正己烷中毒性神经病细胞骨架变性及其机制研究
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摘要
研究目的
正己烷(n-hexane)是一种常用的工业溶剂,主要应用于粘胶配制、除污、干洗、植物油提取、制鞋、印刷、油漆、制药、家具制造及电子元件制造等行业。由于正己烷的急性毒性较低,而使人们忽视了长期慢性低浓度接触正己烷可导致以感觉运动型多发性周围神经病为主要临床特征的慢性中毒。
正己烷中毒患者早期表现为手足发麻、刺痛、蚁走感、手足部痛觉、触觉减退呈手套、袜套样分布等,进一步发展可表现为四肢无力、行走困难以至不能站立甚至出现下肢瘫痪及肌肉萎缩。神经病理检查发现损伤最初发生在远端最长、最粗的感觉和运动神经轴索,并且在郎飞氏节附近形成巨大的充满神经丝(neruofilament,NFs)的肿胀,郎飞氏节解剖结构扭曲且髓鞘从肿胀处回缩,有时伴有节段性脱髓鞘,最后一些远离神经肿胀部位的轴索发生变性。尽管许多工作致力于正己烷中毒性神经病的研究,但其发病机制尚不清楚。基于正己烷中毒的超微结构观察和组织病理学改变,为进一步探讨正己烷中毒性神经病发生的分子机制,我们建立了正己烷终毒物2,5-己二酮(2,5-hexanedione,HD)亚慢性中毒大鼠模型,以细胞骨架蛋白为切入点,观察中毒性神经病发生过程中NFs、微管(α-tubulin、β-tubulin)和微丝(β-actin)蛋白相对含量的变化,确定作用靶点,并在此基础上从细胞骨架蛋白降解的角度进一步探讨细胞骨架蛋白变化的原因,包括磷酸化及其相应蛋白激酶信号通路的变化、泛素蛋白酶体系统(ubiquitin-proteasome system,UPS)的改变等等,为正己烷职业中毒的预防和治疗提供可以借鉴的基础资料。同时,探讨血清中各骨架蛋白含量的变化评价正己烷接触的效应标志物以检测和评价神经系统损伤的可行性,为正己烷等毒物神经毒性的临床监测提供实验依据。
研究方法
1.雄性Wistar大鼠用200和400 mg/kg.bw的HD腹腔注射染毒,染毒时间8周(每周染毒5d,每日1次),建立正己烷中毒性周围神经病模型。
2.取大鼠大脑、脊髓和坐骨神经组织在匀浆缓冲液(1%Triton X-100,50 mM Tris(pH 7.5),25 mM KCl,2 mM MgCl_2,5 mM EGTA,5 mM dithiothreitol,ProteaseInhibitor Cocktail(50ul╱g tissue)and phosphatase inhibitors(5 mM Na_3VO_4,10mM Na_4P_2O_7 and 1 mM iodoacetic acid)中制备匀浆,100,000×g、4℃离心60 min。采用Western-Blotting技术研究大鼠大脑、脊髓、坐骨神经上清和沉淀以及血清中phos-NF-H(pNF-H)、phos-&n-phos-NF-H(p&n-pNF-H)、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等骨架蛋白。
3.神经组织通过匀浆缓冲液A(20 mM Tris-HCL buffer(PH 7.4),1 mM DTT,5mM EGTA,2 mM EDTA,10%glycerol,1 mM MgCl_2,1 mM PMSF,2μg/mlaprotinin,and 2μg/ml leupeptin)和B(100 mM KCl,5 mM NaCl,3 mM MgCl_2,50 mM Hepes,pH 7.4,1 mM dithiothreitol(DTT),0.5μg/ml aprotinin,0.5μg/mlleupeptin,200μM PMSF and 1 mM EGTA)制备组织匀浆,然后27,000×g、4℃离心60 min提取神经组织胞膜和胞浆蛋白组分,分别测定钙调蛋白(calmodulin,CaM)、钙调蛋白激酶Ⅱ(Ca~(2+)/CaM-dependent protein kinaseⅡ,CaMKII)、蛋白激酶A(protein kinase A,PKA)、蛋白激酶C蛋白激酶C(proteinkinase C,PKC)、周期蛋白依赖性的蛋白激酶5(cyclin dependent kinase 5,CDK5)和CDK5相关因子含量的变化。
4.取大鼠大脑、脊髓和坐骨神经组织胞膜和/或胞浆蛋白组分,利用放射性同位素技术研究HD染毒对大鼠神经组织中CaMKII、PKA、PKC和CDK5活性的影响。
5.真核表达Flag-NFM质粒的构建、转化、鉴定、扩增和提纯。
6.培养HEK293细胞,将不同质粒按不同目的需要组合,利用细胞转染技术转染HEK293细胞,采用免疫共沉淀和Western-blotting等技术检测HD染毒情况下NFs与E3连接酶的结合、泛素化以及被蛋白酶体降解情况。
7.神经组织在相应匀浆液(50 mM Tris HCl,pH 7.4,150 mM NaCl,1 mM EDTA,1%Triton X-100 and 1%Protease Inhibitor Cocktail)中8200×g、4℃离心30min,取上清,测定UPS系统中各组分E1、E3泛素连接酶和蛋白酶体的含量和活性。
研究结果
1.动物模型的建立以及神经行为学指标的变化
给予HD染毒后,动物逐渐出现肌肉松弛、肌张力降低、步态异常以至瘫痪等症状,神经行为学测试表明染毒大鼠呈现典型的中毒性周围神经病特征,这提示正己烷中毒性周围神经病模型建立成功。
对照组大鼠体重在实验过程中稳定上升,体重增加了85.9%;HD染毒后动物体重增长较对照组明显缓慢。200 mg/kg HD剂量组大鼠体重增加与对照组相比明显减慢,实验结束时约为对照组的73.8%,差异均有统计学意义(P＜0.01)。400 mg/kg HD剂量组大鼠体重不再增长,反而出现下降现象,8周染毒结束时,平均体重约为对照组的41.4%。
200和400 mg/kg HD染毒后,大鼠均出现明显的步态异常,步态评分增高。染毒8周,200 mg/kg HD剂量组大鼠出现中度的共济失调和后肢无力,评分平均为2.65±0.4。400 mg/kg HD剂量组大鼠症状较重,试验结束时大鼠出现明显的后肢无力、拖拉、完全无法站立,并最终累及到前肢,平均步态评分为4.00±0.00。
2.HD染毒大鼠神经组织和血清中细胞骨架蛋白含量的变化
2.1 NFs亚单位在神经组织上清和沉淀中的变化
与对照组相比,HD中毒大鼠大脑、脊髓和坐骨神经匀浆上清中,NF-L含量在200 mg/kg HD染毒组分别下降了65.9%、78.3%和38.6%,在400 mg/kg HD染毒组分别下降了74.6%、83.6%和58.4%;NF-M含量在200 mg/kg HD染毒组分别下降了24.8%、80.9%和24.9%,在400 mg/kg HD染毒组分别下降了37.6%,89.3%和60.4%;pNF-H含量在200 mg/kg HD染毒组分别下降了64.9%,38.4%and 38.0%,在400 mg/kg HD染毒组分别下降了71.3%,63.1%and 44.1%;p&n-pNF-H在200mg/kg HD染毒组分别下降了37.8%,52.8%and 37.1%,在400 mg/kg HD染毒组分别下降了56.3%,69.5%and 53.4%。
200和400 mg/kg HD染毒导致NF-L含量在大鼠大脑、脊髓和坐骨神经匀浆沉淀中分别下降了28.6%、42.6%、36.1%和42.2%、52.5%、58.4%;NF-M含量分别下降了40.3%、42.1%、29.1%和57.1%、49.4%、52.1%;pNF-H含量分别下降了48.2%、35.0%、43.1%和55.1%、48.1%、65.9%;p&n-pNF-H含量分别下降了41.4%、34.9%、31.2%和57.7%、48.8%、51.4%。
2.2神经组织NFs蛋白的变化与大鼠神经行为异常之间的相关性
神经组织中NF-L、NF-M和pNF-H的改变与大鼠步态评分之间有明显的负相关性。在大脑、脊髓和坐骨神经组织匀浆上清中NF-L与大鼠步态评分的相关系数分别为-0.916、-0.891和-0.935;NF-M与大鼠步态评分的相关系数分别为-0.838、-0.865和-0.895;pNF-H与大鼠步态评分的相关系数分别为-0.850、-0.894和-0.815。在大脑匀浆沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.840、-0.915和-0.874;脊髓沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.885、-0.870和-0.904;坐骨神经组织沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.921、-0.918和-0.903。
