| ObjectiveN-Hexane belongs to one of the most important aliphatic compounds and is widely used in glue indus-try,paints,varnishes,printing inks,shoe manufacturing, and shoe repair.Exposure of humans and experimental animals to the hexacarbons n-hexane or methyl n-butyl ketone produces nerve damage characterized as a central-peripheral distal axonopathy.The earlier intoxicated patients performed a symmetrical,distal sensorimotor neuropathy with a progression of the loss of both sensory and motor function.Other symptoms such as headache,anorexia and dizziness may precede or coincide with the neuropathy.The corresponding pathological alterations observed in this axonopathy include the accumulation of neurolfilaments(NFs) proximal to the nodes of Ranvier,a Wallerian-type degeneration consisting of accumulation of mitochondria,vesicles, microtubules(MT) and dense bodies distal to the axonal swellings,retraction of myelin from Ranvier nodes and segmental demyelination,and distal axon atrophy.The mechanisms of the axonopathy induced by hexane are currently unclear, although multiple hypotheses have been proposed.Based on the pathological alterations,we built HD-intoxicated rats models and investigated the relative levels of cytoskeletal proteins in the tissues of cerebral cortex,spinal cords and sciatic nerves to determine the molecular mechanisms.Furthermore,the mechanisms of alterations of cytoskeletal proteins by degradation including phosphorylation status,related protein knisases and ubiquitin-proteasome system(UPS) were further investigated.In addition,the contents of cytoskelatal proteins in the rat serum were also determined to investigate the possibility that was used as the effect biomarker to evaluate the HD neurotoxicity and nervous injury.MethodsAdult male Wistar rats were treated with HD by intraperitoneal injection at dosages of 200 and 400 mg/kg HD respectively for 8 weeks.The onset and development of neurotoxicity were determined by neurological testing(gait abnormality). The tissues of cerebral cord,spinal cord and sciatic nerve,were homogenized in ice-cold homogenizing buffer containing 1%Triton X-100,50 mM Tris(pH 7.5),25 mM KCl,2 mM MgCl2,5 mM EGTA,5 mM dithiothreitol,Protease Inhibitor Cocktail(50ul/g tissue) and phosphatase inhibitors(5 mM Na3VO4,10mM Na4P2O7 and 1 mM iodoacetic acid) and then centrifuged at 100,000×g for 30 min at 4℃. The relative levels of pNF-H,NF-M,NF-L,p&n-pNF-H,α-tubulin,β-tubulin andβ-actin in the supernatant and pellet of nerve tissues were determined by immunoblotting.In addition,the cytosolic and membrane fractions of cerebral cord,spinal cord and sciatic nerve were also extracted by homogenating in ice-cold homogenizing buffer A(20 mM Tris-HCL buffer(PH 7.4),1 mM DTT,5 mM EGTA,2 mM EDTA, 10%glycerol,1 mM MgCl2,1 mM PMSF,2μg/ml aprotinin,and 2μg/ml leupeptin) and B((100 mM KCl,5 mM NaCl,3 mM MgCl2,50 mM Hepes,pH 7.4,1 mM dithiothreitol(DTT),0.5μg/ml aprotinin,0.5μg/ml leupeptin,200μM PMSF and 1 mM EGTA),and then centrifuging at 27,000×g for 60 min at 4℃.The levels of calmodulin(CAM),Ca2+/CaM-dependent protein kinaseⅡ(CaMKII),protein kinase A (PKA),protein kinase C(PKC),cyclin dependent kinase 5(CDK5) and CDK5-related factors were investigated in both the two fractions using WB.The activities of CaMKII,PKA,PKC and CDK5 were determined by using corresponding radioactivated 32P assay kits in corresponding cytosolic or membrane fractions of cerebral cortex,spinal cord and sciatic nerve of control and experimental group rats.The construction,transformation,identification and purification of Flag-NFM plasmid were performed using molecular biology technology.Plasmids were transfected into HEK293 cells in different combination.The ubiquitination by E3 ligase and degradation by proteasome of NF-M were determined by immunoprecipitation and Western-blotting.The tissues of cerebral cord,spinal