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The subventricular zone (SVZ) is a source of post-natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the post-natal SVZ ependymocytes, radial glia and astrocyte-like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post-natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl-2-chloroethylamide and JWH-056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2-positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post-natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter. © The Authors (2007).

Introduction. The endocannabinoid system consists of cannabinoid receptors, endogenous ligands and the enzymatic elements involved in their synthesis and breakdown. Aim. To report on currently held knowledge about the functioning of the system as a modulator of the neuroinflammatory processes associated with chronic diseases such as multiple sclerosis. Development. Cannabinoids are synthesised and released on demand and their production increases in times of neuroinflammation and neural damage. In this context then, their actions in the microglial cells and in the astrocytes are characterised by a lowered expression of inflammatory mediators and pro-inflammatory cytokines. Furthermore, cannabinoids can play a role as neuroprotectors by means of different types of mechanisms and, in experimental models of multiple sclerosis, they slow down the symptoms, reduce inflammation and can favour remyelination. Conclusions. The clinical use of cannabinoids or pharmacological agents that affect the endogenous cannabinoid system during inflammation of the central nervous system and in multiple sclerosis is currently under consideration and subject to debate. Detailed analysis of the results obtained over the past decade has made it possible to establish the existence of several mechanisms of action of cannabinoids in pathologies affecting the central nervous system that are accompanied by chronic inflammation. Likewise, they also clearly show that the cannabinoid system is an interesting proposal as a new therapeutic tool. © 2006, Revista de Neurología.

Theiler's virus infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids have been shown to exert beneficial effects on animal models of MS and evidence suggests that the endocannabinoid system plays a role in the tonic control of spasticity. In this study we show that OMDM1 [(R)-N-oleoyl-(1′- hydroxybenzyl)-2′-ethanolamine] and OMDM2 [(S)-N-oleoyl-(1′- hydroxybenzyl)-2′-ethanolamine], two selective inhibitors of the putative endocannabinoid transporter and hence of endocannabinoid inactivation, provide an effective therapy for Theiler murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment of TMEV-infected mice with OMDM1 and OMDM2 enhanced anandamide levels in the spinal cord and ameliorated motor symptoms. This was associated with a down-regulation of inflammatory responses in the spinal cord. In addition we show that OMDM1 and OMDM2 down-regulate macrophage function by (i) decreasing the surface expression of major histocompatibility complex (MHC) class II molecules, (ii) inhibiting nitric oxide synthase-2 (NOS-2) expression and (iii) reducing the production of the pro-inflammatory cytokines interleukin-1beta (IL-1β) and interleukin-12 (IL-12p40). Taken together, these results point to the manipulation of the endocannabinoid system as a possible strategy to develop future MS therapeutic drugs. © 2005 International Society for Neurochemistry., This work was supported by grants from the Ministerio de Ciencia y Tecnología (MCYT, Spain, SAF‐2001/1246) and Ministero Italiano per l'Universita′ e la Ricerca (MIUR‐FIRB, to VDM).

[EN]Theiler’s murine encephalomyelitis virus (TMEV) disease is induced following intracerebral inoculation of TMEV, a member of picornavirus family, in susceptible animals. The pathogenesis of paralytic syndrome is associated with a chronic progressive demyelinating disease characterized by perivascular of immune inflammatory cells. Although TMEV induced demyelinating disease (TMEV-IDD) is initiated by virus specific CD4+ T cells targeting CNS-persistent virus, CD4+ T-cell responses against self myelin epitopes activated via epitope spreading contribute to chronic disease pathogenesis. In the present report we delineated possible pathogenic mechanisms related with inflammatory process, leading to demyelination and axonal loss. The importance of proinflammatory cytokines in sustaining the inflammatory process and cause direct oligodendrotoxicity is emphasized. Different approaches in therapeutic strategies affecting cytokines are also presented., [ES]La esclerosis múltiple (EM) es la enfermedad desmielinizante más frecuente en adultos jóvenes, y aunque su etiología es desconocida, estudios epidemiológicos apuntan a una infección vírica como posible causa de la enfermedad. La inoculación de cepas susceptibles de ratones con el virus de la encefalomielitis murina de Theiler (TMEV, del inglés Theiler’s Murine Encephalomyelitis Virus) conduce al desarrollo de una enfermedad crónica desmielinizante caracterizada por lesiones multifocales en la sustancia blanca, con infiltrados inflamatorios similares a la patología observada en la EM en su fase crónica progresiva. Los oligodendrocitos o la mielina son diana de factores solubles citotóxicos liberados por linfocitos CD4+ del subtipo Th1, en respuesta a un fenómeno de hipersensibilidad retardada contra el virus, así como por la microglía y astroglía cerebral activada. En los estadios más avanzados de la infección, se produce una respuesta de inmunidad específica contra antígenos de la mielina, debido al fenómeno de la expansión de epítopos. En la presente revisión, tratamos de delinear los mecanismos patogénicos que conducen a la desmielinización y daño axonal en este modelo. Se destaca la importancia de las citocinas proinflamatorias para el mantenimiento del proceso inflamatorio, y se describen estrategias terapéuticas relacionadas con el sistema de citocinas pro y antiinflamatorias.

Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral inoculation of TMEV, a member of picornavirus family, in susceptible animals. The pathogenesis of paralytic syndrome is associated with a chronic progressive demyelinating disease characterized perivascular of immune inflammatory cells. Although TMEV induced demyelinating disease (TME V-IDD) is initiated by virus specific CD4+ T cells targeting CNS-persistent virus, CD4+ T-cell responses against self myelin epitopes activated via epitope spreading contribute to chronic disease pathogenesis. In the present report we delineated possible pathogenic mechanisms related with inflammatory process, leading to demyelination and axonal loss. The importance of proinflammatory cytokines in sustaining the inflammatory process and cause direct oligodendrotoxicity is emphasized. Different approaches in therapeutic strategies affecting cytokines are also presented.

In brain astrocytes, nuclear factor ΚB ΚB) is activated by stimuli that produce cellular stress causing the expression of genes involved in defence, including the inducible nitric oxide synthase (NOS-2). Theiler's murine encephalomyelitis virus (TMEV) induces a persistent CNS infection and chronic immune-mediated demyelination, similar to human multiple sclerosis. The cytokines interleukin (IL)-4 and IL-10 inhibit the expression of proinflammatory cytokines, counteracting the inflammatory process. Our study reports that infection of cultured astrocytes with TMEV resulted in a time-dependent phosphorylation of IΚBα, degradation of IΚBα and IΚBβ, activation of NF-ΚB and expression of NOS-2. The proteasome inhibitor MG-132 blocked TMEV-induced nitrite accumulation, NOS-2 mRNA expression and phospho-IΚBα degradation, suggesting NF-ΚB-dependent NOS-2 expression. Pretreatment of astrocytes with IL-4 or IL-10 decreased p65 nuclear translocation, NF-ΚB binding activity and NOS-2 transcription. IL-4 and IL-10 caused an accumulation of IΚBα in TMEV-infected astrocytes without affecting IΚBβ levels. The IΚB kinase activity and the degradation rate of both IΚBs were not modified by either cytokine, suggesting de novo synthesis of IΚBα. Indeed, IL-4 or IL-10 up-regulated IΚBα mRNA levels after TMEV infection. Therefore, the accumulation of IΚBα might impair the translocation of the NF-ΚB to the nucleus, mediating the inhibition of NF-ΚB activity. Overall, these data suggest a novel mechanism of action of IL-4 and IL-10, which abrogates NOS-2 expression in viral-infected glial cells.

Proinflammatory mediators have been implicated in demyelinating disorders, including multiple sclerosis, whereas it has been proposed that the anti-inflammatory cytokines interleukin- (IL-) 4 and IL-10 participate in disease recovery. The present study analysed the effect of interferon-γ (IFN-γ) and bacterial endotoxin (lipopolysaccharide, LPS) on proliferation and survival of progenitors and differentiated oligodendrocytes. We also investigated the presence of receptors for IL-4 and IL-10 in oligodendroglial cells and explored a possible protective action of IL-4 and IL-10 in cultures following LPS/IFN-γ. Finally, the role of endogenous nitric oxide (NO) on cell viability and the modulatory action of IL-4 and IL-10 on inducible nitric oxide synthase (iNOS) expression were also analysed. We report that LPS and/or IFN-γ reduced proliferation and viability of oligodendroglial cells. Cell death, presumably by apoptosis as evidence by TUNEL and Annexin V binding, was observed following LPS/IFN-γ, progenitors being more sensitive than differentiated cells. At both developmental stages, LPS/IFN-γ-treated cultures expressed iNOS protein and released micromolar concentrations of NO. In progenitors, LPS/IFN-γ-mediated cell damage was partially dependent on endogenous NO production, whereas NO was fundamental for cytotoxicity of differentiated oligodendrocytes. Both cell types expressed mRNA for IL-4 and IL-10 receptors and expression of IL-10 receptors at the protein level was also demonstrated. Treatment with either cytokine inhibited the expression of iNOS resulting from the proinflammatory stimulation. IL-10 was more effective than IL-4 in suppressing iNOS expression and, interestingly, IL-10 conferred protection against oligodendroglial death evoked by LPS/IFN-γ. Our data raise the question of whether IL-10 may play a protective role in demyelinating diseases, not only downregulating the function of inflammatory cells but also promoting survival of progenitors and differentiated oligodendrocytes.