2.3α-tubulin、β-tubulin和β-actin含量在神经组织上清和沉淀中的变化
HD中毒后α-tubulin、β-tubulin和β-actin蛋白含量在大鼠大脑、脊髓和坐骨神经组织上清和沉淀中发生了不一致的变化。
在脊髓和坐骨神经组织上清中,α-tubulin含量在400 mg/kg HD染毒组分别下降了24.4%和44.8%,而在大脑组织上清中无明显变化。与对照组相比,β-tubulin含量在200mg/kg HD染毒组大鼠脊髓和坐骨神经上清中分别下降了16.8%和17.6%,在400 mg/kg HD染毒组分别下降了21.8%和40.7%,但是在大脑组织上清中200和400 mg/kg HD染毒组分别升高了14.1%和17.1%。β-actin的变化与β-tubulin非常相似。400 mg/kg HD染毒导致大鼠坐骨神经组织上清中β-actin含量下降29.2%,在大脑组织上清中升高17.7%。
α-tubulin在400 mg/kg HD染毒大鼠大脑、脊髓和坐骨神经组织匀浆沉淀中分别下降了19.2%、7.3%和35.8%。β-tubulin含量在200和400 mg/kg HD染毒大鼠坐骨神经沉淀中也分别下降了28.2%和43.9%,但在大脑和脊髓沉淀中保持不变。β-actin含量在400 mg/kg HD染毒大鼠大脑和坐骨神经沉淀中也分别下降了14.7%和38.2%。
2.4神经组织α-tubulin、β-tubulin和β-actin蛋白的变化与大鼠神经行为异常之间的相关性
坐骨神经组织中α-tubulin、β-tubulin和β-actin的改变与大鼠步态评分之间有明显的负相关性,而在大脑和脊髓组织中与步态评分无相关性。坐骨神经上清中α-tubulin、β-tubulin和β-actin与大鼠步态评分之间的相关系数分别为-0.874、-0.851和-0.876;沉淀中与大鼠步态评分之间的相关系数分别为-0.922、-0.870和-0.857。
2.5血清中细胞骨架蛋白的变化
pNF-H含量在200和400 mg/kg HD染毒大鼠血清中分别升高了33.5%和59.8%。NF-L含量在400 mg/kg HD染毒大鼠血清中也明显升高了,但是NF-M、α-tubulin、β-tubulin和β-actin含量未出现明显改变。
2.6血清中NFs蛋白的变化与大鼠步态异常之间的相关性
为确定血清中pNF-H的变化是否可反映HD中毒性神经病的进展,我们检测了中毒大鼠血清中NFs蛋白的变化与大鼠步态评分之间的相关性。血清中pNF-H变化与大鼠步态评分之间有明显的正相关性,相关系数为0.943。
3.NFs相关蛋白激酶在神经组织胞浆和胞膜组分中的变化
3.1 CaM,CaMKII,PKA和PKC的含量和活性在神经组织胞浆和胞膜组分中的变化
HD染毒导致CaM、CaMKII、PKA和PKC含量在大脑、脊髓和坐骨神经胞浆蛋白组分中明显升高。与对照组相比,CaM含量在200 mg/kg HD染毒组大鼠坐骨神经胞浆蛋白组分中升高了22.7%,在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了26.8%、23.9%和46.1%。ELISA试剂盒测定结果与WB结果一致,即CaM含量在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了76.9%、62.9%和112.6%。大脑胞浆组分中CaMKII含量也明显升高,在200和400 mg/kg HD染毒组分别升高了23.7和30.3%,但是在脊髓和坐骨神经中未检测到。PKA含量在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了21.5%、46.1%和21.0%;PKC含量在脊髓和坐骨神经胞浆蛋白组分中也分别升高23.0%和55.5%,但在200和400 mg/kg HD染毒组大鼠大脑胞浆蛋白组分中分别下降22.5%和39.4%。
CaM含量在200 mg/kg HD染毒组大鼠大脑胞膜组分中升高了28.6%,在400mg/kg HD染毒组大鼠大脑和坐骨神经胞膜组分中也分别升高了18.4%和45.1%。ELISA测定CaM浓度也表现为升高,在400 mg/kg HD染毒组大鼠大脑和坐骨神经胞膜组分中分别升高了11.1%和25.6%。CaMKII在400 mg/kg HD染毒组大鼠大脑胞膜组分中也明显升高了26.1%。PKA含量在200和400 mg/kg HD染毒组大鼠脊髓胞膜组分中分别升高了19.3%和25.5%。虽然PKC含量在200和400 mg/kg HD染毒组大鼠大脑胞膜组分中分别下降18.4%和21.5%,但在400 mg/kg HD染毒组大鼠脊髓和坐骨神经胞膜组分中分别升高了13.9%和44.7%,
CaMKII活性在200和400 mg/kg HD染毒组大鼠胞浆蛋白组分中分别升高了63.4%和83.7%,但在脊髓和坐骨神经中未检测到。脊髓胞浆蛋白组分中PKA活性在200和400 mg/kg HD染毒组分别下降了25.0%和25.0%,但在200 mg/kg HD染毒组大脑和400 mg/kg HD染毒组坐骨神经中分别升高36.5%和28.1%。PKC活性在200和400 mg/kg HD染毒组大脑和脊髓胞膜蛋白组分中分别下降24.9%、31.0%和27.9%、32.6%,但在坐骨神经胞膜蛋白组分中分别分别升高了8.1%和20.3%。
3.2 CaMKII,PKA,PKC活性与大鼠神经行为异常之间的相关性
大脑中CaMKII和坐骨神经PKC活性的变化与大鼠步态评分存在明显的正相关性,相关系数分别为0.888和0.765。然而脊髓和坐骨神经中PKA活性与大鼠步态评分存在明显的负相关性,相关系数分别为-0.772和-0.712。大脑和脊髓中PKC活性与大鼠步态评分也存在明显的负相关性,相关系数分别为-0.742和-0.664。
3.3 CDK5和相关激活因子在神经组织胞浆和胞膜蛋白组分中的变化
CDK5含量在400 mg/kg HD染毒组大鼠大脑胞浆和胞膜蛋白组分中分别下降了43.4%和17.3%。CDK5活性的变化与含量变化一致,即在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中分别下降了9.1%和15.2%。CDK5含量在200和400mg/kg HD染毒组大鼠脊髓胞浆(78.2%和21.9%)和胞膜蛋白(26.3%和12.7%)组分中明显升高,但活性却明显下降。200和400 mg/kg HD染毒组大鼠坐骨神经中CDK5含量也明显升高,在胞浆组分中分别升高了26.1%4和41.5%;其活性在胞浆组分中分别升高14.7%和37.6%,在胞膜蛋白组分中分别升高了9.0%和28.8%。
与对照组相比,p35前体含量在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中分别下降17.0%和37.6%,在胞膜蛋白组分中分别下降19.6%和23.0%;p25含量在大脑胞浆组分中分别下降26.5%和51.2%,在胞膜蛋白组分中分别下降12.6%和36.4%;p35含量在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中也分别下降了11.9%和39.9%。然而p35前体、p35和p25在脊髓胞浆和胞膜蛋白组分中改变不一致,即在胞浆蛋白组分中升高,在胞膜蛋白组分中下降。另外,p35前体含量在200和400 mg/kg HD染毒组大鼠坐骨神经胞浆组分中分别升高38.1%和16.9%,在胞膜蛋白组分中分别下降59.2%和26.3%;p35和p25在400 mg/kg HD染毒组大鼠坐骨神经胞浆组分中分别升高28.3%和14.4%。
3.4 CDK5活性与大鼠神经行为异常之间的相关性
在大脑、脊髓和坐骨神经胞浆蛋白组分中CDK5活性与大鼠步态评分之间存在明显的相关性,相关系数分别为-0.746、-0.860和0.864。坐骨神经胞膜蛋白组分中CDK5活性与大鼠步态评分之间存在明显的正相关性,相关系数为0.851,大脑和脊髓中CDK5活性与大鼠步态评分之间无明显相关性。
4.神经组织中NFs亚单位聚合率的变化
NF-L的聚合率在200 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经中分别增加27.1%、38.0%和11.2%;在400 mg/kg HD染毒组分别增加了63.7%、51.6%和15.6%。NF-M聚合率在200和400 mg/kg HD染毒组大鼠大脑明显下降了11.7%和18.9%;而在脊髓中分别升高了51.5%和65.0%;坐骨神经中分别升高了-3.2%(P＞0.05)和9.5%。pNF-H聚合率在200和400 mg/kg HD染毒组大鼠大脑分别升高了16.4%和23.5%;脊髓中分别升高了2.3%(P＞0.05)和7.9%。p&n-pNF-H聚合率在400 mg╱kg HD染毒组大鼠大脑、脊髓和坐骨神经中也明显升高,分别升高了16.6%、24.0%和13.7%。
