RESUMO
CNP::EGFP transgenic mice, genetically engineered to express the enhanced green fluorescent protein (EGFP) under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase) promoter in oligodendroglial and Schwann cells, constitute a very important and widely used tool for the study of oligodendrocyte (OLG) development and function in young mice. Our results showed that CNP::EGFP mice were significantly more susceptible to CPZ-induced demyelination, as evaluated by MBP immunostaining, oligodendroglial progenitor cell (OPC) recruitment and astroglial, microglial and nestin response. This enhanced vulnerability was a consequence of their hypomyelination. CNP::EGFP control mice also displayed a significant decrease in corpus callosum (CC) thickness and MBP immunoreactivity. Morphometric analysis further showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio carried out in the optic nerve (ON) and CC of CNP::EGFP, as compared to WT mice. Moreover, our results showed a decrease in the number of axons of small caliber, concomitantly with an increase in the number of axons of bigger size with more and enlarged mitochondria, which suggests a high energy demand. These findings and those displaying that MBP+ cells and NF200 staining in the CNP::EGFP cortex were more sparsely distributed provide evidence of axonal loss, which was supported by a decreased number of NeuN+ cells in the CA3 fields of the hippocampus. Transgenic mice also showed an increase in microglial and astroglial activation, accompanied by enhanced lipid peroxidation and recruitment of morphologically altered OPC. Finally, CNPase protein levels proved to be lower than MBP in the CC, which might indicate an altered pattern in myelin proteins with a CNPase deficiency. Behavioral analysis of adult CNP::EGFP transgenic mice supported our results, as it revealed a decrease in locomotion, exploratory activity and motor impairment, as compared to their WT littermates. Our data highlight the relevance of confronting results obtained in adult CNP::EGFP mice with those observed in WT mice. According to our findings, CNP::EGFP hypomyelination might be triggered by the cellular stress induced by the high level of EGFP expression in mature OLG. Adult CNP::EGFP mice could be considered a useful tool to evaluate future therapies for demyelinating diseases such as multiple sclerosis (MS), since these animals present chronic demyelination with axonal degeneration, a characteristic of such pathologies.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Inibidores da Monoaminoxidase/toxicidade , Proteínas da Mielina/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Aldeídos/metabolismo , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Antígeno CD11b/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Ectodisplasinas/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Interleucina-1beta/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.
Assuntos
Diferenciação Celular , Bainha de Mielina/fisiologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Axônios/metabolismo , Comportamento Animal , Células Cultivadas , Cuprizona/toxicidade , Galectina 1/metabolismo , Galectina 3/deficiência , Galectina 3/genética , Galectina 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Polissacarídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos WistarRESUMO
In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNgamma and/or TNFalpha having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFalpha-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines.