Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.
Animals ; Fluorescent Antibody Technique ; Mice ; Mutation/genetics ; Neurofilament Proteins/genetics/*metabolism ; Phosphatidylinositol Phosphates/*metabolism ; Phospholipase C gamma/metabolism ; *Protein Multimerization

OBJECTIVETo screen the proteins with differential expression levels in the cerebral tissue of hens exposed to tri-ortho-cresyl phosphate (TOCP), and to provide target proteins for studying the mechanism of organophosphoms ester-induced delayed neurotoxicity (OPIDN).

METHODSThirty two adult Roman hens were randomly divided into four groups: TOCP group was exposed to 1000 mg/kg TOCP, PMSF group was exposed to 40 mg/kg PMSF, PMSF plus TOCP group was exposed to 40 mg/kg PMSF and after 24 h exposed to 1000 mg/kg TOCP, control group was exposed to normal saline. All hens exposed to chemicals by gastro-intestine for 5 days were sacrificed, and the cerebral tissue were dissected and homogenized in ice bath. Total proteins extracted from the cerebral tissue were separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The 2-DE maps were visualized after silver staining and analyzed by Image Master 2D software. At last ,the expressed protein spots were identified by Mass spectrometry.

RESULTSFrom total proteins in TOCP group, the PMSF plus TOCP group and PMSF group, 1185, 1294 and 1063 spots were detected, respectively. One thousand three hundred thirty two spots from total proteins in control group were detected. The match rates of protein spots in TOCP group, the PMSF plus TOCP group and PMSF group were 78.32 %, 79.56 % and 80.93%, respectively. There were 235 protein spots with differential expression levels between TOCP group and control group, which included 158 up regulation spots and 77 down regulation spots. According to the PMSF features, there were 102 spots with differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF plus TOCP group, among them there were 13 spots with 4 fold differential expression levels between TOCP group and control group and without differential expression levels between TOCP group and PMSF group. Seven protein spots (homer-1b, Destrin, heat shock protein 70, eukaryotic translation initiation factors, proteasome alpha1 subunit, lactate dehydrogenase B, glutamine synthetase) were detected by Mass spectrometry.

CONCLUSIONThere are 112 protein spots with differential expression levels of the cerebral tissue in TOCP group, which may be related to OPIDN, among them 13 protein spots with differential expression levels are associated closely with OPIDN. Seven protein spots detected by Mass spectrometry may be related to the mechanism induced by OPIDN.

Animals ; Brain ; metabolism ; Cerebrum ; drug effects ; metabolism ; Chickens ; Neurofilament Proteins ; metabolism ; Phenylmethylsulfonyl Fluoride ; toxicity ; Proteome ; analysis ; Tritolyl Phosphates ; toxicity

OBJECTIVETo investigate the dynamic alterations of neurofilament subunits (NF) in sciatic nerve of hens with organophosphorus ester induced the delayed neurotoxicity or neuropathy (OPIDN).

METHODSHens with OPIDN were produced by giving 30 mg/kg methamidophos subcutaneously to the 10-month-old Roman hens daily for 15 days, and sacrificed after manifesting neurotoxic clinical signs on the 2nd, 10th, and 23rd day respectively. The sciatic nerves were dissected, homogenized and centrifuged. The levels of NF in supernatant and pellet of sciatic nerves were examined by Western blotting respectively at different time from 2 to 23 days.

RESULTSIntegrated optional density (IOD) of high molecular weight neurofilament (NF-H) in sciatic nerve pellet of hens on the day 2, 10, 23 after appearance of OPIDN were 145,117 +/- 17,038, 55,917 +/- 17,333 and 45,038 +/- 6,662 respectively. As compared with the control group (78,875 +/- 22,569), the contents of NF-H in pellet were increased by 84% on day 2, and decreased by 29% and 43% on day 10 and 23 respectively. IOD of NF-H in supernatant of sciatic nerves were 4,709 +/- 1,739, 12,337 +/- 3,205 and 16,745 +/- 931, which were reduced significantly as compared with the control (44,083 +/- 6,895) at three different times. There was no significant difference in IOD of middle molecular weight neurofilament (NF-M) between control group (27,925 +/- 2,660) and on day 2 (31,493 +/- 4,625) in pellet. Those were 19,367 +/- 2,746 and 6,612 +/- 1,119 respectively on day 10 and day 23 in pellet of hen's sciatic nerve, which were much less than that in control. Little were detected in supernatant on day 10, and the IOD of NF-M were 3,196 +/- 269 and 5,206 +/- 1,292 on day 2 and day 23 respectively, which were lessened by 81% and 70% as compared with the control (17,243 +/- 3,232). In sciatic nerve pellet of hens, IOD of low molecular weight neurofilament (NF-L) on day 2 was 39,211 +/- 3,800, which was much higher than that in the control (28,749 +/- 9,319). There were no significant differences between IOD on day 10 (27,974 +/- 3,611), day 23 (21,507 +/- 2,286) and the control. There was no detection both on day 2 and 10 in supernatant of sciatic nerve, and IOD of NF-L were 5,962 +/- 1,929 on day 23, which were reduced significantly compared with the control (11,897 +/- 352).