5.UPS在HD中毒性神经病NFs含量下降中的作用
5.1 MG132部分地抑制了HD促进NFs降解
在表达NF-M的HEK293细胞中(转染0.5 ug Flag-NFM质粒)中,NF-M含量在加入CHX 0、1、4和8小时后分别下降了4.2%、15.7%、23.2%和47.9%,可见NF-M蛋白半衰期大约是12 h。加入4 mM和8 mM HD后NF-M含量降低更加明显,说明HD中毒可加速NFM的降解。HD染毒30 min后,加入蛋白酶体抑制剂MG132,NF-M含量与未加MG132以前比较降解明显减缓。
5.2 HD作用下C末端Hsc70反应蛋白(carboxyl terminus of the Hsc70-interactingprotein,CHIP)作为含U-box结构域的E3连接酶增强NF-M泛素化,并促进其通过蛋白酶体降解
Flag-NFM、Myc-CHIP和/或HA-Ub质粒分别/同时转染HEK293细胞,经EZview Red ANTI-FLAG M2 Affinity Gel免疫沉淀后,在同时转染Flag-NFM、HA-Ub和Flag-NFM、HA-Ub、Myc-CHIP抗HA反应阳性,且同时转染Myc-CHIP质粒的细胞裂解液中NF-M蛋白的泛素化程度较转染Flag-NFM和HA-Ub的细胞裂解液强,加入HD后可见泛素化程度更强的NF-M蛋白。在同时转染Flag-NFM和增加量的Myc-CHIP质粒的细胞裂解液中,随着CHIP蛋白表达量的升高,NF-M蛋白的含量逐渐下降,HD染毒后降低更加明显,蛋白酶体抑制剂MG132可部分地阻断NF-M蛋白含量的下降,表明HD可增强CHIP促NF-M泛素化并通过蛋白酶体降解。
5.3神经组织中泛素活化酶E1、CHIP和蛋白酶体含量的变化
与对照组相比,200mg/kg HD组大鼠E1含量在大脑和脊髓中分别下降了15.8%和24.3%(P＜0.01),但在400mg/kg组分别升高了7.2%(P＞0.05)和29.1%(P＜0.01);E1含量在200和400mg/kg染毒组大鼠坐骨神经组织匀浆中均明显升高,分别升高了14.9%和59.8%,与对照组相比差异有统计学意义。
HD染毒后坐骨神经组织匀浆中CHIP含量明显升高。与对照组相比,200和400mg/kg组大鼠坐骨神经中CHIP含量分别升高了18.3%和55.3%,与对照组相比差异有统计学意义;但在大脑和脊髓中未检测到CHIP蛋白的存在。
HD染毒后,大脑、脊髓和坐骨神经组织匀浆中蛋白酶体含量均出现明显升高的趋势。与对照组相比,200mg/kg组大鼠蛋白酶体含量在大脑、脊髓和坐骨神经中分别升高了37.7%、25.8%和18.4%,与对照组相比差异有统计学意义;在400mg/kg组中分别升高了56.4%、86.3%和37.9%,与对照组相比差异有统计学意义。
结论
1.NFs比微管和微丝更加敏感,易受HD损伤,可能是正己烷中毒性神经病的分子靶点。
2.pNF-H含量在HD中毒大鼠血清中明显升高,并与大鼠神经行为异常呈明显的相关性,表明pNF-H可作为HD中毒性神经病早期诊断的生物标志物。
3.HD中毒导致大鼠神经组织CaM、PKA、PKC和CDK5的含量和活性以及NFs聚合率的变化,表明NFs相关蛋白信号通路和NFs磷酸化状态改变可能参与了HD中毒性神经病的发生。
4.由于CaMKII在脊髓和坐骨神经组织中为检测到,说明Ca~(2+)-CaM-CaMKII-NFs磷酸化信号通路可能部分地参与了HD中毒性神经病发生。
5.PKA、PKC和CDK5活性在中枢神经系统和外周神经系统能够变化不一致,可能决定了NFs在中枢神经系统和外周神经系统通过不同的途径降解。
6.CHIP作为一种含U-box结构域的E3连接酶促进NFs泛素化并通过蛋白酶体降解。
7.蛋白酶体抑制剂MG132部分地抑制了HD促进NFs降解,说明UPS通路在HD中毒性神经病中NFs含量下降中发挥了部分作用。
Objective
N-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.
Methods
Adult 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 MgCl_2,5 mM EGTA,5 mM dithiothreitol,Protease Inhibitor Cocktail(50ul/g tissue) and phosphatase inhibitors(5 mM Na_3VO_4,10mM Na_4P_2O_7 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 MgCl_2,1 mM PMSF,2μg/ml aprotinin,and 2μg/ml leupeptin) and B((100 mM KCl,5 mM NaCl,3 mM MgCl_2,50 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),Ca~(2+)/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 ~(32)P 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 changes
Age-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 serum
2.1 Alterations of NFs subunits in the supernatant and pellet fractions of nerve tissues
The 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 tissues
A 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 tissues
The 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 tissues
Significant(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 serum
The 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 serum
To 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 tissues
3.1 Changes of CaM,CaMKII,PKA,PKC contents and activities in the cytosolic and membrane fractions of nerve tissues
HD 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 abnormality
Significant 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 tissues
The 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 abnormality
The 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 SN
The 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 neuropathy
5.1 MG132 partly inhibits HD-induced NFs degradation
After 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 proteasome
Flag-NFM,Myc-CHIP and HA-Ub were co-transfected into HEK293 cells and then immunoprecipitation was performed with EZview~(TM) 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 tissues
Compared 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.
Conclusions
1.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 Ca~(2+)-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.
