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

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.
Animals ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Disease Models, Animal ; Female ; Ganglia, Spinal ; cytology ; Gene Expression Regulation ; genetics ; physiology ; HEK293 Cells ; Humans ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Male ; MicroRNAs ; genetics ; metabolism ; Motor Endplate ; genetics ; Myelin P0 Protein ; metabolism ; Nerve Regeneration ; genetics ; physiology ; Nerve Tissue Proteins ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Sciatic Neuropathy ; metabolism ; surgery ; therapy

OBJECTIVETo analyze mutation of the PMP22 gene in a pedigree affected with Charcot-Marie-Tooth disease.

METHODSGenomic DNA was extracted from peripheral blood samples of the proband and members from his family, and fetal DNA was extracted from amniotic fluid sample. Multiplex ligation-dependent probe amplification (MLPA) and array-based comparative genomic hybridization (array-CGH) analyses were carried out to determine the copy number of the PMP22 gene. Sanger sequencing was carried out to detect point mutations of the PMP22 gene.

RESULTSA heterozygous duplication of the PMP22 gene was detected in the proband and his father, while no point mutation, insertion or deletion was found in them. No duplication or deletion of the PMP22 gene was found in other family members.

CONCLUSIONBased on clinical symptoms and genetic findings, the heterozygous duplication of the PMP22 gene is probably the cause of the disease in the proband. The fact that the father has carried the same duplication but with no detectable symptom may be due to irregular transmission pattern of the mutation. Genetic counseling for the family should therefore be with caution.

Adult ; Charcot-Marie-Tooth Disease ; genetics ; Comparative Genomic Hybridization ; methods ; DNA Mutational Analysis ; Family Health ; Female ; Gene Dosage ; Gene Duplication ; Genetic Predisposition to Disease ; genetics ; Heterozygote ; Humans ; Male ; Multiplex Polymerase Chain Reaction ; methods ; Myelin Proteins ; genetics ; Pedigree

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

OBJECTIVE: DNA methylation has been shown to be a potential biomarker for early cancer detection. The aim of this study was to evaluate DNA methylation profiles according to liquid-based Pap (LBP) test results and to assess their diagnostic value in a Korean population. METHODS: A total of 205 patients with various Papanicolaou test results were enrolled to this study (negative, 26; atypical squamous cells of undetermined significance, 39; low grade squamous intraepithelial lesion, 44; high grade squamous intraepithelial lesion (HSIL), 48; and cancer, 48). DNA methylation analysis of four genes, ADCYAP1, PAX1, MAL, and CADM1, was performed on residual cervical cells from LBP samples using a quantitative bisulfite pyrosequencing method. To evaluate the diagnostic performance of the four methylated genes for cancer detection, receiver operating characteristic (ROC) curves were drawn. Sensitivities and specificities were also tested at cutoffs determined from the ROC curves. RESULTS: Cervical cancer cells showed dramatically increased methylation levels for the four genes analyzed. ADCYAP1 and PAX1 also trended toward elevated methylation levels in HSIL samples, although the levels were much lower than those in cancer cells. The sensitivities of methylated ADCYAP1, PAX1, MAL, and CADM1 for the detection of cancer were 79.2%, 75.0%, 70.8%, and 52.1%, and the specificities were 92.0%, 94.0%, 94.7%, and 94.0%, respectively. Methylated ADCYAP1 and PAX1 demonstrated relatively better discriminatory ability than did methylated MAL and CADM1 (area under the curves 0.911 and 0.916 vs. 0.854 and 0.756, respectively). CONCLUSION: DNA methylation status, especially in the ADCYAP1 and PAX1 genes, showed relatively good specificity, ranging from 90% to 94%. The possible additive and complementary roles of DNA methylation testing with respect to conventional cervical cancer screening programs will need to be validated in prospective population-based studies.
Alphapapillomavirus/genetics ; *Atypical Squamous Cells of the Cervix/pathology/virology ; Cell Adhesion Molecules/genetics ; *DNA Methylation ; Female ; Genotype ; Humans ; Immunoglobulins/genetics ; Myelin and Lymphocyte-Associated Proteolipid Proteins/genetics ; Paired Box Transcription Factors/genetics ; Papanicolaou Test ; Pituitary Adenylate Cyclase-Activating Polypeptide/genetics ; ROC Curve ; Squamous Intraepithelial Lesions of the Cervix/*genetics/pathology/virology ; Uterine Cervical Neoplasms/*genetics/pathology/virology ; Vaginal Smears

OBJECTIVETo study the effects of Jisuikang (Chinese characters) on Nogo-NgR gene expression, and to explore the protective effects and mechanism of Jisuikang (Chinese characters) on spinal cord injury in rats.

METHODSOne hundred eighty female rats were randomly assigned to 6 groups(30 rats per group). Sham group: T10 lamina was resected only and spinal cord was untreated. Model group: spine cord injury (SCI) was created with a modified impinger of Allen's by impacting on the T10 spinal cord. Prednisolone group: Prednisolone (0.06 g/kg) was given by intragastric administration at a time interval of 24 hours after operation. The Jisuikang (Chinese characters) high, moderate and low dose groups: Jisuikang (Chinese characters) was supplied with different dose (50 g/kg, 25 g/kg, 12.5 g/kg) by intragastric administration in rats after operation,for the first time at 30 min after surgery. Animals were killed 3, 7, 14 days after surgery. The expression levels of Nogo-A and NgR were observed by Western Blot and Real-time PCR.

