myelin-basic-protein and thiazolyl-blue

Ischemic brain injury is widely modeled in vitro with paradigms of oxygen-glucose deprivation (OGD), which leads to cell death. The prevention and attenuation of brain injury by the tetracycline antibiotic minocycline has been attributed largely to suppression of microglial activation, but its benefits in oligodendrocyte cells have not been well characterized. Using primary cultures of rat oligodendroglial precursor cells (OPC) exposed to OGD, we investigated the direct effects of minocycline on the survival, proliferation, and maturation of oligodendroglial lineage cells. OGD for 2 hr caused a decrease in the total number of OPC and the amount of proliferating progenitors by 50%, which was attenuated by inclusion of minocycline. The reduced numbers of immature oligodendroglial cells at 72 hr and of mature oligodendrocytes at 120 hr after OGD were partially restored by minocycline. In OPC, OGD caused an increase of reactive oxygen species (ROS) and production of TUNEL-positive cell numbers, which was abolished by minocycline. Minocycline preferentially increased the expression of superoxide dismutase under OGD but not in control OPC. Minocycline also prevented the OGD-induced downregulation of the transcription factors Sox10 and Olig2 and of myelin-specific genes 2'3' cyclic nucleotide phosphodiesterase (CNP) and myelin basic protein (MBP) in response to OGD. These studies demonstrate direct protective actions of minocycline on oligodendroglial-lineage cells, suggesting potential benefit in white matter injury involving OGD.

Topics: Analysis of Variance; Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; Embryo, Mammalian; Embryonic Stem Cells; Female; Gangliosides; Gene Expression Regulation; Glucose; Hypoxia; Ki-67 Antigen; Minocycline; Myelin Basic Protein; O Antigens; Oligodendroglia; Oxidative Stress; Pregnancy; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factors

Damage to central nervous system white matter is observed following hypoglycemia, raising the possibility that hypoglycemia influences oligodendrocytes and myelination. To examine effects of hypoglycemia on oligodendrocytes and myelin formation, we studied cultured oligodendrocyte precursor cells and cerebellar slice cultures. We observed that with decreasing concentrations of glucose, oligodendrocyte precursor cell proliferation, maturation, and migration decreased. We also observed that hypoglycemia induced apoptotic cell death and activation of caspase-3 in oligodendrocyte precursor cells. Slice culture studies showed that glucose is required for myelinated fiber formation, as with reduction in the glucose concentration, the density of myelinated fibers decreased. Collectively, these data show that hypoglycemia inhibits oligodendrocyte development and myelination and that hypoglycemia triggers apoptotic cell death in oligodendrocyte precursor cells.

Topics: Animals; Animals, Newborn; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Differentiation; Cell Movement; Cells, Cultured; Cerebellum; Dose-Response Relationship, Drug; Glucose; Hypoglycemia; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro Techniques; Mice; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; Rats; Tetrazolium Salts; Thiazoles; Time Factors

We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT-PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA-binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor-kappaB (NFkappaB). For these cells we found that an inductor of NFkappaB (tumor necrosis factor-alpha) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db-cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF-I and NFkappaB activation, but, aside from aTf, only db-cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.

Topics: Apoproteins; Blotting, Western; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA, Complementary; Electrophoretic Mobility Shift Assay; Genes, Reporter; Lac Operon; Myelin Basic Protein; NF-kappa B; Nuclear Proteins; Oligodendroglia; Plasmids; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sp1 Transcription Factor; Tetrazolium Salts; Thiazoles; Thyroid Hormones; Transcription Factors; Transferrin; Tumor Necrosis Factor-alpha