staining and Western blotting were used to assess the protein level of β-amyloid precursor protein (β-APP) and glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. The protein levels of the myelin-derived growth inhibitory proteins, neurite growth inhibitor-A (Nogo-A), myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMG) were also examined by Western blot after MCAO.Results: The survival rate of the vehicle group 14 days after cerebral ischemia-reperfusion was 50%, which increased to 80% after EPO treatment at the start of reperfusion. EPO improved neurobehavioral outcomes at days 3 and 7 after MCAO was compared with the vehicle group (P < 0.05). Furthermore, EPO ameliorated demyelination, demonstrated by upregulation of the MBP/NF-200 ratio. Meanwhile, increased levels of β-APP, GFAP, Nogo-A, and MAG after MCAO were reduced by EPO treatment (P < 0.05).Conclusion: EPO treatment attenuates axonal injury and improves neurological function after cerebral ischemia in adult mice.

Schwann cell proliferation and differentiation that is induced by ferulic acid through MEK1/ERK1/2 signalling promotes peripheral nerve remyelination following crush injury in rats.

Schwann cell proliferation and differentiation is critical for the remyelination of injured peripheral nerves. Ferulic acid (FA) is a widely used antioxidant agent with neuroprotective properties. However, the potentially beneficial effects of FA on Schwann cells are unknown. Therefore, the present study was designed to examine the effects of FA on Schwann cell proliferation and differentiation. By using the cultured primary Schwann cells and proliferation assay, the results identified that FA was capable of increasing Schwann cell proliferation and expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) in vitro. It was also observed that the beneficial effect of FA treatment on Schwann cells was mainly dependent on the activation of MEK1/ERK1/2 signalling. Furthermore, FA was intraperitoneally administered to rats with sciatic nerve crush injury, and the results revealed an increase in Schwann cell proliferation and differentiation, while the MAG and MBP expression levels in sciatic nerves were markedly upregulated following FA administration. In conclusion, the current results demonstrate that Schwann cell proliferation and differentiation is induced by FA through MEK1/ERK1/2 signalling and that FA may accelerate injured peripheral nerve remyelination.

GSK249320, A Monoclonal Antibody Against the Axon Outgrowth Inhibition Molecule Myelin-Associated Glycoprotein, Improves Outcome of Rodents with Experimental Stroke

Myelin-associated glycoprotein (MAG) is an inhibitor of axon growth. MAG levels increase after stroke. GSK249320 is a monoclonal antibody that neutralizes MAG-mediated inhibition and so may promote axon outgrowth and improve post-stroke outcomes. The current study tested the hypothesis that GSK249320 initiated 24 hours or 7 days after experimental stroke improves behavioural outcomes. Rats with right middle cerebral artery occlusion for 90 minutes were randomized to receive 6 weeks of intravenous (a) GSK249320 starting 24 hours post-stroke, (b) GSK249320 starting 7 days post-stroke, or (c) vehicle. Behavioral testing was performed over 7 weeks. Serial MRI demonstrated no differences in infarct volume across groups. Animals treated with GSK249320 24 hours post-stroke showed larger increases in Neuroscore (time X group, p = 0.0008) and staircase test (main effect of group, p = 0.0214) as compared to controls, but animals treated 7 days post-stroke showed no significant behavioral benefit. No significant results were found for the sticky tape or cylinder tests. A separate set of animals with experimental stroke received a single intravenous dose of GSK249320 or vehicle at 1 hour, 24 hours, 48 hours or 1 week post-stroke, and immunohistochemistry methods were used to measure GSK249320 distribution; GSK249320 was found in the ipsilesional hemisphere only, the extent of which increased with later times of injection. These data suggest that intravenous GSK249320 penetrates the lesion site and is associated with a small effect on functional outcomes when initiated 24 hours post-stroke and so support the translational potential of this monoclonal antibody as a restorative therapy for patients with stroke.

Associations between brain microstructures, metabolites, and cognitive deficits during chronic HIV-1 infection of humanized mice.

