The Function And Mechanism Of FA/DHFR Regulation On Oligodendrocytes Development And Remyelination

Objectives:Folate (FA) plays an important role during the central nervous system (CNS) development, lacking of folate may increase fetal risk of neural tube defects, which is the main cause of perinatal child mortality. Current research focuses on the role of folate towards neurons, while the role of folate on oligodendrocytes (OLs) was rarely reported. Our previous studies have found that folate deficiency causes an obvious delay in oligodendrocytes development in vivo, while folate supplement enhances oligodendrocytes maturation both in vitro and in vivo, which is accompanied with the upregulation of DHFR expression. These results suggest that FA/DHFR acts a key role in oligodendrogenesis. Based on these, this study aims to explore the regulation of FA/DHFR on oligodendrocytes development and remyelination, and then investigate the mechanism under it. Taken together, our study is not only to investigate the effects of FA/DHFR on oligodendrocytes development, but also to provide new vision for the prevention and treatment of diseases caused by folate deficiency or related to demyelination in the CNS.Methods:1. The effects of FA/DHFR on oligodendrocytes development and remyelination1) Female mice were received different dose of folate (0.2 mg/kg and 40 mg/kg) in the diet from 2 weeks prior to mating until animals sacrifice, in situ hybridazation and immunostaining analysis were carried out to detect oligodendrocytes development in spinal cords.2) qRT-PCR, western blot and immunofluorescence were used to evaluate the effect of folate on oligodendrocytes in vitro.3) DHFR expression was detected after folate treatment by qRT-PCR and western blot. 4) Immunostaining was used to detect DHFR expression in oligodendrocyte-enriched regions of the CNS.5) Methotrexate (MTX) was used as a specific DHFR inhibitor to estimate the effect on oligodendrocytes by qRT-PCR and western blot in vitro.6) Injection by i.p of MTX was used for mimicking a specific FA/DHFR-inhibited model, Elisa and qRT-PCR was applied for the establishment of the model, then immunostaining and transmission electron microscope were applied to observe the development of oligodendrocytes and myelin sheathintegrity.7) Immunostaining was carried out to see whether folate antagonized oligodendrocytes defect caused by DHFR inhibition.8) Demyelination and remyelination model induced by cuprizone (CPZ) was used to detect the treatment of folate and the injury of MTX.2. The mechanism of FA/DHFR regulation on oligodendrocytes development1) Immunofluorescence and western blot were used to detect the apoptosis and differentiation of oligodendrocytes when DHFR was inhibited.2) Immunofluorescence and H&E staining were used to detect the proliferation of oligodendrocytes and inflammation when DHFR was inhibited.3) p-AMPKa expression in oligodendrocytes was determined by immunostaining.4) Detection of AMPK signaling pathway after FA/MTX treatment in vivo and in vitro by western blot.5) Overexpression or knock down of AMPKal by transfection with AMPKal plasmid or sh-RNA into Oli-neu cells, qRT-PCR was used to test the effect of AMPKa signal on oligodendrocytes development.6) In vitro and in vivo models were used to evaluate whether AMPKa activitors reversed oligodendrocytes defects caused by DHFR inhibition.Results:1. The effects of FA/DHFR on oligodendrocytes development and remyelination1) Folate was required for oligodendrocytes developmentFemale mice were received low-folate (0.2 mg/kg) or high-folate (40 mg/kg) in the diet from 2 weeks prior to mating until animals sacrifice, and oligodendrocytes development stage in the CNS of the neuronal mice was examined. Results showed that low-folate slowered oligodendrocytes development process when compared to the mice feed with normal diet, while high-folate promoted oligodendrocytes development significantly. Then the effect of folate was verified by primary OPCs culture in vitro, and the results revealed that high concentration of folate (50 ?g/ml) increased the oligodendrocytes marker Olig2 expression and the myelin protein MBP and CNP expression notably in contrast to low concentration of folate (0.02 ?g/ml). Together, these data suggested that folate played an important role in oligodendrocytes development.2) Folate downstream DHFR participated in regulation of oligodendrocytes development and myelinationExpression of key factors in folate metabolism in spinal cords from mice feed with high-folate diet was detected by qRT-PCR, and results showed that only dhfr gene expression was up-regulated. Meanwhile Oli-neu cells were cultured with different concentration of folate, and results of PCR and western blot showed that the mRNA and protein levels of oligodendrocytes markers increased notably as well as DHFR protein expression after folate