RESULTSThe expression of Nogo-A and NgR was at the basic level at all time points in sham group. Compared with model group, the protein expression levels of Nogo-A and NgR in sham, prednisolone, Jisuikang (Chinese characters) moderate dose groups were statistically significant at all time points (P < 0.05). No difference was found in Jisuikang (Chinese characters) high and low dose groups (P > 0.05). Three days after surgery, the mRNA levels of Nogo-A and NgR in treatment group were significantly lower than that in model group (P < 0.01); 7 days after surgery,Nogo-A and NgR mRNA expression were dramatically upregulated and peaked; 14 days after operation, the expression was decreased, but still significantly higher than that in other treatment groups (P < 0.01). Prednisolone and Jisuikang (Chinese characters) moderate dose groups showed the most significant effects among all groups,but there was no statistically significant difference between two groups (P > 0.05).

CONCLUSIONThe decoction Jisuikang (Chinese characters) can promote the nerve cell regeneration by regulating Nogo-A and NgR gene expression, activating Nogo- NgR signaling pathways after acute spinal cord injury.

Animals ; Female ; GPI-Linked Proteins ; analysis ; genetics ; physiology ; Medicine, Chinese Traditional ; Myelin Proteins ; analysis ; genetics ; physiology ; Nerve Regeneration ; drug effects ; Nogo Proteins ; Nogo Receptor 1 ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis ; genetics ; physiology ; Signal Transduction ; drug effects ; Spinal Cord Injuries ; drug therapy ; metabolism

OBJECTIVETo delineate the clinical, electrophysiological and genetics features of a family where 4 members were affected with hereditary neuropathy with liability to pressure palsies (HNPP).

METHODSClinical features of the 4 patients were summarized. Electrophysiological examination and genetic analysis were carried out.

RESULTSAll of the patients showed recurrent motor and sensory disturbances after minor traction or constriction. Electrophysiology study revealed that the prolonged latency and reduced conduction velocity of peripheral nerve were general and with multiple sites of affection. The nerve locations liable to entrapment showed conduction block. A deletion mutation of peripheral myelin protein 22 (PMP22) gene was identified by genetic analysis.

CONCLUSIONHNPP usually affects areas where nerves are liable to entrapment, and presents with motor and sensory disturbances of the innervated areas. Electrophysiological study reveals general nervous demyelination. Genetic analysis can clarify the diagnosis of HNPP.

Adult ; Arthrogryposis ; genetics ; physiopathology ; Hereditary Sensory and Motor Neuropathy ; genetics ; physiopathology ; Humans ; Male ; Myelin Proteins ; genetics ; Neural Conduction

OBJECTIVE: To suppress NgR gene expression in neural stem cells and observe differentiation of neural stem cells in vitro after interfered which provide nutritional support for the facial nerve repair in vivo. METHOD: PCR amplification, restriction endonuclease digestion, T4DNA ligase connections were used to connected NgR with rector pGCsi, and constructed recombinant vector (NgR shRNA). Lipofectamine 2000 were used to transfect the NSC. The expression of NgR was examined by Western Blot. The proportion of neural stem cells transformed into neurons after transfection was tested by Immunocytochemistry. Neural stem cells were planted in PLGA tubes after transfected, and were scanned by electron microscopy. RESULT: NgR shRNA plasmid was constructed and infected neural stem cells successfully. Western Blot showed that the expression of NgR decreased in neural stem cells after interference. Immunocytochemistry showed that the rate of the neural stem cells transformed into neurons after interfered was significantly higher (P < 0.01). CONCLUSION Neural stem cells were transformed into neurons after NgR shRNA plasmid infected neural stem cells, which promoted axonal regeneration more effectively and provided a efficient and stable gene platform for facial nerve repair.
Animals ; Cell Differentiation ; Cells, Cultured ; Facial Nerve ; surgery ; GPI-Linked Proteins ; genetics ; metabolism ; Myelin Proteins ; genetics ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Nogo Receptor 1 ; RNA Interference ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; genetics ; metabolism

BACKGROUNDOne of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo. Here, we tested the inhibition of Nogo by RNA interference both in vitro and in vivo, using recombinant adenovirus-mediated transfection of short hairpin RNAs, to explore a new method of treatment for spinal cord injury.

METHODSWe designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA, packaged the clones into adenovirus, and amplified the recombinant virus in 293 cells. We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats. We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.

RESULTSIn vitro, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%, respectively, compared with Nogo expression in cells transfected with the unrelated control small hairpin RNA (P < 0.005). Similarly, Nogo protein expression decreased by 50% and 48%, respectively (P < 0.005). In vivo, in spinal cord injury model rats, the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%, respectively, compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA (P < 0.005). The Nogo protein level was similarly decreased.

CONCLUSIONSWe were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs, both in vitro and in vivo. This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery. Thus gene therapy may be an effective treatment for spinal cord injury.

Adenoviridae ; genetics ; Animals ; Blotting, Western ; Humans ; Immunohistochemistry ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; therapy

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