正己烷(n-hexane)是一种常用的工业溶剂,主要应用于粘胶配制、除污、干洗、植物油提取、制鞋、印刷、油漆、制药、家具制造及电子元件制造等行业。由于正己烷的急性毒性较低,而使人们忽视了长期慢性低浓度接触正己烷可导致以感觉运动型多发性周围神经病为主要临床特征的慢性中毒。
正己烷中毒患者早期表现为手足发麻、刺痛、蚁走感、手足部痛觉、触觉减退呈手套、袜套样分布等,进一步发展可表现为四肢无力、行走困难以至不能站立甚至出现下肢瘫痪及肌肉萎缩。神经病理检查发现损伤最初发生在远端最长、最粗的感觉和运动神经轴索,并且在郎飞氏节附近形成巨大的充满神经丝(neruofilament,NFs)的肿胀,郎飞氏节解剖结构扭曲且髓鞘从肿胀处回缩,有时伴有节段性脱髓鞘,最后一些远离神经肿胀部位的轴索发生变性。尽管许多工作致力于正己烷中毒性神经病的研究,但其发病机制尚不清楚。基于正己烷中毒的超微结构观察和组织病理学改变,为进一步探讨正己烷中毒性神经病发生的分子机制,我们建立了正己烷终毒物2,5-己二酮(2,5-hexanedione,HD)亚慢性中毒大鼠模型,以细胞骨架蛋白为切入点,观察中毒性神经病发生过程中NFs、微管(α-tubulin、β-tubulin)和微丝(β-actin)蛋白相对含量的变化,确定作用靶点,并在此基础上从细胞骨架蛋白降解的角度进一步探讨细胞骨架蛋白变化的原因,包括磷酸化及其相应蛋白激酶信号通路的变化、泛素蛋白酶体系统(ubiquitin-proteasome system,UPS)的改变等等,为正己烷职业中毒的预防和治疗提供可以借鉴的基础资料。同时,探讨血清中各骨架蛋白含量的变化评价正己烷接触的效应标志物以检测和评价神经系统损伤的可行性,为正己烷等毒物神经毒性的临床监测提供实验依据。
研究方法
1.雄性Wistar大鼠用200和400 mg/kg.bw的HD腹腔注射染毒,染毒时间8周(每周染毒5d,每日1次),建立正己烷中毒性周围神经病模型。
2.取大鼠大脑、脊髓和坐骨神经组织在匀浆缓冲液(1%Triton X-100,50 mM Tris(pH 7.5),25 mM KCl,2 mM MgCl_2,5 mM EGTA,5 mM dithiothreitol,ProteaseInhibitor Cocktail(50ul╱g tissue)and phosphatase inhibitors(5 mM Na_3VO_4,10mM Na_4P_2O_7 and 1 mM iodoacetic acid)中制备匀浆,100,000×g、4℃离心60 min。采用Western-Blotting技术研究大鼠大脑、脊髓、坐骨神经上清和沉淀以及血清中phos-NF-H(pNF-H)、phos-&n-phos-NF-H(p&n-pNF-H)、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等骨架蛋白。
3.神经组织通过匀浆缓冲液A(20 mM Tris-HCL buffer(PH 7.4),1 mM DTT,5mM EGTA,2 mM EDTA,10%glycerol,1 mM MgCl_2,1 mM PMSF,2μg/mlaprotinin,and 2μg/ml leupeptin)和B(100 mM KCl,5 mM NaCl,3 mM MgCl_2,50 mM Hepes,pH 7.4,1 mM dithiothreitol(DTT),0.5μg/ml aprotinin,0.5μg/mlleupeptin,200μM PMSF and 1 mM EGTA)制备组织匀浆,然后27,000×g、4℃离心60 min提取神经组织胞膜和胞浆蛋白组分,分别测定钙调蛋白(calmodulin,CaM)、钙调蛋白激酶Ⅱ(Ca~(2+)/CaM-dependent protein kinaseⅡ,CaMKII)、蛋白激酶A(protein kinase A,PKA)、蛋白激酶C蛋白激酶C(proteinkinase C,PKC)、周期蛋白依赖性的蛋白激酶5(cyclin dependent kinase 5,CDK5)和CDK5相关因子含量的变化。
4.取大鼠大脑、脊髓和坐骨神经组织胞膜和/或胞浆蛋白组分,利用放射性同位素技术研究HD染毒对大鼠神经组织中CaMKII、PKA、PKC和CDK5活性的影响。
5.真核表达Flag-NFM质粒的构建、转化、鉴定、扩增和提纯。
6.培养HEK293细胞,将不同质粒按不同目的需要组合,利用细胞转染技术转染HEK293细胞,采用免疫共沉淀和Western-blotting等技术检测HD染毒情况下NFs与E3连接酶的结合、泛素化以及被蛋白酶体降解情况。
7.神经组织在相应匀浆液(50 mM Tris HCl,pH 7.4,150 mM NaCl,1 mM EDTA,1%Triton X-100 and 1%Protease Inhibitor Cocktail)中8200×g、4℃离心30min,取上清,测定UPS系统中各组分E1、E3泛素连接酶和蛋白酶体的含量和活性。
研究结果
1.动物模型的建立以及神经行为学指标的变化
给予HD染毒后,动物逐渐出现肌肉松弛、肌张力降低、步态异常以至瘫痪等症状,神经行为学测试表明染毒大鼠呈现典型的中毒性周围神经病特征,这提示正己烷中毒性周围神经病模型建立成功。
对照组大鼠体重在实验过程中稳定上升,体重增加了85.9%;HD染毒后动物体重增长较对照组明显缓慢。200 mg/kg HD剂量组大鼠体重增加与对照组相比明显减慢,实验结束时约为对照组的73.8%,差异均有统计学意义(P＜0.01)。400 mg/kg HD剂量组大鼠体重不再增长,反而出现下降现象,8周染毒结束时,平均体重约为对照组的41.4%。
200和400 mg/kg HD染毒后,大鼠均出现明显的步态异常,步态评分增高。染毒8周,200 mg/kg HD剂量组大鼠出现中度的共济失调和后肢无力,评分平均为2.65±0.4。400 mg/kg HD剂量组大鼠症状较重,试验结束时大鼠出现明显的后肢无力、拖拉、完全无法站立,并最终累及到前肢,平均步态评分为4.00±0.00。
2.HD染毒大鼠神经组织和血清中细胞骨架蛋白含量的变化
2.1 NFs亚单位在神经组织上清和沉淀中的变化
与对照组相比,HD中毒大鼠大脑、脊髓和坐骨神经匀浆上清中,NF-L含量在200 mg/kg HD染毒组分别下降了65.9%、78.3%和38.6%,在400 mg/kg HD染毒组分别下降了74.6%、83.6%和58.4%;NF-M含量在200 mg/kg HD染毒组分别下降了24.8%、80.9%和24.9%,在400 mg/kg HD染毒组分别下降了37.6%,89.3%和60.4%;pNF-H含量在200 mg/kg HD染毒组分别下降了64.9%,38.4%and 38.0%,在400 mg/kg HD染毒组分别下降了71.3%,63.1%and 44.1%;p&n-pNF-H在200mg/kg HD染毒组分别下降了37.8%,52.8%and 37.1%,在400 mg/kg HD染毒组分别下降了56.3%,69.5%and 53.4%。
200和400 mg/kg HD染毒导致NF-L含量在大鼠大脑、脊髓和坐骨神经匀浆沉淀中分别下降了28.6%、42.6%、36.1%和42.2%、52.5%、58.4%;NF-M含量分别下降了40.3%、42.1%、29.1%和57.1%、49.4%、52.1%;pNF-H含量分别下降了48.2%、35.0%、43.1%和55.1%、48.1%、65.9%;p&n-pNF-H含量分别下降了41.4%、34.9%、31.2%和57.7%、48.8%、51.4%。
2.2神经组织NFs蛋白的变化与大鼠神经行为异常之间的相关性
神经组织中NF-L、NF-M和pNF-H的改变与大鼠步态评分之间有明显的负相关性。在大脑、脊髓和坐骨神经组织匀浆上清中NF-L与大鼠步态评分的相关系数分别为-0.916、-0.891和-0.935;NF-M与大鼠步态评分的相关系数分别为-0.838、-0.865和-0.895;pNF-H与大鼠步态评分的相关系数分别为-0.850、-0.894和-0.815。在大脑匀浆沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.840、-0.915和-0.874;脊髓沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.885、-0.870和-0.904;坐骨神经组织沉淀中NF-L、NF-M和pNF-H的变化与大鼠步态评分的相关系数分别为-0.921、-0.918和-0.903。
2.3α-tubulin、β-tubulin和β-actin含量在神经组织上清和沉淀中的变化