BackgroundHost-species specificity of the human immunodeficiency virus (HIV) limits pathobiologic, diagnostic and therapeutic research investigations to humans and non-human primates. The emergence of humanized mice as a model for viral infection of the nervous system has overcome such restrictions enabling research for HIV-associated end organ disease including behavioral, cognitive and neuropathologic deficits reflective of neuroAIDS. Chronic HIV-1 infection of NOD/scid-IL-2Rgcnull mice transplanted with human CD34+ hematopoietic stem cells (CD34-NSG) leads to persistent viremia, profound CD4+ T lymphocyte loss and infection of human monocyte-macrophages in the meninges and perivascular spaces. Murine cells are not infected with virus.MethodsChanges in mouse behavior were measured, starting at 8 weeks after viral infection. These were recorded coordinate with magnetic resonance spectroscopy metabolites including N-acetylaspartate (NAA), creatine and choline. Diffusion tensor magnetic resonance imaging (DTI) was recorded against multispectral immunohistochemical staining for neuronal markers that included microtubule associated protein-2 (MAP2), neurofilament (NF) and synaptophysin (SYN); for astrocyte glial fibrillary acidic protein (GFAP); and for microglial ionized calcium binding adaptor molecule 1 (Iba-1). Oligodendrocyte numbers and integrity were measured for myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) antigens.ResultsBehavioral abnormalities were readily observed in HIV-1 infected mice. Longitudinal open field activity tests demonstrated lack of habituation indicating potential for memory loss and persistent anxiety in HIV-1 infected mice compared to uninfected controls. End-point NAA and creatine in the cerebral cortex increased with decreased MAG. NAA and glutamate decreased with decreased SYN and MAG. Robust inflammation reflected GFAP and Iba-1 staining intensities. DTI metrics were coordinate with deregulation of NF, Iba-1, MOG and MAG levels in the whisker barrel and MAP2, NF, MAG, MOG and SYN in the corpus callosum.ConclusionsThe findings are consistent with some of the clinical, biochemical and pathobiologic features of human HIV-1 nervous system infections. This model will prove useful towards investigating the mechanisms of HIV-1 induced neuropathology and in developing novel biomarkers and therapeutic strategies for disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1750-1326-9-58) contains supplementary material, which is available to authorized users.

Assessing White Matter Ischemic Damage in Dementia Patients by Measurement of Myelin Proteins

White matter ischemia is difficult to quantify histologically. Myelin-associated glycoprotein (MAG) is highly susceptible to ischemia, being expressed only adaxonally, far from the oligodendrocyte cell body. Myelin-basic protein (MBP) and proteolipid protein (PLP) are expressed throughout the myelin sheath. We compared MAG, MBP, and PLP levels in parietal white matter homogenates from 17 vascular dementia (VaD), 49 Alzheimer's disease (AD), and 33 control brains, after assessing the post-mortem stability of these proteins. Small vessel disease (SVD) and cerebral amyloid angiopathy (CAA) severity had been assessed in paraffin sections. The concentration of MAG remained stable post-mortem, declined with increasing SVD, and was significantly lower in VaD than controls. The concentration of MBP fell progressively post-mortem, limiting its diagnostic utility in this context. Proteolipid protein was stable post-mortem and increased significantly with SVD severity. The MAG/PLP ratio declined significantly with SVD and CAA severity. The MAG and PLP levels and MAG/PLP did not differ significantly between AD and control brains. We validated the utility of MAG and MAG/PLP measurements on analysis of 74 frontal white matter samples from an Oxford cohort in which SVD had previously been scored. MAG concentration and the MAG/PLP ratio are useful post-mortem measures of ante-mortem white matter ischemia.

Neurotrophin‐3 targets the translational initiation machinery in oligodendrocytes

Neurotrophin-3 (NT-3) regulates oligodendrocyte (OLG) differentiation by mechanisms that remain poorly understood. Exposure of OLGs to NT-3 induces a significant increase in the levels of myelin basic protein (MBP). However, we found that this stimulation occurs in the absence of measurable effects on MBP gene promoter activation or mRNA expression, suggesting that NT-3 upregulates MBP protein expression by a posttranscriptional mechanism. Furthermore, NT-3 also causes an increase in the levels of myelin-associated glycoprotein (MAG) and myelin OLG glycoprotein (MOG), raising the possibility of a more general effect on myelin protein synthesis. Surprisingly, (35)S-methionine incorporation into total OLG proteins demonstrated a 50% increase in labeling following only a brief, 15-min treatment with NT-3. Such a remarkably fast response is unlikely due to transcriptional activation, reinforcing the possibility that NT-3 may play a crucial role in regulating protein expression by a posttranscriptional mechanism. In support of this idea, we found that NT-3 stimulates the phosphorylation of essential regulators of the initiation machinery, eukaryotic initiation factor 4E (eIF4E), and its inhibitory binding partner 4E binding protein 1 (4EBP1), two crucial players in controlling cap-dependent protein synthesis. This stimulation involves the activation of pathways mediated by ERK1/2 and PI3K/mTOR, implicating these two kinase systems as modulators of protein synthesis in developing OLGs. Altogether, these observations show for the first time that NT-3 has the capacity of targeting the translational machinery and suggest a potential stimulatory effect of this neurotrophin on myelination by direct action on protein translation in the OLGs.