cord and sciatic nerve,were homogenized in ice-cold homogenizing buffer containing 50 mM Tris HCl,pH 7.4,150 mM NaCl,1 mM EDTA,1%Triton X-100 and 1%Protease Inhibitor Cocktail and then centrifuged at 8,200×g for 30 min at 4℃.The relative levels of E1,CHIP and proteasome in nerve tissues were determined by immunoblotting.Results 1.The establishment of the animal model and corresponding behavioral changesAge-matched control rats had a starting mean body weight of 234.0±12 g,which increase steadily to 435.2±29 g at endpoint(8 weeks).This represents a 85.9% increase in body weight during the experiment period.Rats in the 200 mg/kg HD-treated group had a similar starting weight,but gained only 37%of their origi-nal weight,i.e.at the 8 weeks endpoint,intoxicated rats weighed 73.8%of control. However,rats in the 400 mg/kg HD-intoxicated group had an opposite pattern in body weight and decreased by 58.6%compared to that of control rats.Intoxication of rats at the lower and the higher dose-rate caused progressive development of gait abnormalities,rats in the 200 mg/kg exposure group developed an unsteady walking pattern with a slight ataxia,and hindlimb weakness(2.65±0.4 of mean gait score),and rats in the 400 mg/kg HD-treated group developed frank hindlimb weakness and an inability to rear(4.00±0.0of mean gait score).Finally,the weakness of the most severely intoxicated rats included the forelimbs.2.Changes of cytoskeletal proteins in HD-intoxicated rats' nerve tissues and serum2.1 Alterations of NFs subunits in the supernatant and pellet fractions of nerve tissuesThe exposure to HD resulted in a significant decrease in NF-L,NF-M and pNF-H in the supernatant fraction of nerve tissues.In comparison with the control rats,the levels of NF-L significantly decreased(P＜0.01) by 65.9%,78.3%and 38.6%in cerebral cortex,spinal cord and sciatic nerve of rats treated with 200 mg/kg HD and by 74.6%,83.6%and 58.4%in cerebral cortex,spinal cord and sciatic nerve of rats treated with 400 mg/kg HD,respectively.Those of NF-M decreased by 24.8%,80.9% and 24.9%and by 37.6%,89.3%and 60.4%,respectively(P＜0.01).pNF-H contents reduced by 64.9%,38.4%and 38.0%and 71.3%,63.1%and 44.1%,respectively(P＜0.01).And the p&n-pNF-H decreased by 37.8%,52.8%and 37.1%and 56.3%,69.5% and 53.4%,respectively.In addition to determining NFs subunits in the supernatant fraction,the NFs subunits in the insoluble fractions obtained while preparing the supernatant fractions were also determined.The immunoblotting results showed that exposure to HD of 200 and 400 mg/kg resulted in a significant decrease(P＜0.01) of NF-L levels in cerebral cortex,spinal cord and sciatic nerve by 28.6%,42.6%and 36.1%and 42.2%,52.5% and 58.4%respectively.In the 200 and 400 mg/kg treatment groups,the levels of NF-M in the pellet decreased by 40.3%,42.1%and 29.1%and 57.1%,49.4%and 52.1%,respectively(P＜0.01),while the levels of pNF-H decreased by 48.2%,35.0% and 43.1%and 55.1%,48.1%and 65.9%,respectively(P＜0.01).And the p&n-pNF-H decreased by 41.4%,34.9%and 31.2%and 57.7%,48.8%and 51.4%, respectively.2.2 Relationship of neurobehavioral abnormality and alterations of NFs in nerve tissuesA significant correlation was observed between gait abnormality and NFs changes (NF-L,NF-M and pNF-H) in both fractions.In the supernatant fraction of cerebral cortex,spinal cord and sciatic nerve,the correlation coefficients of NF-L with gait score were -0.916,-0.891 and -0.932,respectively(P＜0.01).The correlation coefficients of NF-M with gait score in cerebral cortex,spinal cord and sciatic nerve were -0.838,-0.865 and -0.895 in the supernatant fraction,respectively(P＜0.01), and pNF-H with gait score,-0.850,-0.894 and -0.815,respectively(P＜0.01).In the pellet fraction of cerebral cortex,spinal cord and sciatic nerve,significant correlations were also observed between NF-L,NF-M,pNF-H and gait score, respectively.The correlation coefficients of NF-L with gait score were -0.840,-0.885 and -0.921,respectively(P＜0.01).