HD中毒后α-tubulin、β-tubulin和β-actin蛋白含量在大鼠大脑、脊髓和坐骨神经组织上清和沉淀中发生了不一致的变化。
在脊髓和坐骨神经组织上清中,α-tubulin含量在400 mg/kg HD染毒组分别下降了24.4%和44.8%,而在大脑组织上清中无明显变化。与对照组相比,β-tubulin含量在200mg/kg HD染毒组大鼠脊髓和坐骨神经上清中分别下降了16.8%和17.6%,在400 mg/kg HD染毒组分别下降了21.8%和40.7%,但是在大脑组织上清中200和400 mg/kg HD染毒组分别升高了14.1%和17.1%。β-actin的变化与β-tubulin非常相似。400 mg/kg HD染毒导致大鼠坐骨神经组织上清中β-actin含量下降29.2%,在大脑组织上清中升高17.7%。
α-tubulin在400 mg/kg HD染毒大鼠大脑、脊髓和坐骨神经组织匀浆沉淀中分别下降了19.2%、7.3%和35.8%。β-tubulin含量在200和400 mg/kg HD染毒大鼠坐骨神经沉淀中也分别下降了28.2%和43.9%,但在大脑和脊髓沉淀中保持不变。β-actin含量在400 mg/kg HD染毒大鼠大脑和坐骨神经沉淀中也分别下降了14.7%和38.2%。
2.4神经组织α-tubulin、β-tubulin和β-actin蛋白的变化与大鼠神经行为异常之间的相关性
坐骨神经组织中α-tubulin、β-tubulin和β-actin的改变与大鼠步态评分之间有明显的负相关性,而在大脑和脊髓组织中与步态评分无相关性。坐骨神经上清中α-tubulin、β-tubulin和β-actin与大鼠步态评分之间的相关系数分别为-0.874、-0.851和-0.876;沉淀中与大鼠步态评分之间的相关系数分别为-0.922、-0.870和-0.857。
2.5血清中细胞骨架蛋白的变化
pNF-H含量在200和400 mg/kg HD染毒大鼠血清中分别升高了33.5%和59.8%。NF-L含量在400 mg/kg HD染毒大鼠血清中也明显升高了,但是NF-M、α-tubulin、β-tubulin和β-actin含量未出现明显改变。
2.6血清中NFs蛋白的变化与大鼠步态异常之间的相关性
为确定血清中pNF-H的变化是否可反映HD中毒性神经病的进展,我们检测了中毒大鼠血清中NFs蛋白的变化与大鼠步态评分之间的相关性。血清中pNF-H变化与大鼠步态评分之间有明显的正相关性,相关系数为0.943。
3.NFs相关蛋白激酶在神经组织胞浆和胞膜组分中的变化
3.1 CaM,CaMKII,PKA和PKC的含量和活性在神经组织胞浆和胞膜组分中的变化
HD染毒导致CaM、CaMKII、PKA和PKC含量在大脑、脊髓和坐骨神经胞浆蛋白组分中明显升高。与对照组相比,CaM含量在200 mg/kg HD染毒组大鼠坐骨神经胞浆蛋白组分中升高了22.7%,在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了26.8%、23.9%和46.1%。ELISA试剂盒测定结果与WB结果一致,即CaM含量在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了76.9%、62.9%和112.6%。大脑胞浆组分中CaMKII含量也明显升高,在200和400 mg/kg HD染毒组分别升高了23.7和30.3%,但是在脊髓和坐骨神经中未检测到。PKA含量在400 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经胞浆蛋白组分中分别升高了21.5%、46.1%和21.0%;PKC含量在脊髓和坐骨神经胞浆蛋白组分中也分别升高23.0%和55.5%,但在200和400 mg/kg HD染毒组大鼠大脑胞浆蛋白组分中分别下降22.5%和39.4%。
CaM含量在200 mg/kg HD染毒组大鼠大脑胞膜组分中升高了28.6%,在400mg/kg HD染毒组大鼠大脑和坐骨神经胞膜组分中也分别升高了18.4%和45.1%。ELISA测定CaM浓度也表现为升高,在400 mg/kg HD染毒组大鼠大脑和坐骨神经胞膜组分中分别升高了11.1%和25.6%。CaMKII在400 mg/kg HD染毒组大鼠大脑胞膜组分中也明显升高了26.1%。PKA含量在200和400 mg/kg HD染毒组大鼠脊髓胞膜组分中分别升高了19.3%和25.5%。虽然PKC含量在200和400 mg/kg HD染毒组大鼠大脑胞膜组分中分别下降18.4%和21.5%,但在400 mg/kg HD染毒组大鼠脊髓和坐骨神经胞膜组分中分别升高了13.9%和44.7%,
CaMKII活性在200和400 mg/kg HD染毒组大鼠胞浆蛋白组分中分别升高了63.4%和83.7%,但在脊髓和坐骨神经中未检测到。脊髓胞浆蛋白组分中PKA活性在200和400 mg/kg HD染毒组分别下降了25.0%和25.0%,但在200 mg/kg HD染毒组大脑和400 mg/kg HD染毒组坐骨神经中分别升高36.5%和28.1%。PKC活性在200和400 mg/kg HD染毒组大脑和脊髓胞膜蛋白组分中分别下降24.9%、31.0%和27.9%、32.6%,但在坐骨神经胞膜蛋白组分中分别分别升高了8.1%和20.3%。
3.2 CaMKII,PKA,PKC活性与大鼠神经行为异常之间的相关性
大脑中CaMKII和坐骨神经PKC活性的变化与大鼠步态评分存在明显的正相关性,相关系数分别为0.888和0.765。然而脊髓和坐骨神经中PKA活性与大鼠步态评分存在明显的负相关性,相关系数分别为-0.772和-0.712。大脑和脊髓中PKC活性与大鼠步态评分也存在明显的负相关性,相关系数分别为-0.742和-0.664。
3.3 CDK5和相关激活因子在神经组织胞浆和胞膜蛋白组分中的变化
CDK5含量在400 mg/kg HD染毒组大鼠大脑胞浆和胞膜蛋白组分中分别下降了43.4%和17.3%。CDK5活性的变化与含量变化一致,即在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中分别下降了9.1%和15.2%。CDK5含量在200和400mg/kg HD染毒组大鼠脊髓胞浆(78.2%和21.9%)和胞膜蛋白(26.3%和12.7%)组分中明显升高,但活性却明显下降。200和400 mg/kg HD染毒组大鼠坐骨神经中CDK5含量也明显升高,在胞浆组分中分别升高了26.1%4和41.5%;其活性在胞浆组分中分别升高14.7%和37.6%,在胞膜蛋白组分中分别升高了9.0%和28.8%。
与对照组相比,p35前体含量在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中分别下降17.0%和37.6%,在胞膜蛋白组分中分别下降19.6%和23.0%;p25含量在大脑胞浆组分中分别下降26.5%和51.2%,在胞膜蛋白组分中分别下降12.6%和36.4%;p35含量在200和400 mg/kg HD染毒组大鼠大脑胞浆组分中也分别下降了11.9%和39.9%。然而p35前体、p35和p25在脊髓胞浆和胞膜蛋白组分中改变不一致,即在胞浆蛋白组分中升高,在胞膜蛋白组分中下降。另外,p35前体含量在200和400 mg/kg HD染毒组大鼠坐骨神经胞浆组分中分别升高38.1%和16.9%,在胞膜蛋白组分中分别下降59.2%和26.3%;p35和p25在400 mg/kg HD染毒组大鼠坐骨神经胞浆组分中分别升高28.3%和14.4%。
3.4 CDK5活性与大鼠神经行为异常之间的相关性
在大脑、脊髓和坐骨神经胞浆蛋白组分中CDK5活性与大鼠步态评分之间存在明显的相关性,相关系数分别为-0.746、-0.860和0.864。坐骨神经胞膜蛋白组分中CDK5活性与大鼠步态评分之间存在明显的正相关性,相关系数为0.851,大脑和脊髓中CDK5活性与大鼠步态评分之间无明显相关性。
4.神经组织中NFs亚单位聚合率的变化
NF-L的聚合率在200 mg/kg HD染毒组大鼠大脑、脊髓和坐骨神经中分别增加27.1%、38.0%和11.2%;在400 mg/kg HD染毒组分别增加了63.7%、51.6%和15.6%。NF-M聚合率在200和400 mg/kg HD染毒组大鼠大脑明显下降了11.7%和18.9%;而在脊髓中分别升高了51.5%和65.0%;坐骨神经中分别升高了-3.2%(P＞0.05)和9.5%。pNF-H聚合率在200和400 mg/kg HD染毒组大鼠大脑分别升高了16.4%和23.5%;脊髓中分别升高了2.3%(P＞0.05)和7.9%。p&n-pNF-H聚合率在400 mg╱kg HD染毒组大鼠大脑、脊髓和坐骨神经中也明显升高,分别升高了16.6%、24.0%和13.7%。
5.UPS在HD中毒性神经病NFs含量下降中的作用
5.1 MG132部分地抑制了HD促进NFs降解
在表达NF-M的HEK293细胞中(转染0.5 ug Flag-NFM质粒)中,NF-M含量在加入CHX 0、1、4和8小时后分别下降了4.2%、15.7%、23.2%和47.9%,可见NF-M蛋白半衰期大约是12 h。加入4 mM和8 mM HD后NF-M含量降低更加明显,说明HD中毒可加速NFM的降解。HD染毒30 min后,加入蛋白酶体抑制剂MG132,NF-M含量与未加MG132以前比较降解明显减缓。
5.2 HD作用下C末端Hsc70反应蛋白(carboxyl terminus of the Hsc70-interactingprotein,CHIP)作为含U-box结构域的E3连接酶增强NF-M泛素化,并促进其通过蛋白酶体降解
Flag-NFM、Myc-CHIP和/或HA-Ub质粒分别/同时转染HEK293细胞,经EZview Red ANTI-FLAG M2 Affinity Gel免疫沉淀后,在同时转染Flag-NFM、HA-Ub和Flag-NFM、HA-Ub、Myc-CHIP抗HA反应阳性,且同时转染Myc-CHIP质粒的细胞裂解液中NF-M蛋白的泛素化程度较转染Flag-NFM和HA-Ub的细胞裂解液强,加入HD后可见泛素化程度更强的NF-M蛋白。在同时转染Flag-NFM和增加量的Myc-CHIP质粒的细胞裂解液中,随着CHIP蛋白表达量的升高,NF-M蛋白的含量逐渐下降,HD染毒后降低更加明显,蛋白酶体抑制剂MG132可部分地阻断NF-M蛋白含量的下降,表明HD可增强CHIP促NF-M泛素化并通过蛋白酶体降解。
5.3神经组织中泛素活化酶E1、CHIP和蛋白酶体含量的变化
与对照组相比,200mg/kg HD组大鼠E1含量在大脑和脊髓中分别下降了15.8%和24.3%(P＜0.01),但在400mg/kg组分别升高了7.2%(P＞0.05)和29.1%(P＜0.01);E1含量在200和400mg/kg染毒组大鼠坐骨神经组织匀浆中均明显升高,分别升高了14.9%和59.8%,与对照组相比差异有统计学意义。
HD染毒后坐骨神经组织匀浆中CHIP含量明显升高。与对照组相比,200和400mg/kg组大鼠坐骨神经中CHIP含量分别升高了18.3%和55.3%,与对照组相比差异有统计学意义;但在大脑和脊髓中未检测到CHIP蛋白的存在。
HD染毒后,大脑、脊髓和坐骨神经组织匀浆中蛋白酶体含量均出现明显升高的趋势。与对照组相比,200mg/kg组大鼠蛋白酶体含量在大脑、脊髓和坐骨神经中分别升高了37.7%、25.8%和18.4%,与对照组相比差异有统计学意义;在400mg/kg组中分别升高了56.4%、86.3%和37.9%,与对照组相比差异有统计学意义。
结论
1.NFs比微管和微丝更加敏感,易受HD损伤,可能是正己烷中毒性神经病的分子靶点。
2.pNF-H含量在HD中毒大鼠血清中明显升高,并与大鼠神经行为异常呈明显的相关性,表明pNF-H可作为HD中毒性神经病早期诊断的生物标志物。
3.HD中毒导致大鼠神经组织CaM、PKA、PKC和CDK5的含量和活性以及NFs聚合率的变化,表明NFs相关蛋白信号通路和NFs磷酸化状态改变可能参与了HD中毒性神经病的发生。
4.由于CaMKII在脊髓和坐骨神经组织中为检测到,说明Ca~(2+)-CaM-CaMKII-NFs磷酸化信号通路可能部分地参与了HD中毒性神经病发生。
5.PKA、PKC和CDK5活性在中枢神经系统和外周神经系统能够变化不一致,可能决定了NFs在中枢神经系统和外周神经系统通过不同的途径降解。
6.CHIP作为一种含U-box结构域的E3连接酶促进NFs泛素化并通过蛋白酶体降解。
7.蛋白酶体抑制剂MG132部分地抑制了HD促进NFs降解,说明UPS通路在HD中毒性神经病中NFs含量下降中发挥了部分作用。
Objective
N-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.
Methods
Adult 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 MgCl_2,5 mM EGTA,5 mM dithiothreitol,Protease Inhibitor Cocktail(50ul/g tissue) and phosphatase inhibitors(5 mM Na_3VO_4,10mM Na_4P_2O_7 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 MgCl_2,1 mM PMSF,2μg/ml aprotinin,and 2μg/ml leupeptin) and B((100 mM KCl,5 mM NaCl,3 mM MgCl_2,50 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),Ca~(2+)/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 ~(32)P 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 changes
Age-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 serum
2.1 Alterations of NFs subunits in the supernatant and pellet fractions of nerve tissues
The 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 tissues
A 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 tissues
The 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 tissues
Significant(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 serum
The 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 serum
To 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 tissues
3.1 Changes of CaM,CaMKII,PKA,PKC contents and activities in the cytosolic and membrane fractions of nerve tissues
HD 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 abnormality
Significant 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 tissues
The 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 abnormality
The 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 SN
The 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 neuropathy
5.1 MG132 partly inhibits HD-induced NFs degradation
After 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 proteasome
Flag-NFM,Myc-CHIP and HA-Ub were co-transfected into HEK293 cells and then immunoprecipitation was performed with EZview~(TM) 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 tissues
Compared 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.
Conclusions
1.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 Ca~(2+)-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.