Alterations of myelin‐specific proteins and sphingolipids characterize the brains of acid sphingomyelinase‐deficient mice, an animal model of Niemann–Pick disease type A

Niemann-Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD-A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM'knock-out' (ASMKO) mice, an animal model of NPD-A, develop a phenotype largely mimicking that of NPD-A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin-specific proteins, myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin-enriched sphingolipids in the brains of ASMKO and wild-type mice in early stages of post-natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post-natal weeks (wk-pn), the expression levels of MAG, CNP, and MBP were below those observed in wild-type mice and the same applied to PLP at 10 wk-pn. Moreover, at 4 wk-pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP-galactose ceramide galactosyltransferase and galactose-3-O-sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk-pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD-A.

Autoimmune-induced preferential depletion of myelin-associated glycoprotein (MAG) is genetically regulated in relapsing EAE (B6 × SJL) F1 mice

BackgroundExperimental autoimmune encephalomyelitis (EAE) is commonly used to investigate mechanisms of autoimmune-mediated damage to oligodendrocytes, myelin, and axons in multiple sclerosis (MS). Four distinct autoimmune mechanisms with subsequently distinct patterns of demyelination have been recognized in acute MS lesions. EAE correlates for those distinct patterns of MS lesions are unknown. An excessive loss of myelin-associated glycoprotein (MAG), as a result of distal oligodendrogliopathy, is found exclusively in the subtype III lesion. We sought to answer if types of demyelination in acute lesions during onset and relapse of EAE can replicate the specific patterns observed in MS acute lesions.MethodsIn parental H-2b (C57BL/6, B6) and hybrid H-2b/s [(B6 × SJL) F1] EAE mice, we examined spinal cord levels of MOG, MAG, and myelin basic protein (MBP), and compared to levels of axonal neurofilament (NF160) to assess axonal function, and levels of PARPp85 as an indicator of irreversible apoptosis.ResultsDuring disease onset, levels of MOG significantly dropped in both strains, although more profoundly in H-2b/s mice. Levels of MOG recovered in relapsing mice of both strains. Regulation of MAG was dissimilar to MOG. Modest loss of MAG was found at disease onset in both strains of mice. Unexpectedly, in relapsing H-2b/s mice, a major depletion of MAG and NF160, accompanied with sharp elevation of PARPp85 levels, was measured. PARPp85 immunoreactivity was observed in cytoplasm and nuclei of some MBP containing cells.ConclusionTaken together, our results show genetically controlled distinct patterns of MOG and MAG depletion, in MOG35–55 induced EAE in H-2b and H-2b/s mice. The data also suggest distinctive immune regulation of acute lesions that develop in relapsing compared to disease onset. A profound depletion of MAG, concomitant with marked depletion of axonal NF160, and sharp elevation of PARPp85 levels, occurred exclusively in relapsing H-2b/s mice. Our findings suggest concurrence of sharp decrease of MAG levels, axonal dysfunction and irreversible apoptosis with severe relapsing disease in H-2b/s mice. We propose that MOG-induced EAE in H-2b/s mice may prove as a useful model in studying mechanisms, which govern autoimmune-induced preferential loss of MAG, and its impact on oligodendroglial pathology.