-0.915,-0.866 and -0.918 were the correlation coefficients of NF-M with gait score,respectively(P＜0.01).pNF-H also significant correlated with gait score in cerebral cortex(-0.874),spinal cord(-0.904) and sciatic nerve(-0.903),respectively(P＜0.01).2.3 Alterations ofα-tubulin,β-tubulin andβ-actin in the supernatant and pellet fractions of nerve tissuesThe contents ofα-tubulin,β-tubulin andβ-actin proteins in cerebral cortex,spinal cord and sciatic nerve were inconsistently affected.In the supernatant fractions of spinal cord and sciatic nerve,the levels ofα-tubulin significantly decreased(P＜0.01) by 24.4%and 44.8%in 400 mg/kg HD treated rats,respectively,while remained unchanged in cerebral cortex.Compared to that of the control rats,densitometric analysis showed the relative amount ofβ-tubulin was also markedly decreased(P＜0.01) in spinal cord and sciatic nerve of rats treated with 200(by 16.8%and 17.6%, respectively) and 400 mg/kg(by 21.8%and 40.7%,respectively) HD,but increased in cerebral cortex of both HD-treated rats(by 14.1%and 17.1%,respectively).The changes ofβ-actin were similar with that ofβ-tubulin.Exposure to 400 mg/kg HD resulted in a significant decrease ofβ-actin in sciatic nerve(by 29.2%,P＜0.01) and increase in cerebral cortex(by 17.7%,P＜0.01).The levels ofα-tubulin,β-tubulin andβ-actin were also assayed in the pellet fraction to reveal any contribution of microtubule(MT) and microfilament(MF) proteins on neuropathy induced by HD.Significant reduction(P＜0.01) ofα-tubulin contents in cerebral cortex(by 19.2%),spinal cord(by 7.3%) and sciatic nerve(by 35.8%) were observed in rats treated with 400 mg/kg HD.β-tubulin levels reduced by 28.2%and 43.9%in sciatic nerve of 200 and 400 mg/kg treated rats,respectively,but remained unchanged in cerebral cortex and spinal cord of both group rats.Cerebral cortex and sciatic nerve also showed a significant decrease(P＜0.01) ofβ-actin contents(by 14.7%and 38.2%,respectively) in 400 mg/kg HD treated rats.2.4 Relationship of neurobehavioral abnormality and alterations ofα-tubulin,β-tubulin andβ-actin protein in nerve tissuesSignificant(P＜0.01) negative correlations were observed betweenα-tubulin,β-tubulin andβ-actin and gait abnormality in the supernatant(r=-0.874,-0.851 and -0.876,respectively) and pellet fraction(r=-0.922,-0.870 and -0.857,respectively) of sciatic nerve.However,the alterations ofα-tubulin,β-tubulin andβ-actin in cerebral cortex and spinal cord had no significant correlation(P＞0.05) with gait abnormality.2.5 Alterations of cytoskeletal proteins in serumThe levels of pNF-H in serum significantly increased by 33.5%and 59.8%(P＜0.01) in 200 and 400 mg/kg HD rats,respectively.NF-L contents also increased in 400 mg/kg HD group rats,while the levels of NF-M,α-tubulin,β-tubulin andβ-actin remain unchanged.2.6 Relationship of neurobehavioral abnormality and alterations of NFs in serumTo investigate whether the alterations of pNF-H could reflect the neuropathy progression,we examined the relationships between gait score and pNF-H and NF-L levels in serum of HD-intoxicated rats.A significant positive(P＜0.01) correlation was observed between gait abnormality and pNF-H level in serum as the intoxication went on,while NF-L correlated poorly(P＞0.05) with gait score.The correlation coefficient of pNF-H contents with gait score of HD-treated rat was 0.943(P＜0.01).3.Aterations of NFs-related protein kinases contents and activities in nerve tissues3.1 Changes of CaM,CaMKII,PKA,PKC contents and activities in the cytosolic and membrane fractions of nerve tissuesHD intoxication resulted in a significant increase of CaM,CaMKII,PKA and PKC contents in the cytosolic fractions of cerebral cortex,spinal cord and sciatic nerve.In comparison with the control rats,the levels of CaM significantly