引文
1.Couri D,Milks M.Toxicity and metabolism of the neurotoxic hexacarbons n-hexane,2-hexanone,and 2,5-hexanedione[J].Annu Rev Pharmacol Toxicol,1982,22:145-166.
2.Spencer PS,Bischoff MC,Schaumburg HH.On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central--peripheral distal axonopathy[J].Toxicol Appl Pharmacol,1978,44(1):17-28.
3.Abbritti G,Siracusa A,Cianchetti C,et al.Shoe-makers' polyneuropathy in Italy:the aetiological problem[J].Br J Ind Med,1976,33(2):92-99.
4.Echaniz AM,Castro A,de Miguel J,et al.Toxic polyneuropathy caused by occupational use of glues[J].Med Clin(Bare),1985,85(16):685-685.
5.Wang JD,Chang YC,Kao KP,et al.An outbreak of n-hexane induced polyneuropathy among press proofing workers in Taipei[J].Am J Ind Med,1986,10(2):111-118.
6.林斯星,陈嘉斌.一起严重的正己烷职业中毒事故报告[J].中国工业医学杂志,1997,10(3):172-173.
7.杨玉珍.14例正己烷中毒患者的观察及护理[J].中国职业医学,1995,22(2):63-64.
8.张永汉,周奇,黄俊超.慢性正己烷中毒9例[J].中华劳动卫生职业病杂志,1999,17(2):118-120.
9.邝守仁,黄汉林,刘惠芳,等.慢性正己烷中毒102例临床分析[J].中华内科杂志,2001,40(5):329-331.
10.Graham DG,Amamath V,Valentine WM,et al.Pathogenetic studies of hexane and carbon disulfide neurotoxicity[J].Crit Rev Toxicol,1995,25(2):91-112.
11.Carden MJ,Lee VM,Schlaepfer WW.2,5-Hexanedione neuropathy is associated with the covalent crosslinking of neurofilament proteins[J].Neurochem.Pathol,1986,5:25-35.
12.Spencer PS,Sabri MI,Schaumburg HH,et al.Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies[J]?Ann.Neurol,1979,5:501-507.
13.Sahenk Z,Mendell JR.Acrylamide and 2,5-hexanedione neuropathies:abnormal bidirectional transport rate in distal axons[J].Brain Res,1981,219(2):397-405.
14.Monaco S,Autilio-Gambetti L,Zabel D,et al.Giant axonal neuropathy: acceleration of neurofilament transport in optic axons[J].Proe.Natl Acad.Sci.USA,1985,82:920-924.
15.Monaco S,Jacob J,Jenich H,et al.Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication[J].Brain Res,1989,491:328-334.
16.Monaco S,Wongrnongkolrit T,Shearson CM,et al.Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport[J].Brain Res,1990,519:73-81.
17.Pyle S J,Amarnath V,Graham DG,et al.Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure[J].Cell Motil Cytoskeleton,1993,26(2):133-143.
18.Genter MB,Szakal-Quin G,Anderson CW,et al.Evidence that pyrrole formation is a pathogenetic step in gamma-diketone neuropathy[J].Toxicol Appl Pharmacol,1987,87(2):351-362.
19.Genter St,Clair MB,Amamath V,et al.Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy[J].Chem Res Toxicol,1988,1(3):179-185.
20.Sayre LM,Shearson CM,Wongmongkolrit T,et al.Structural basis of gamma-diketone neurotoxicity:non-neurotoxicity of 3,3-dimethyl-2,5-hexanedione,a gamma-diketone incapable of pyrrole formation[J].Toxicol Appl Pharmacol,1986,84(1):36-44.
21.Sickles DW.Toxic neurofilamentous axonopathies and fast anterograde axonal transport.Ⅲ.Recovery from single injections and multiple dosing effects of acrylamide and 2,5-hexanedione[J].Toxicol.Appl.Pharmacol,1991,108(3):390-396.
22.Sickles DW.Toxic neurofilamentous axonopathies and fast anterograde axonal transport.Ⅳ.In vitro analysis of transport following acrylamide and 2,5-hexanedione[J].Toxicol.Lett,1992,61(2-3):199-204.
23.Papasozomenos SC,Yoon M,Crane R,et al.Redistribution of proteins of fast axonal transport following administration of beta, beta'-iminodipropionitrile: a quantitative autoradiographic study[J]. J. Cell. Biol, 1982,95(2Pt1): 672-675.
24. Xu Z, Cork LC, Griffin JW, et al. Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease[J]. Cell, 1993,73(1): 23-33.
25. Lehning EJ, Jortner BS, Fox JH, et al. y-diketone peripheral neuropathy. I. Quality morphometric analyses of axonal atrophy and swelling[J]. Toxicol Appl Pharmacol, 2000,165(2): 127-140.
26. Lapadula DM, Suwita E, Abou-Donia MB. Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy [J]. Brain Res, 1988, 458(1): 123-131.
27. Lehning EJ, Dyer KS, Jortner BS, et al. Axonal atrophy is a specific component of 2,5-hexanedione peripheral neuropathy [J]. Toxicol Appl Pharmacol, 1995, 135(1): 58-66.
28. Stone JD, Peterson AP, Eyer J, et al. Neurofilaments are nonessential to pathogenesis of toxicant-induced axonal degeneration[J]. J Neurosci, 2001, 21(7): 2278-2287.
29. Elder GA, Friedrich VL Jr, Bosco P, et al. Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content[J]. J Cell Biol, 1998,141(3): 727-39.
30. Xu Z, Marszalek JR, Lee MK, et al. Subunit composition of neurofilaments specifies axonal diameter[J]. J Cell Biol, 1996,133(5): 1061-9.
31. Rao MV, Houseweart MK,Williamson TL, et al. Neurofilament -dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation[J]. J Cell Biol, 1998,143(1): 171-81.
1.Lapadula DM,Suwita E,Abou-Donia MB.Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy[J].Brain Res,1988,458(1):123-131.
2.Chiu FC,Opanashuk LA,He DK,et al.gamma-diketone peripheral neuropathy.Ⅱ.Neurofilament subunit content[J].Toxicol Appl Pharmacol.2000,165(2):141-147.
3.LoPachin RM,He D,Reid ML.2,5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve[J].NeuroToxicology.2005,26(2):229-240.
4.Opanashuk LA,He DK,Lehning E J,et al.Gamma-diketone peripheral neuropathy Ⅲ.Neurofilament gene expression[J].Neurotoxicology.2001,22(2):215-220.
5.Vosler PS,Brennan CS,Chen J.Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration[J].Mol Neurobiol.2008,38(1):78-100.
6.Seet BT,Dikic I,Zhou MM,et al.Reading protein modifications with interaction domains[J].Nat Rev Mol Cell Biol.2006,7(7):473-483.
7.Hunter T.The age of crosstalk:phosphorylation,ubiquitination,and beyond[J].Mol Cell.2007,28(5):730-738.
8.温萍.蛋白质的泛素/蛋白酶体降解[J].宜宾学院学报.2004,6(3):168-170.
9.Wilkinson KD.Ubiquitination and deubiquitination:tar2 geting of proteins for degradation by the proteasome[J].Semin.Cell Dev.Biol.2000,11(3):141-148.
10.Hershko A,Ciechanover A.The ubiquitin system[J].Annu.Rev.Biochem.1998,67:425-479.
11.Haglund K,Dikic I.Ubiquitylation and cell signaling[J].EMBO J.2005,24(19): 3353-3359.
12.Sun L,Chen ZJ.The novel functions of ubiquitination in signaling[J].Curr Opin Cell Biol.2004,16(2):119-126.
13.佟向军,陈建国,翟中和.神经丝的结构和功能[J].细胞生物学杂志,1998,20(2):63-67.
14.Elhanany E,Jaffe H,Link WT,et al.Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein[J].J Neurochem.1994,63(6):2324-2335.
