Myelin-associated glycoprotein activation triggers glutamate uptake by oligodendrocytes in vitro and contributes to ameliorate glutamate-mediated toxicity in vivo.

BACKGROUND: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. PURPOSE: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. RESULTS: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. CONCLUSIONS: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.

Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75(NTR) receptor-mediated activation of RhoA signaling pathways.

Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75(NTR)-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75(NTR), Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75(NTR)-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75(NTR)-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75(NTR)-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75(NTR)/RhoA/ROCK pathway, or overexpression of a p75(NTR) mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75(NTR)/RhoA/ROCK signaling pathway. Also, our results highlight the relevance of the nurture/protective effects of myelin on neurons.

White matter injury induced by perinatal exposure to glutaric acid.

Glutaric acid (GA) is a neurotoxic metabolite that accumulates in the CNS of patients with glutaric acidemia-I (GA-I), a neurometabolic disease caused by deficient activity of glutaryl-CoA dehydrogenase. Most GA-I patients display characteristic CNS lesions, mainly in the gray and white matter of basal ganglia and cerebral cortex. Neurons and astrocytes are believed to be vulnerable to millimolar concentrations of GA. However, little is known about the effects of GA on oligodendrocytes (OL) and the myelination process in the postnatal brain. Here, we show that a single intracerebroventricular administration of GA to rat neonatal pups induced a selective and long-lasting myelination failure in the striatum but no deleterious effect in the myelination of the corpus callosum. At 45 days post-GA injection, the myelinated area of striatal axonal bundles was decreased by 35 %, and the expression of myelin basic protein and myelin-associated glycoprotein (MAG) reduced by 25 and 60 %, respectively. This was accompanied by long lasting cytopathology features in MAG and CC-1-expressing OLs, which was confirmed by transmission electron microscopy. Remarkably, GA did not induce acute loss of pre-OLs in the striatum as assessed by NG2 or PDGFRα immunohistochemistry, suggesting an indirect and progressive mechanism for OL damage. In accordance, GA-induced white matter injury was restricted to the striatum and associated to GA-induced astrocytosis and neuronal loss. In conclusion, the current evidence indicates a pathogenic mechanism by which GA can permanently affect myelin status.

Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors, and myelin proteins following spinal cord injury.

Progesterone is emerging as a myelinizing factor for central nervous system injury. Successful remyelination requires proliferation and differentiation of oligodendrocyte precursor cells (OPC) into myelinating oligodendrocytes, but this process is incomplete following injury. To study progesterone actions on remyelination, we administered progesterone (16 mg/kg/day) to rats with complete spinal cord injury. Rats were euthanized 3 or 21 days after steroid treatment. Short progesterone treatment (a) increased the number of OPC without effect on the injury-induced reduction of mature oligodendrocytes, (b) increased mRNA and protein expression for the myelin basic protein (MBP) without effects on proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), and (c) increased the mRNA for Olig2 and Nkx2.2 transcription factors involved in specification and differentiation of the oligodendrocyte lineage. Furthermore, long progesterone treatment (a) reduced OPC with a concomitant increase of oligodendrocytes; (b) promoted differentiation of cells that incorporated bromodeoxyuridine, early after injury, into mature oligodendrocytes; (c) increased mRNA and protein expression of PLP without effects on MBP or MOG; and (d) increased mRNA for the Olig1 transcription factor involved in myelin repair. These results suggest that early progesterone treatment enhanced the density of OPC and induced their differentiation into mature oligodendrocytes by increasing the expression of Olig2 and Nkx2.2. Twenty-one days after injury, progesterone favors remyelination by increasing Olig1 (involved in repair of demyelinated lesions), PLP expression, and enhancing oligodendrocytes maturation. Thus, progesterone effects on oligodendrogenesis and myelin proteins may constitute fundamental steps for repairing traumatic injury inflicted to the spinal cord.

Overexpression of human transferrin in two oligodendroglial cell lines enhances their differentiation.

We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O(4), GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co-cultured N19 and N20.1 Tf-transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf-overexpressing clones, while all the isoforms were present in the co-cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon-glia interaction, such as MAG, N-CAM, and F3/Contactin. This co-culture system represents a potentially powerful tool to study neuron-glia interactions that occur during myelinogenesis and the role of Tf in this process.

Distribution of sodium and potassium channels as well as myelin associated glycoprotein (MAG) during the early stages of Wallerian degeneration.

The distribution of sodium and potassium channel proteins and the myelin associated glycoprotein (MAG) was studied by immunofluorescence during the early stages of Wallerian degeneration. Routine electron microscopy was also performed in order to investigate the success of the lesion in producing degeneration and also to evaluate the integrity of the axolemma and cytoskeleton. Sural nerves from Sprague-Dawley rats were submitted to surgical crush and analyzed after 30, 36 and 48 h. The preparations were observed by light microscopy and the amount of labeled and unlabeled sites was quantified using a computer linked microscope. The number of sodium and potassium labeled nodes was dramatically reduced 30 h after crushing. However, a small number of labeled nodes was still present even after 48 h. These remaining nodal channel proteins are probably responsible for the maintenance of the nerve's electrical activity during the first 4 days of Wallerian degeneration. Ultrastructural analyses of longitudinal sections revealed areas of intact axolemma in the presence of a partially or completely disrupted cytoskeleton. These results are in disagreement with the generally accepted view that axolemma and cytoskeleton disruption occur simultaneously in the peripheral nervous system. Our results also concern the mechanism underlying the disappearance of these channel proteins during the degeneration of the peripheral nerve fibres, since in this pathology the axons are affected before myelin and Schwann cells become affected.