increased by 22.7%in cytosolic fractions of sciatic nerve of rats treated with 200 mg/kg HD and by 26.8%,23.9%and 46.1%in cerebral cortex,spinal cord and sciatic nerve of rats treated with 400 mg/kg HD,respectively.CaM levels were also determined using commercial ELISA kits,and the results showed the similar and more magnitude pattern in the cytosolic fractions of CC(by 76.9%,P＜0.01),SC(by 62.9%,P＜0.01) and SN(by 112.6%,P＜0.01) of 400 mg/kg HD intoxicated rats.The levels of CaMKII were also significantly increased in the cytosolic fractions of cerebral cortex of 200(by 23.7%,P＜0.01) and 400(by 30.3%,P＜0.01) mg/kg HD-treated rats, while CaMKII could not be detected in spinal cord and sciatic nerve of rats.Those of PKA increased by 21.5%,46.1%and 21.0%in the cytosolic fractions of cerebral cortex,spinal cord and sciatic nerve of rats treated with 400 mg/kg HD,respectively (P＜0.01).PKC contents also increased by 23.0%and 55.5%in the cytosolic fractions of spinl cord and sciatic nerve of rats treated with 400 mg/kg HD, respectively.While a significant decrease(by 22.5%and 39.4%,respectively) was observed in the cytosolic fractions of cerebral cortex of 200 and 400 mg/kg HD-treated rats.In addition to determining CaM,CaMKII,PKA and PKC in the cytosolic fraction, CaM,CaMKII,PKA and PKC levels in the membrane fractions were also determined. The immunoblotting results showed that exposure to HD of 200 and 400 mg/kg resulted in a significant increase(P＜0.01) of CaM levels in cerebral cortex(by 28.6%and 18.4%,respectively) and sciatic nerve(by 10.0%(P＞0.05) and 45.1%, respectively).In ELISA,the changes of CaM concentrations showed the similar pattern and increased by 11.1%and 25.6%in cerebral cortex and sciatic nerve of 400 mg/kg HD intoxicated rats,rspectively.CaMKII contents were also increased by 26.1%in the membrane fractions of 400 mg/kg HD-treated rats' cerebral cortex.In the 200 and 400 mg/kg treatment groups,the levels of PKA in the membrane fractions of spinal cord increased by 19.3%and 25.5%,respectively(P＜0.01).Although PKC contents were decreased by 18.4%and 21.5%in cerebral cortex of 200 and 400 mg/kg HD groups,respectively,a significant increase in spinal cord(by 13.9%,P＜0.01) and sciatic nerve(by 44.7%,P＜0.01) of 400 mg/kg HD-treated rats were observed.CaMKII,PKA and PKC activities in the cytosolic or membrane fractions of cerebral cortex,spinal cord and sciatic nerve were also investigated using SignaTECT assay system(Promega).The activities of CaMKII were significantly increased in cerebral cortex cytosolic fractions of 200(by 63.4%,P＜0.01) and 400(by 83.7%,P＜0.01) mg/kg HD-intoxicated rats.But CaMKII activities could not be detected in spinal cord and sciatic nerve of both HD-treated rats.Although PKA activities were significantly decreased in the cytosolic fractions of spinal cord of 200(by 25.0%,P＜0.01) and 400(by 50.0%,P＜0.01) mg/kg HD group rats,significant increases were observed in cerebral cortex(by 36.5%,P＜0.01) and sciatic nerve(by 28.1%,P＜0.01) of 200 or 400 mg/kg HD-treated rats.The activities of PKC significantly decreased in the membrane fraction of cerebral cortex(by 24.9%and 31.0%, respectively,P＜0.01) and spinal cord(by 27.9%and 32.6%,respectively,P＜0.01) of both HD-treated rats.Significant increases of PKC activities in sciatic nerve were observed in 200(by 8.1%,P＜0.05) and 400(by 20.3%,P＜0.01) mg/kg HD-treated rats.3.2 Correlations of CaMKII,PKA,PKC and neurobehavioral abnormalitySignificant positive correlations were observed between CaMKII activities in cerebral cortex(R=0.888,P＜0.01) or PKC in sciatic nerve(R=0.765,P＜0.01) and gait abnormality.However,PKA activities in spinal cord(R=-0.772,P＜0.01) and sciatic nerve(R=-0.712,P＜0.01) were significant negative correlated with gait abnormality.PKC activities in cerebral cortex(R=-0.742,P＜0.01) and spinal cord (R=-0.664,P＜0.01) were also negatively correlated with gait abnormality.3.3 Changes of CDK5 and related factors contents and activities in the cytosolic and membrane fractions of nerve tissuesThe levels of CDK5 in cerebral cortex were significantly decreased(P＜0.01) by 43.4%and 17.3%,respectively,in the cytosolic and membrane fractions of 400 mg/kg HD-treated rats.CDK5 activities showed the same pattern and decreased(P＜0.01) by 9.1%and 15.2%,respectively,in the cytosolic fraction of both HD-treated rats, while remain unchanged in the membrane fractions.However,the CDK5 levels in spinal cord were significantly(P＜0.01) increased in the cytosolic and membrane fractions of 200(78.2%and 26.3%,respectively) and 400(by 21.9%and 12.7%, respectively) mg/kg HD-treated rats.The CDK5 activities were significantly decreased in the cytosolic fractions of spinal cord.Significant increases of CDK5 levels were also observed in the cytosolic fractions of sciatic nerve in 200(by 26.1%, P＜0.01) and 400(by 41.5%,P＜0.01) mg/kg HD-intoxicated rats.CDK5 activities showed the same pattern and increased(P＜0.01) in both the cytosolic and membrane fractons of sciatic nerve of 200(by 14.7%and 9.0%,respectively) and 400(by 37.6%and 28.8%,respectively) mg/kg HD-treated rats.In comparison with the control rats,the levels of p35 precursor in cerebral cortex were significantly decreased(P＜0.01) by 17.0%and 19.6%in both the cytosolic and membrane fractions of 200 mg/kg HD-treated rats and by 37.6%and 23.0%of 400mg/kg HD-intoxicated rats,respectively.Significant decreases(by 11.9%and 39.9%,P＜0.01,respectively) of p35 levels were observed in the cytosolic fraction of rats treated with 200 and 400 mg/kg HD.Those of p25 were also decreased in 200(by 26.5%and 12.6%,P＜0.01,respectively) and 400(by 51.2%and 36.4%,P＜0.01, respectively) mg/kg HD-treated rats.However,p35 precursor,p35 and p25 levels were inconsistently affected in the cytosolic and membrane fractions of spinal cord. The levels of p35 and p25 decreased in the cytosolic fractions,while increased in the membrane fractions.Furthermore,significant increases of p35 precursor levels were observed in both the cytosolic and membrane fractions of sciatic nerve of rats treated with 200(by 38.1%and 59.2%,P＜0.01,respectively) and 400(by 16.9%and 26.3%, P＜0.01,respectively) mg/kg HD.P35 and p25 were also elevated(P＜0.01) by 28.3%and 14.4%in the cytosolic and membrane fractions of 400 mg/kg HD-intoxicated rats,respectively.3.4 Correlations of CDK5 and neurobehavioral abnormalityThe activities of CDK5 were significantly correlated(P＜0.01) with gait abnormality in the cytosolic fractions of cerebral cortex(R=-0.746),SC(R=-0.840) and SN(R=0.864).Significant positive correlation(R=0.851,P＜0.01) was observed between gait abnormality and CDK5 activities in the membrane fractions of SN. However,the alterations of CDK5 activities in the membrane fractions of cerebral cortex and spinal cord had no significant correlation(P＞0.05) with gait abnormality.4.Alterations NFs subunits polymerization ratios in CC,SC and SNThe polymerization ratios of NF-L significantly increased(P＜0.01) by 27.1%, 38.0%,11.2%and 63.7%,51.6%,15.6%in cerebral cortex,spinal cord and sciatic nerve of rats treated with 200 and 400 mg/kg HD,respectively.Although those of NF-M decreased by 11.7%and 18.9%in cerebral cortex of 200 and 400 mg/kg HD-treatd rats,the ratios of NFM were significantly increased by 51.5%and 65.0%in spinal cord and by -3.2%(P＞0.05) and 9.5%in sciatic nerve,respectively.pNF-H polymerization ratios were elevated in cerebral cord(by 16.4%and 23.5%, respectively) and sciatic nerve(by 2.3%,P＞0.05 and 7.9%,respectively) of 200 and 400 mg/kg HD-treated rats.And the polymerization ratios of p&n-pNF-H increased by 6.3%(P＞0.05),14.3%and 6.7%(P＞0.05) and 16.6%,24.0%and 13.7%in cerebral cortex,spinal cord and sciatic nerve of rats treated with 200 and 400 mg/kg HD,respectively.5.Role