CONCLUSIONThe alterations of NF in sciatic nerve might contribute to the occurrence and development of OPIDN.

Animals ; Chickens ; Female ; Insecticides ; toxicity ; Neurofilament Proteins ; metabolism ; Organothiophosphorus Compounds ; toxicity ; Sciatic Nerve ; drug effects ; metabolism ; pathology ; Toxicity Tests

Adolescent ; Cerebellar Cortex ; metabolism ; pathology ; Cerebellar Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Ganglioneuroma ; metabolism ; pathology ; surgery ; Humans ; Neurofilament Proteins ; metabolism ; Synaptophysin ; metabolism

Brain ; Choristoma ; metabolism ; pathology ; surgery ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Infant ; Neurofilament Proteins ; metabolism ; Orbit ; pathology ; surgery ; Orbital Diseases ; metabolism ; pathology ; surgery

OBJECTIVETo investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells.

METHODSBone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry.

RESULTSMononuclear cells separated by Percoll's were passaged 10 times by trypsin/ethylenediaminetetraacetic acid (EDTA) digestion in 40 days, and BMSCs increased about 6x10(7) times in this short period. Immunohistochemistry identified that BMSCs were CD34- and CD31-, but they expressed neuron specific enolase; 0.01%-0.02% of total cells expressed nestin, the marker for neural progenitor cells; 40%-50% cells stained heavily by neurofilament 200; and no glial fibrillary acidic protein (GFAP) positive cells were identified; S100 expression was detected among 0.1%-0.2% cells.

CONCLUSIONSBone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; Humans ; Immunohistochemistry ; Intermediate Filament Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurofilament Proteins ; metabolism ; Phosphopyruvate Hydratase ; metabolism ; S100 Proteins ; metabolism ; Schwann Cells ; cytology

OBJECTIVETo study the changes in the levels of autophagy-related proteins, Atg1, Atg5, and Beclin1, in organophosphate-induced delayed neuropathy (OPIDN) caused by tri-ortho-cresyl phosphate (TOCP), and to investigate the molecular pathogenic mechanism of OPIDN.

METHODSThirty adult Roman hens were randomly and equally divided into control group and 1, 5, 10, and 21 d intoxication groups. Each hen in the intoxication group was administered TOCP by gavage at a single dose of 750 mg/kg, while each hen in the control group was administered the same volume of corn oil. The hens were killed at the corresponding time points, and their tibial nerves and spinal cords were collected. The levels of Atg1, Atg5, and Beclin1 in the tibial nerves and spinal cords were measured by immunoblotting.

RESULTSCompared with those in the control group, the levels of Atg1 in tibial nerves decreased by 29.8%, 64.4%, 43.5%, and 19.8% at 1, 5, 10, and 21 d, respectively, after intoxication ((P < 0.05); the levels of Atg5 in tibial nerves decreased by 36.8%, 49.6%, 51.2%, and 31.5% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05); the levels of Beclin1 in tibial nerves decreased by 68.5%, 66.3%, and 32.2% at 1, 5, and 10 d, respectively, after intoxication (P < 0.05). Compared with those in the control group, the levels of Atg1 in spinal cords decreased by 23.5%, 48.7%, and 20% at 1, 5, and 10 d, respectively, after intoxication (P < 0.05); the levels of Atg5 in spinal cords decreased by 32.7%, 51.5%, 47.3%, and 39.6% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05); the levels of Beclin1 in spinal cords decreased by 28.9%, 50.2%, 43.2%, and 28.3% at 1, 5, 10, and 21 d, respectively, after intoxication (P < 0.05).

CONCLUSIONThe intoxication of TOCP is associated with the significant changes in the levels of autophagy-related proteins in the nervous tissues of hens, which might be involved in the pathogenesis of OPIDN.

Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; Chickens ; Female ; Intracellular Signaling Peptides and Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Nervous System Diseases ; chemically induced ; metabolism ; Neurofilament Proteins ; metabolism ; Spinal Cord ; metabolism ; Tibial Nerve ; metabolism ; Tritolyl Phosphates ; toxicity

OBJECTIVETo investigate the effect of 2,5-hexanedione (HD) on degradation of low-molecular-weight neurofilaments (NF-L) in nervous tissue of rats, and to explore the molecular mechanism of n-hexane neuropathy.

METHODSFifty male Wistar rats were randomly divided into one-week poisoning group (n = 10), two-week poisoning group (n = 10), three-week poisoning group (n = 10), four-week poisoning group (n = 10), and control group (n = 10). In the four poisoning groups, a rat model of n-hexane neuropathy was established by intraperitoneal injection of HD (400 mg/kg/d). The change in the sciatic nerve ultrastructure of each rat was observed under an electron microscope. The progression of HD-induced peripheral neuropathy was evaluated using a gait scoring system. The degradation rates of NF-L in the sciatic nerve and spinal cord of each rat were measured by Western Blotting.

