OBJECTIVE: To investigate the auditory characteristics of guinea pigs immunized with purified P0 protein from inner ear of guinea pigs. METHOD: Purified inner ear P0 protein was made by Preparative SDS-PAGE. To evaluate the results of Auditory Brainstem Response (ABR), Compound Action Potention (CAP), and Distortion Product Oto-acoustic Emissions (DPOAE) in guinea pigs immunized with purified guinea pigs inner ear myelin protein P0. RESULT: Seven ears of the guinea pigs immunized with purified inner ear P0 protein developed hearing loss. In the ABR study, peak latencies of wave I, III and the interpeak latency of I -III, I-IV were elevated in this group of guinea pigs compared with the control group (P < 0.01). But the interpeak latency of III-IV did not change. In the CAP study, the threshold elevated and latency prolonged (P < 0.01). No significant change of DPOAE was found in the P0-sensitized guinea pigs when compared to controls (P > 0.05). Even though the contra-lateral suppressive effect had the depressed tendency, but there was no significant different when compared with the controls (P > 0.05). CONCLUSION Purified inner ear P0 protein is an important autoimmune inner ear antigen and can develop autoimmune disease of the auditory nerve.
Animals ; Auditory Threshold ; Evoked Potentials, Auditory, Brain Stem ; physiology ; Guinea Pigs ; Immunization ; Myelin P0 Protein ; immunology

BACKGROUND: Mutations in the myelin protein zero (MPZ) gene, which is located on chromosome 1q21-q22, is present in Charcot-Marie-Tooth disease type 1B (CMT1B), CMT type 2, Dejerine-Sottas syndrome, and congenital hypomyelination neuropathy. It is proposed that the nature and position of the MPZ mutations mainly determine the axonal and demyelinating phenotypes. In this study, we investigated to determine the clinical and electrophysiological characteristics in CMT patients with mutations in the MPZ gene. METHODS: We examined mutations of MPZ, in 62 Korean families diagnosed as having CMT disease. Mutations were confirmed by through both strands sequencing. Nerve conduction studies were carried out in CMT patients having each mutation. RESULTS: The three mutations (Asp118Asn, c.449-1G>T (3'-splice site), Lys236Glu), determined to be novel, were not detected in the 105 healthy controls. The mutation frequency of MPZ was similar as those found in several European populations. Electrophysiologically, 3'-splice site mutation (449-1G>T) showed the conduction block and moderate slowing nerve conduction velocities like that of CMT1B. However, the other mutations represented the electrophysiological features of CMT type 2. CONCLUSIONS: We report the identified three novel MPZ mutations in Korean CMT patients and the phenotype-genotype correlations based on nerve conduction studies.
Axons ; Charcot-Marie-Tooth Disease ; Hereditary Sensory and Motor Neuropathy ; Humans ; Mutation Rate ; Myelin P0 Protein* ; Myelin Sheath* ; Neural Conduction ; Phenotype

OBJECTIVE: To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and to study the possible role it may play in the etiology of autoimmune inner ear disease. METHOD: A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30,000 was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freunds adjuvant, another 10 guinea pigs were immunized with complete Freunds adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique. RESULT: The purity of the protein was demonstrated by a single band at the 30000 site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F = 6.48, P<0.01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only. CONCLUSION P0 protein from guinea pigs in ner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein was successfully established.
Animals ; Autoimmune Diseases ; immunology ; Disease Models, Animal ; Guinea Pigs ; Labyrinth Diseases ; immunology ; Myelin P0 Protein ; immunology ; isolation & purification

BACKGROUNDChronic exposure to n-hexane can lead to peripheral neuropathy that no effective treatment regimen could be applied presently. This study investigated whether myelin protein zero (P0) protein and its antibody could be used to distinguish n-hexane intoxication and protect workers from peripheral neuropathy.

METHODSWe compared P0 protein and its antibody among three levels of n-hexane-exposed groups, which included 18 patients with n-hexane-induced peripheral neuropathy as case group, 120 n-hexane-exposed workers as n-hexaneexposed control group, and 147 non-hexane-exposed participants used as control group. ELISA method was applied to detect P0 protein and its antibody.

RESULTSP0 protein in serum was significantly higher in the case group and n-hexane-exposed control group in comparison with the control group (P < 0.01). Compared with the n-hexane-exposed control group, the case group also had significant increase of P0 protein (P < 0.01). After 6 months therapy, P0 protein was observed to decrease significantly in the case group (P < 0.01). The P0 antibody in serum was significantly higher in the n-hexane-exposed control group than in the control group (P < 0.01), but not significantly different between cases and controls.