Exercise normalizes levels of MAG and Nogo‐A growth inhibitors after brain trauma

Myelin is a major obstacle for axonal growth after CNS injury, to the extent that it is crucial to develop interventions to counteract postinjury growth inhibition and foster neural repair. We have studied the effects of the fluid percussion injury (FPI) model of traumatic brain injury (TBI) on protein levels of two myelin-associated molecules, myelin-associated glycoprotein (MAG) and Nogo-A, in the adult rat. We found that FPI elevated hippocampal levels of MAG and Nogo-A. Given the beneficial effects of exercise on CNS function, we evaluated the capacity of exercise to reduce these myelin-derived proteins after FPI. One week of voluntary running wheel exercise overcame the injury-related increase in MAG and Nogo-A. The action of brain-derived neurotrophic factor (BDNF) has been associated with exercise as well as with the modulation of growth inhibition in vitro. We found that the selective blockade of BDNF using the immunoadhesive chimera TrkB-IgG abolished the effects of exercise on MAG and Nogo-A. FPI reduced levels of growth-associated protein 43 (GAP-43), a marker of axonal growth, and synaptophysin (SYP), an indicator of synaptic growth. Exercise counteracted the effects of FPI on GAP-43 and SYP, while BDNF blockade abolished these effects of exercise. Protein kinase A (PKA) has been related to the ability of BDNF to overcome growth inhibition. In agreement, we found that exercise increased PKA levels and this effect was prevented by BDNF blockade. These results indicate that exercise promotes a permissive cellular environment for repair after TBI, in a process in which BDNF plays a central role.

Alterations in myelination in the central nervous system of dystonia musculorum mice

Dystonia musculorum (dt) is an autosomal recessive sensory neuropathy in mice resulting from a mutation in the gene encoding the cytoskeletal linker protein Bpag1. In addition to neurodegeneration, dt mice display myelination abnormalities in the peripheral nervous system. In this report we investigated whether myelination abnormalities are also present in the central nervous system of dt(Tg4) mice. Transcripts for both neural isoforms of Bpag1 (a1 and a2) were detected in optic nerves and spinal cords of wild-type mice. Light microscopy of resin-embedded thin sections revealed a reduction in myelinated axons in both optic nerves and spinal cords in dt(Tg4) mice. As well, hypermyelinated axons were detected in these tissues. Ultrastructural analysis of optic nerves and spinal cords from dt(Tg4) mice revealed an increase in the number of amyelinated axons, the presence of hypo- and hypermyelinated axons, and redundant myelin that course away from axons. Changes in the level of myelin proteins accompanied the morphological alterations. Myelin-associated glycoprotein levels were reduced in optic nerves of dt(Tg4) mice, and myelin basic protein levels were altered in optic nerves, sciatic nerves, and spinal cords of affected mice. Short-term cultures of oligodendrocytes derived from dt(Tg4) mice did not show morphological alterations.

Brain gangliosides: Functional ligands for myelin stability and the control of nerve regeneration

Gangliosides, sialylated glycosphingolipids which are the predominant glycans on vertebrate nerve cell surfaces, are emerging as components of membrane rafts, where they can mediate important physiological functions. Myelin associated glycoprotein (MAG), a minor constituent of myelin, is a sialic acid binding lectin with two established physiological functions: it is involved in myelin-axon stability and cytoarchitecture, and controls nerve regeneration. MAG is found selectively on the myelin membranes directly apposed to the axon surface, where it has been proposed to mediate myelin-axon interactions. Although the nerve cell surface ligands for MAG remain to be established, evidence supports a functional role for sialylated glycoconjugates. Here we review recent studies that reflect on the role of gangliosides, sialylated glycosphingolipids, as functional MAG ligands. MAG binds to gangliosides with the terminal sequence ‘NeuAcα3Gal β3GalNAc’ which is found on the major nerve gangliosides GD1a and GT1b. Gangliosides lacking that terminus (e.g., GM1 or GD1b), or having any biochemical modification of the terminal NeuAc residue fail to support MAG binding. Genetically engineered mice lacking the GalNAc transferase required for biosynthesis of the ‘NeuAcα3Gal β3GalNAc’ terminus have grossly impaired myelination and progressive neurodegeneration. Notably the MAG level in these animals is dysregulated. Furthermore, removal of NeuAc residues from nerve cells reverses MAG-mediated inhibition of neuritogenesis, and neurons from mice lacking the ‘NeuAcα3Gal β3GalNAc’ terminus have an attenuated response to MAG. Cross-linking nerve cell surface gangliosides can mimic MAG-mediated inhibition of nerve regeneration. Taken together these observations implicate gangliosides as functional MAG ligands.

Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

Biochemical Analysis of Myelin Proteins in a Novel Neurological Mutant: The Taiep Rat

Hemispheres, spinal cords, and sciatic nerves were taken from taiep, carrier, and control rats at ages ranging from 1 day to 16 months. Absolute myelin yields from CNS taiep tissues peaked at approximately 2 months and then decreased until they reached a low but stable level. Myelin yield from the affected hemispheres expressed as a percentage of age-matched controls decreased continuously from 2 weeks until it reached a stable level of approximately 10-15%. The same was true for the spinal cords, but here the myelin yield reached a plateau at a slightly higher percentage of 20-25%. In comparison with control rats, isolated CNS myelin fractions from the affected rats had a greater content of high molecular weight proteins. Western blot analyses of CNS homogenates revealed that myelin basic protein (MBP), proteolipid protein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase were all present but decreased to levels generally consistent with the deficiencies of myelin. However myelin-associated glycoprotein (MAG) levels always were reduced much more than those of the other three myelin proteins, and at younger ages the apparent molecular weight for MAG was increased in the mutants. Western blot analyses of sciatic nerve homogenates showed that the levels of MBP, MAG, and P0 were not significantly different in control and mutant animals. These results suggested an early hypomyelination of the CNS, with peak levels of myelin at 2 months, followed by a prolonged period of myelin loss, until a very low but stable myelin level was reached. The consistently greater loss of MAG, in comparison with other CNS myelin proteins, is different from most other hypomyelinating mutants in which MAG is relatively preserved in comparison with the proteins of compact myelin. This might be due to microtubular abnormalities in the taiep mutant interfering with transport of myelin proteins and having the greatest effect on MAG because of its most distal location in the periaxonal oligodendroglial membranes.

Myelin deficiency in female rats due to a mutation in the PLP gene

Myelin deficiency ( md) in female rats due to a mutation in the X-linked proteolipid protein (PLP) gene is caused by X-chromosome monosomy. Cytogenetic analysis revealed a single X karyotype [41,X(md/0)]. An immunocytochemical, electron microscopic, and biochemical study was performed on male and female md rats. The central nervous system (CNS) of the female md rat [41,X(md/0)] revealed the same total lack of PLP as the CNS of the affected male littermate [42,XY(md/Y)]. Immunocytochemistry for myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNP) revealed “islands” of myelin sheath-like reaction product in both. Electron microscopy showed great paucity of compact myelin sheaths in 41,X(md/0) and 42,XY(md/Y). Reduced levels of MPB, MAG, and CNP were confirmed for both sexes but MAG and CNP were substantially higher in 41,X(md/0). Sexual differentiation of the brain may account for the observed differences since normal female reproductive organs are present in the md female rat.

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination.

Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells.

Myelin-associated glycoprotein (MAG) is an integral membrane protein expressed by myelinating glial cells that occurs in two developmentally regulated forms with different carboxyterminal cytoplasmic domains (L-MAG and S-MAG). To investigate the role of MAG in myelination a recombinant retrovirus was used to introduce a MAG cDNA (L-MAG form) into primary Schwann cells in vitro. Stably infected populations of cells were obtained that constitutively expressed MAG at the cell surface without the normal requirement for neuronal contact to induce expression. Constitutive expression of L-MAG did not affect myelination. In long term co-culture with purified sensory neurons, the higher level of MAG expression on infected Schwann cells was reduced to control levels on cells that formed myelin. On the other hand, unlike normal Schwann cells, infected Schwann cells associated with nonmyelinated axons or undergoing Wallerian degeneration expressed high levels of MAG. This suggests that a posttranscriptional mechanism modulates MAG expression during myelination. Immunostaining myelinating cultures with an antibody specific to L-MAG showed that L-MAG was normally transiently expressed at the earliest stages of myelination. In short term co-culture with sensory neurons, infected Schwann cells expressing only L-MAG segregated and ensheathed larger axons after 4 d in culture provided that an exogenous basal lamina was supplied. Similar activity was rarely displayed by control Schwann cells correlating with the low level of MAG induction after 4 d. These data strongly suggest that L-MAG promotes the initial investment by Schwann cells of axons destined to be myelinated.