treatment. These observations revealed that DHFR may participate in regulation of oligodendrocytes development.Immunostaining results suggested that DHFR could be expressed in oligodendrocytes rather than astrocytes. In addition, DHFR was strongly expressed in CC1+ differentiated oligodendrocytes but weakly in PDGFRa+ OPCs in spinal white matter. Transfection with DHFR plasmid could up-regulate expression of oligodendrocytes-associated genes as well as down-regulate negative regulators of oligodendrocyte in Oli-neu cells. On the contrary, qRT-PCR and western blot results showed that gene and protein expression of oligodendrocytes-related regulators reduced in a dose-dependent manner in use of DHFR-special inhibitor MTX.Injection by i.p of MTX was used to inhibit DHFR expression in vivo, and results showed that mRNA and protein levels of DHFR decreased significantly after MTX treatment. Besides, folate level in serum also reduced in a dose-dependent manner. Furthermore, immunostaining results showed that oligodendrocytes markers decreased significantly after DHFR inhibition and it could not be repaired automatically as the age increased. The TEM results showed that DHFR inhibition clearly damaged the integrity of myelin sheath. qRT-PCR and immunostaining results suggested that folate could partially reverse oligodendrocytes defects induced by DHFR inhibition. Taken together, these data revealed that DHFR participated in regulation of oligodendrocytes development.3) The effects of folate and MTX on cuprizone-induced demyelinationImmunohistochemistry and LFB staining results revealed that folate (10 mg/kg/d) injected by i.g. in cuprizone-induced demyelination model can efficiently alleviate symptoms of demyelination, suggesting a protection of folate on demyelination. Consistent with this, MTX (4 mg/kg/3d) injected by i.p. in remyelination stage of cuprizone-induced demyelination model delayed the automatic repair of myelin obviously, indicating that DHFR may play a role in remyelination.2. The mechanism of FA/DHFR regulation on oligodendrocytes development1) DHFR inhibition caused oligodendrocytes apoptosis and differentiation disordersTUNEL and immunostaining results showed that DHFR inhibition induced oligodendrocytes apoptosis and differentiation disorders, western blot results further verified that DHFR inhibition led most of oligodendrocytes in undifferentiated stage. In addition, BrdU staining and quantitative results showed that DHFR inhibition had no effect on the proliferation of oligodendrocytes. H&E staining results showed that no significant inflammation was detected after DHFR inhibition.2) FA/DHFR regulated oligodendrocytes development through AMPKa signaling pathwayImmunofluorescence results showed that p-AMPKa was expressed in oligodendrocytes. Western blot results suggested that folate supplement could activate AMPKa pathway, while DHFR inhibition could block AMPKa pathway. AMPKa activation by transfection with AMPKal plasmid in Oli-neu cells promoted expression of oligodendrocytes-associated regulators. On the contrary, AMPKa inhibition by transfection with AMPKal sh-RNA in Oli-neu cells decreased expression of oligodendrocytes-associated regulators.Meanwhile AMPKa activators metformin (MET) and AICAR were applied in Oli-neu cells to test the effect of AMPKa on oligodendrocytes. The results showed that both of them could antagonize the decrement of oligodendrocytes-related regulators expression induced by DHFR inhibition. Furthermore, MET (100 mg/kg/d) was injected by i.p. to observe its protection against MTX. qRT-PCR results showed that the mRNA level of AMPKal had increased in MET+MTX compared with MTX. Immunohistochemistry and LFB staining results showed that MET increased the expression of oligodendrocytes markers and myelin sheath region notably. Taken together, these data indicated that AMPKa took part in FA/DHFR regulation of oligodendrocytes development.Conclusion:This study discovered that FA/DHFR promoted oligodendrocytes development and myelination in vitro and in vivo for the first time, which was verified in demyelination model induced by CPZ. Furthermore we investigated the mechanism under FA/DHFR regulation of oligodendrocytes development. We found that DHFR inhibition caused oligodendrocytes appotosis and differentiation disorders, and AMPKa pathway participated in FA/DHFR regulation of oligodendrocytes development. Interestingly, AMPKa activator was able to reverse oligodendrocytes defects in vitro and in vivo caused by FA/DHFR inhibition partially. Taken together, we suggested here a novel role of FA/DHFR/AMPKa pathway for oligodendrocytes survival and differentiation, which not only gives evidence for the function of folate in the CNS develoment, bur also enriches the regulatory factors network of oligodendrocytes, broaden the vision and direction for the clinical treatment of demyelinating disease.