Maturation of oligodendrocyte progenitors (O2A) is characterized by morphological changes and the sequential expression of specific antigens leading to the formation of myelin membrane. Monoclonal antibodies A2B5, A007, anti-vimentin, and anti-galactocerebroside, recognize oligodendroglia at different stages of development. The neuroepithelial precursor marker nestin is also expressed by the oligodendroglial lineage; we have used enriched populations of progenitors isolated from neonatal rat brain cultures to further examine the cellular distribution of this intermediate filament protein. The phenotypic distribution of nestin positive cells among the oligodendrocyte lineage showed that 65% reacted with A2B5, whereas only 5% were A007+, and 4% galactocerebroside+. The remaining 25% of the cells were not labeled and had small cellular bodies devoid of processes, characteristic of the pre-O2A progenitor. Further analysis of the nestin+ population showed that the majority of the cells were also vimentin+. Antibody-dependent complement mediated cytolysis of A2B5+ (O2A cells) and galactocerebroside+ (mature oligodendrocytes) cells left a population of nestin+ cells that were induced to proliferate in the presence of growth factors and to differentiate into A2B5+ and galactocerebroside+ cells. Proliferating cells maintained in the presence of platelet-derived growth factor or basic fibroblast growth factor retained nestin expression along with A2B5. By contrast, in serum-free medium nestin expression decreased while postmitotic cells acquired A007 and galactocerebroside. Our results suggest that nestin expression is a marker of pre-O2A cells that is maintained in proliferating glial progenitors, but is quickly down-regulated in postmitotic oligodendrocytes (A007+/galacto-cerebroside+) along with A2B5 and vimentin. However, other glial cells including type 2 astrocytes and some amoeboid microglia also share nestin expression. © 2001 Wiley-Liss, Inc.

1. Interleukin-1 (IL-1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E2 (PGE2) production after IL-1β stimulation is dependent upon activation of protein kinases in astroglial cells. 2. Astrocyte cultures stimulated with IL-1β or the phorbol ester, PMA significantly increased PGE2 secretion. The stimulatory action of IL-1β on PGE2 production was totally abolished by NS-398, a specific inhibitor of cyclo-oxygenase-2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL-1β induced the expression of COX-2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL-1 β treatment also induced the expression of COX-2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. 3. Treatment of cells with protein kinase C inhibitors (H-7, bisindolylmaleimide and calphostin C) inhibited IL-1β stimulation of PGE2. In addition, PKC-depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL-1. The ablation of the effects of PMA and IL-1β on PGE2 production, likely results from down-regulation of phorbol ester sensitive-PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC-α from cytosol to membrane following treatment with IL-1β. 4. In addition, IL-1β treatment led to activation of extracellular signal-regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL-1β-induced PGE2 release. 5. ERK1/2 activation by IL-1β was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. 6. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE2, likely by regulating the induction of cyclo-oxygenase-2, in IL-1β-stimulated astroglial cells.

The Na,K-ATPase, which catalyzes the active transport of Na+ and K+, has two principal subunits (α and β) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the β isoforms, β1 predominates in neurons and β2 in astrocytes, although there are some exceptions. Here we demonstrate that β3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified β3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. β3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, β3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the β1 and β2 subunits, showing distinct patterns of labelling of axons (β1) and astrocytes (β2). The C6 glioma cell line was also found to express the β3 isoform preferentially. Since β3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage. (C) 2000 Wiley-Liss, Inc.