CONCLUSIONSP0 antibodies in serum may be a short-term effect biomarker for n-hexane exposure. P0 protein in serum may be an early effective biomarker for peripheral nerve neuropathy and its biological limit value needs investigation in the future study.

Adult ; Antibodies ; blood ; immunology ; Cross-Sectional Studies ; Female ; Hexanes ; toxicity ; Humans ; Male ; Myelin P0 Protein ; blood ; immunology ; Peripheral Nervous System Diseases ; blood ; chemically induced ; immunology ; Young Adult

Objective: To investigate the role of CD4⁺CD25⁺regulatory cell (CD4⁺CD25⁺Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4⁺CD25⁺Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4⁺CD25⁺Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4⁺CD25⁺Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4⁺CD25⁺Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4⁺CD25⁺Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4⁺CD25⁺Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4⁺CD25⁺Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.
Animals ; Rats ; Forkhead Transcription Factors ; Myelin P0 Protein ; Rats, Sprague-Dawley ; T-Lymphocytes, Regulatory ; CD4 Antigens/immunology* ; Interleukin-2 Receptor alpha Subunit/immunology*

OBJECTIVETo study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord.

METHODSGreen fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope.

RESULTSTransplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100 distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope.

CONCLUSIONSCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords.

Animals ; Immunohistochemistry ; Microscopy, Electron ; Myelin Basic Protein ; metabolism ; Myelin P0 Protein ; metabolism ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; S100 Proteins ; metabolism ; Schwann Cells ; physiology ; ultrastructure ; Spinal Cord Injuries ; metabolism ; physiopathology

PURPOSE: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. MATERIALS AND METHODS: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. RESULTS: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. CONCLUSION: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.
Cell Death ; Cell Survival ; Charcot-Marie-Tooth Disease* ; Endoplasmic Reticulum ; Endoplasmic Reticulum Stress ; Genotype ; Mass Screening* ; Mutant Proteins ; Myelin P0 Protein ; Myelin Sheath ; Pathology, Molecular ; Peripheral Nervous System Diseases ; Phenotype ; Schwann Cells ; Thapsigargin

OBJECTIVETo investigate the role of myelin protein zero (P(0)) in 2,5-hexanedione (2,5-HD)-induced peripheral nerve injury, and the protective effect of Ginkgo biloba extract (Egb761) on 2,5-HD-induced toxic peripheral neuropathy.

METHODSAfter 4 weeks of treatment with 2,5-HD at different doses (50, 100, 200, 400 mg/kg) in rats, changes in the levels of P(0) in rat sciatic nerves was investigated, and the effect of Egb761 on 2,5-HD-induced toxic peripheral neuropathy was studied.

RESULTSThe blood-nerve barrier (BNB) permeability of the sciatic nerve increased, and the expression of P(0) mRNA and P(0) protein decreased in a dose-dependent manner after treatment with 2,5-HD for 4 weeks. Pretreatment with Egb761 protected against BNB interruption, and inhibited P(0) mRNA and protein reduction during 2,5-HD treatment. Pretreatment with Egb761 significantly reduced loss of body weight (P<0.01) and mitigated gait abnormalities (2.85±0.22) induced by 400 mg/kg 2,5-HD (P<0.01). It also reduced the signs of neurotoxicity induced by 2,5-HD.

CONCLUSION2,5-HD inhibited the expression of P(0) in a dose-dependent manner, and this may be an important mechanism by which toxic peripheral neuropathy is induced by 2,5-HD. Egb761 has a protective effect against 2,5-HD-induced peripheral neurotoxicity in rats.

Animals ; Dose-Response Relationship, Drug ; Environmental Pollutants ; toxicity ; Gene Expression Regulation ; drug effects ; Hexanones ; toxicity ; Male ; Myelin P0 Protein ; genetics ; metabolism ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects

OBJECTIVE: To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and study the possible role it may play in the etiology of autoimmune inner ear disease. METHOD: A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30kd was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund's adjuvant, another 10 guinea pigs were immunized with complete Freund 's adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique. RESULT: The purity of the protein was demonstrated by a single band at the 30 kD site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F =6.48, P <0. 01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only. CONCLUSION P0 protein from guinea pig's inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein is successfully established.
Adenoviridae ; genetics ; Animals ; Cochlea ; metabolism ; Disease Models, Animal ; Electrophoresis, Polyacrylamide Gel ; Gene Expression ; Gene Transfer Techniques ; Genes, Homeobox ; Genes, Reporter ; Genetic Vectors ; Guinea Pigs ; Myelin P0 Protein ; isolation & purification

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