of UPS in NFs reduction of HD-induced neuropathy5.1 MG132 partly inhibits HD-induced NFs degradationAfter 1,4,8 and 12 h of CHX treatment in Flag-NFM transfected HEK293 cells, NF-M levels were decreased by 4.2%,15.7%,23.2%and 47.9%.So the half life of NF-M was about 12 h.Further reductions of NF-M contents were observed after 4 mM and 8 mM HD treatment,respectively.Furthermore,NF-M contents were partly rescued when MG132(20uM) were added to the cultures after 30 mins of HD treatment.5.2 CHIP,one of U-box E3 ligase,enhances NF-M ubiquitination and degradation by proteasomeFlag-NFM,Myc-CHIP and HA-Ub were co-transfected into HEK293 cells and then immunoprecipitation was performed with EZviewTM Red ANTI-FLAG? M2 Affinity Gel.Immunoprecipitated NF-M showed prominent anti-HA immunoreactivity,with Ub positive species appearing as mulitple higher molecular weight species,possibly representing oligomeric and multimeric ligations.The amount of ubiquitinated NF-M was more markly after Myc-CHIP transfection.More amount of multimeric ubiquitinated NF-M were observed after HD intoxication.At the same time,CHIP over-expression caused a dose-dependent decrease of NF-M contents in Flag-NFM and Myc-CHIP co-transfected HEK293 cells and additional HD greatly elevated such effect of CHIP.Furthermore,NF-M contents were partly rescued when MG132(20uM) were added to the cultures.The results suggested that HD could enhance ubiquitination of NF-M by CHIP and degradation by proteasome.5.3 Alterations of E1,CHIP and proteasome contents in nerve tissuesCompared to that of the control group rats,the contents of E1 were significantly decreased by 15.8%and 24.3%in cerebral cortex and spinal cord of 200 mg/kg HD treated rats,respectively,while increased by 15.8%(P＞0.05) and 24.3%in 400mg/kg HD group rats,respectively.Sciatic nerve showed a significant increase of E1 contents.E1 levels were increased by 14.9%and 59.8%in sciatic nerve of 200 and 400 mg/kg HD treated rats,respectively. Significant increase of CHIP levels in sciatic nerve were observed.CHIP levels were increased by 18.7%and 55.3%in sciatic nerve of 200 and 400 mg/kg HD treated rats,respectively.CHIP protein could not be detected at the present condition.Proteasome contents were significantly increased in cerebral cortex,spinal cord and sciatic nerve of rats after HD intoxication.Compared to that of the control group rats,proteasome contents were significantly elevated by 37.7%,25.8%and 18.4%in cerebral cortex,spinal cord and sciatic nerve of 200 mg/kg HD treated rats, respectively,and by 56.4%,86.3%and 37.9%in 400mg/kg HD group rats, respectively.Conclusions1.NFs is more sensitive than MT and MF proteins,which might be served as the target of HD-induced neuropathy.2.The contents of pNF-H were significantly increased in serum of HD-treated rats and highly correlated with neurobehavioral abnormality,which suggested pNF-H might be served as one of the biomarkers of HD-induced neuropathy for earlier diagnosis.3.The contents and activities of CaM,PKA,PKC,CDK5 and NFs phosphorylation ratios were changed in rats' nerve tissues after HD treatment,which suggested alterations of NFs-related protein kinases and phosphorylation status might be involved in HD-induced neuropathy.4.The pathway of Ca2+-CaM-CaMKII were partly involved in HD-induced neuropathy because CaMKII contents and activities could not be detected in spinal cord and sciatic nerve.5.The pattern of PKA,PKC and CDK5 activities alterations were different,i.e. decrease in CNS and increase in PNS,which might determine how NFs would be degraded in HD-induced neuropathy.6.CHIP,one of the U-box E3 ligase,enhances NFs ubiquitination and degradation by proteasome.7.MG132 partly inhibits NFs degradation induced by HD,which suggests UPS,at least partly,is involved in HD-induced NFs reduction.
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