Endothelin (ET)-1 and its receptors (ETA and ETB receptor) are present in the central nervous system. ET exerts biological effects on gliogenesis and glial cell functions. In order to define a possible mechanism of ETA receptor signaling, the distribution of the ETA receptor in developing oligodendrocytes and the effects of ET-1 on the myelination of oligodendrocytes were examined. ETA receptor immunoreactivity was confined to the perivascular elements of the blood vessels during early postnatal development. However later in development, ETA receptor immunoreactivity was no longer observed in the vessels but became localized to the myelinating oligodendrocytes of the primitive corpus callosum of the white matter, apart from the vessels. ET-1 induced myelin basic protein (MBP) in primary oligodendrocyte precursor cell culture though the ETA receptor and was blocked by an ETA receptor antagonist. In addition, ET-1 evoked the release of Ca2+ which is a central regulator of oligodendrocyte differentiation. Our results provide a link between ET-1 and its ETA receptor and myelination during oligodendrocyte differentiation.
Animals ; Brain/pathology ; Calcium/metabolism ; Calcium Signaling ; Cells, Cultured ; Endothelin-1/metabolism/physiology ; Mice ; Mice, Inbred ICR ; Myelin Basic Proteins/genetics/metabolism ; Myelin Sheath/*physiology ; Oligodendroglia/cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/metabolism/*physiology

We constructed the expression vector by inserting 21.5 KDa MBP human brain full-length cDNA coding sequence digested with restriction enzyme EcoR I and Sal I into downstream of pGEX-5T expression vector. The recombinant vector p5TMP was transformed into E. coli and the positive clonies were selected and incubated in LB medium induced by IPTG (isopropyl- -D-thiogalactoside). A new polypeptide band with apparent molecular weight 42 KDa was detected in transformed cell lysates by SDS-PAGE. Western blotting analysis confirmed that this fusion protein reacted specifically with antibodies to MBP, the expression level of MBP was about 414.6 mg/L medium estimated by immuno-dot blot, ELISA and absorbance scanning. Newzealand rabbits were immunized by subcutaneous injection of the purified recombinant MBP. The titer was obtained at 1:16 after 5 injections. The specificity of the antibody to MBP was confirmed by immuno-blot and Western blotting.
Animals ; Antibody Formation ; Autoantibodies ; Brain ; immunology ; metabolism ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Myelin Basic Protein ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; immunology

Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.
Animals ; Animals, Newborn ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; genetics ; Cells, Cultured ; Cuprizone ; toxicity ; Demyelinating Diseases ; chemically induced ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Regulation, Developmental ; genetics ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Myelin Proteolipid Protein ; metabolism ; Myelin Sheath ; drug effects ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effects of Bushen Yisui Capsule (, BSYSC) on the oligodendrocyte lineage genes (Olig) 1 and Olig2 in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE) in order to explore the remyelination effect of BSYSC.

METHODSThe mice were randomly divided into normal control (NC), EAE model (EAE-M), prednisone acetate (PA, 6 mg/kg), BSYSC high-dose (3.02 g/kg) and BSYSC low-dose (1.51 g/kg) groups. The mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55. The neurological function scores were assessed once daily. The pathological changes in mice brains were observed with hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). The protein expressions of myelin basic protein (MBP), Olig1 and Olig2 in brains were measured by immunohistochemistry. The mRNA expressions of Olig1 and Olig 2 was also determined by quantitative real-time polymerase chain reaction.

RESULTSCompared with the EAE-M mice, (1) the neurological function scores were significantly decreased in BSYSC-treated mice on days 22 to 40 (P<0.01); (2) the inflammatory cells and demyelination in brains were reduced in BSYSC-treated EAE mice; (3) the protein expression of MBP was markedly increased in BSYSC-treated groups on day 18 and 40 respectively (P<0.05 or P<0.01); (4) the protein expression of Olig1 was increased in BSYSC (3.02 g/kg)-treated EAE mice on day 40 (P<0.01). Protein and mRNA expression of Olig2 was increased in BSYSC-treated EAE mice on day 18 and 40 (P<0.01).

CONCLUSIONThe effects of BSYSC on reducing demyelination and promoting remyelination might be associated with the increase of Olig1 and Olig2.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Brain ; drug effects ; pathology ; ultrastructure ; Bromodeoxyuridine ; metabolism ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; genetics ; pathology ; physiopathology ; Female ; Fluorescent Antibody Technique ; Mice, Inbred C57BL ; Myelin-Oligodendrocyte Glycoprotein ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Oligodendrocyte Transcription Factor 2 ; RNA, Messenger ; genetics ; metabolism

Intradermal gene administration was found to induce a more profound immune response than direct intramusclular gene injection. We performed intradermal vaccination of B10.PL mice with DNA encoding for the V 8.2 region of the T-cell receptors (TCR). Three weeks later, these mice were immunized with rat myelin basic protein (MBP). Daily mean clinical scores and mortality rate were lower in this group compared with controls. The proliferative responses of lymph node cells to rat MBP were slightly less in the vaccination groups than in the control groups (p<0.05). However, we detected no differences between the two groups with regard to the production of MBP-specific IgG, IgG1, & IgG2a antibodies. The levels of cytokine mRNA expression in the vaccination groups were observed higher than in the control groups without antigen-specific stimulation, but all of cytokine expressions between the vaccination and control groups after antigen-specific stimulation were identical. These results demonstrate that intradermal DNA vaccines encoding for TCR might prove to be useful in the control of autoimmune disease.
Animals ; Autoantibodies/blood ; Base Sequence ; Cytokines/genetics ; DNA, Complementary/genetics ; Encephalomyelitis, Autoimmune, Experimental/etiology/immunology/*prevention and control ; Female ; Gene Expression ; *Genes, T-Cell Receptor beta ; In Vitro ; Injections, Intradermal ; Lymphocyte Activation ; Mice ; Myelin Basic Proteins/immunology ; RNA, Messenger/genetics/metabolism ; Rats ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Vaccines, DNA/*administration and dosage/genetics