RESULTSThe rats showed decrease in muscle strength and abnormal gait after two weeks of HD poisoning and mild or moderate paralysis after four weeks of HD poisoning. The sciatic nerve showed degenerative change, according to electron microscope observation. Compared with the control group, the two-week poisoning group, three-week poisoning group, and four-week poisoning group had the NF-L degradation rates decreased by 25.8%, 70.4%, and 69.7%, respectively, in the supernatant fraction of sciatic nerve, and by 14.7%, 64.6%, and 67.3%, respectively, in the sediment fraction of sciatic nerve, all showing a significant difference (P < 0.01). Compared with the control group, the one-week poisoning group had the NF-L degradation rate decreased by 33.87% in the supernatant fraction of spinal cord, the four-week poisoning group had the NF-L degradation rate increased by 16.2% in the supernatant fraction of spinal cord, and the one-week poisoning group and two-week poisoning group had the NF-L degradation rates decreased by 46.3% and 13.0% in the sediment fraction of spinal cord, all showing a significant difference (P < 0.01).

CONCLUSIONHD poisoning significantly inhibits NF-L degradation in the sciatic nerve, which may be associated with NF degeneration and accumulation in the axons of patients with n-hexane neuropathy.

Animals ; Hexanes ; poisoning ; Hexanones ; pharmacology ; Male ; Nerve Tissue ; drug effects ; metabolism ; physiopathology ; Neurofilament Proteins ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; metabolism ; physiopathology

OBJECTIVETo investigate the dynamic changes of neurofilament contents in rat's spinal cord induced by 2, 5-hexanedione (2, 5-HD), and explore the molecular mechanism of n-hexane neuropathy.

METHODSMale Wistar rats were administered at a dosage of 400 mg/kg/day 2, 5-HD for 2, 4 and 8 weeks respectively. HD-induced neurological defects were detected and quantified using gait score, and the relative lev-els of NF-H, NF-M, and NF-L in spinal cords of rats were determined by Western Blotting.

RESULTSExposure to 2, 5-HD produced progressive gait abnormalities, which suggested that the rat model of 2, 5-HD-induced neurotoxicity was established successfully. Western-Blotting results showed that NFs content in spinal cord demonstrated a progressive decline as the intoxication continued. In the supernatant fraction, compared to the controls, NF-H con-tent decreased by 15.7%, 57.0%, and 58.0% respectively after 2, 4, and 8-week treatment with 2, 5-HD (P < 0.01); accordingly, NF-M decreased by 36.0%, 61.3%, and 65.2% respectively (P < 0.01); NF-L decreased by 20.8%, 43.9%, and 44.3% respectively (P < 0.01). In the pellet fraction, the contents of NF-H in groups of 4 and 8 weeks' exposure to HD decreased by 35.6% and 43.2%, respectively (P < 0.01), and those of NF-L decreased by 26.4% and 42.1%, respectively (P < 0.01) when compared to the control. Further-more, NF-M contents in groups of 2, 4 and 8 weeks' exposure decreased by 23.3%, 33.9%, and 63.7% respectively (P < 0.01). The NFs level in spinal cords was highly correlated with gait abnormality of treated rats as the intoxication went on. Multiple correlation coefficients of NF-H, NF-M, and NF-L content with gait score of HD-treated rat were 0.8912, 0.9282 and 0.8981 (P < 0.01) respectively.

CONCLUSIONThe declines of NFs are high-ly related to neurobehavioral abnormality of 2, 5-HD-treated animals, and involved in the development of n-hexane neuropathy.

Animals ; Disease Models, Animal ; Gait ; drug effects ; Hexanones ; toxicity ; Male ; Neurofilament Proteins ; metabolism ; Rats ; Rats, Wistar ; Spinal Cord ; drug effects ; metabolism

OBJECTIVETo observe the cellular expression of (R127W) HSPB1 and its influence on neurofilament light chain (NFL) self-assembly and co-localization with NFL.

METHODSEukaryotic expression vectors pEGFPN1-(wt) HSPB1 and pEGFPN1- (R127W) HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-(wt) HSPB1 or pEGFPN1- (R127W) HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with Pcl-NFL and pEGFPN1-(wt)HSPB1, or pCL-NFL and pEGFPN1-(R127W)HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/ (R127W)HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique.

RESULTSThe aggregates formed by EGFP-(R127W)HSPB1 predominantly located around the nucleus, and EGFP-(wt)HSPB1 showed diffusion pattern in Hela cells. When co expressed with EGFP-(wt)HSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-(R127W)HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL co-localized with (R127W)HSPB1 in these aggregates.

CONCLUSIONThe R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.

Base Sequence ; Charcot-Marie-Tooth Disease ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; genetics ; HSP27 Heat-Shock Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Intracellular Space ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Neurofilament Proteins ; metabolism ; Protein Binding ; genetics ; Protein Transport ; Transfection