15.米瑞发,范明,周长满.神经丝磷酸化及其生理病理作用[J].神经解剖学杂志,1998,14(2):199-202.
16.Elhanany E.Identification of endogenously phosphorylated KSP sites in rat neurofilament protein[J].J Neurochem.1992,58(4):1975-1984.
17.Balastik M,Ferraguti F,Pires-da Silva A,et al.Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration[J].Proc Natl Acad Sci USA,2008,105(33):12016-12021.
1.Simpson PB,Russell JT.Role of mitochondrial Ca~(2+) regulation in neuronal and glial cell signalling[J].Brain Res Brain Res Rev,1998,26(1):72-81.
2.Bofill-Cardona E,Kudlacek O,Yang Q,et al.Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch[J].J Biol Chem,2000,275(42):32672-32680.
3.Drum CL,Yan SZ,Sarae R,et al.An extended conformation of ealmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis[J].J Biol Chem,2000,275(46):36334-36340.
4.Shen Y,Zhukovskaya NL,Guo Q,et al.Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.EMBO J,2005,24(5):929-941.
5.Hollander BA,Bennett GS,Shaw G.Localization of sites in the tail domain of the middle molecular mass neurofilament subunit phosphorylated by a neurofilament-associated kinase and by casein kinase Ⅰ[J].J Neurochem,1996,66(1):412-420.
6.Zhang GY,Wang JH,Seharma RK.Purification and characterization of bovine brain calmodulin-dependent protein kinase.Ⅱ.The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme[J].Mol Cell Bioehem,1993,122(2):159-169.
7.Darios F,Muriel MP,Khondiker ME,et al.Neurotoxic calcium transfer from endoplasmic reticulum to mitoehondria is regulated by cyclin-dependent kinase 5-dependent phosphorylation oftau[J].J Neurosci,2005,25(16):4159-4168.
8.鲁吉波,黄金祥,张寿林,等.二硫化碳对大鼠神经细胞内游离钙离子、蛋白激酶C和细胞骨架的影响[J].职业卫生与应急救援,1996,14(4):4-6.
9.孙鹂.铅对细胞内钙离子及钙调蛋白影响的研究进展[J].国外医学卫生学分册,2002,29(6):328-330.
10.王英鹏,宋俊峰,饶志仁.CDK5与神经退行性疾病[J].生理科学进展,2004,350):45-48.
11.Borshi R,Giliberto L,Assim A,et al.Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclearpalsy[J].Neurology,2002,58(4):589-592.
12.Thomas CP,Dunn MJ,Mattera R.Ca~(2+) signalling in K562 human erythroleukaemia cells:effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways[J].Biochem J,1995,312(Ptl),151-158.
13.Mahadev K,Chetty CS,Vemuri MC.Effect of prenatal and postnatal ethanol exposure on Ca~(2+) /calmodulin-dependent protein kinase Ⅱ in rat cerebral cortex[J].Alcohol,2001,23(3):183-188.
14.Chin D,Means AR.Calmodulin:a prototypical calcium sensor.Trends Cell Biol,2000,10(8):322-8.
15.Fukui Y,De Lozanne A,Spudich JA.Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant[J].J Cell Biol,1990,110(2):367-378.
16.Hahn K,DeBiasio R,Taylor DL.Patterns of elevated free calcium and calmodulin activation in living cells[J].Nature,1992,359(6397):736-738.
17.Craske M,Takeo T,Gerasimenko O,et al.Hormone-induced secretory and nuclear translocation of calmodulin:oscillations of calmodulin concentration with the nucleus as an integrator[J].Proc Natl Acad Sci USA,1999,96(8):4426-4431.
18.张艳淑,刘强,刘清君,等.2,5一己二酮对运动神经细胞钙稳态的影响[J].中化劳动卫生职业病杂志,2006,24(5):270-272.
19.Gupta RP,Abou-Donia MB.Tau proteins-enhanced Ca2+/calmodulin (CaM)-dependent phosphorylation by the brain supematant of diisopropyl phosphorofluoridate(DFP)-treated hen:tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II.Brain Res,1998,813(1):32-43.
20.Gupta RP,Bing G,Hong JS,et al.cDNA cloning and sequencing of Ca~(2+) /calmodulin-dependent protein kinase subunit and its mRNA expression in diisopropyl phosphorofluoridate(DFP)-treated hen central nervous system.Mol Cell Biochem,1998,181(1-2):29-39.
21.宋力,黄巧冰,赵克森.蛋白激酶C与内皮细胞骨架蛋白和血管通透性研究进展[J].中国微循环.2003,7(3):184-187.
22.Kishimoto A,Mikawa K,Hashimoto K,et al.Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease(calpaln).J Bio Chem,1989,264(7):4088-4092.
23.Gonda Y,Nishizawa K,Ando S,et al.Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro[J].Biochem Biophys Res Commun,1990,167(3):1316-1325.
24.Bressler J,Kim KA,Chakraborti T,et al.Molecular mechanisms of lead neurotoxicity[J].Neurochem Res.1999,24(4):595-600.
25.Ase K.Limited proteolysis and differential down-regulation of protein kinase C subspecies in KM3 cells[J].Kobe J Med Sci,1989,35(3):137-149.
26.Wieloch T,Cardell M,Bingren H,et al.Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia[J].J Neurochem,1991,56(4):1227-1235.
27.陈康宁,董为伟.蛋白激酶C和脑缺血[J].国外医学·内科学分册,1996,23(4):164-167.
28.Hisanaga S,Gonda Y,Inagaki M,et al.Effects of phosphorylation of the neurofilament L protein on filamentous structures[J].Cell Regul,1990,1(2):237-248.
29.孙莉,刘声远,王建枝,等.蛋白激酶A与阿尔茨海默病[J].生命的化学,2002,22(1):45-47.
30.Kaur A,Gill KD.Disruption of neuronal calcium homeostasis after chronic aluminium toxicity in rats[J].Basic Clin Pharmacol Toxicol.2005,96(2):118-122.
31.Connell-Crowley L,Le Gall M,Vo DJ,et al.The cyclin-dependent kinase CDK5control multiple aspects of axon patterning in vivo[J].Curr Biol,2000,10(10):599-602.
32.Tsai LH,Delalle I,Caviness VS Jr,et al.p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5[J].Nature,1994,371(6496):419-423.
33.Humbert S,Dhavan R,Tsai L.p39 activates cdk5 in neurons,and is associated with the actin cytoskeleton[J].J Cell Sci,2000,113(Pt 6):975-983.
34.Ko J,Humbert S,Bronson RT,et al.p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment[J].J Neurosci,2001,21(17):6758-6771.
35.Cruz JC,Tseng HC,Goldman JA,et al.Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles[J].Neuron,2003,40(3):471-483.
36.Sharma M,Sharma P,Pant HC.CDK-5-mediated neurofilament phosphorylation in SHSY5Y human neuroblastoma cells[J].J Neurochem,1999,73(1):79-86.
37.Elhanany E,Jaffe H,Link WT,et al.Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein[J].J Neurochem,1994,63(6):2324-2335.
38.Borshi R,Giliberto L,Assim A,et al.Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy[J].Neurology,2002,58(4):589-592.
39.Nguyen MD,Lariviere RC,Julien JP.Deregulation of CDK5 in a monse model of ALS.Toxicity alleviated by perikaryal neurofilament inclusions[J].Neuron,2001,30(1):135-147.
40.Wang YP,Mou DL,Song JF,et al.Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN[J].J Neurochem,2006,99(1):186-197.
41.Otth C,Concha Ⅱ,Arendt T,et al.AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576[J].J Alzheimers Dis,2002,4(5):417-430.
42.Okabe S,Hirokawa N.Turnover of fluorescently labelled tubulin and actin in the axon[J].Nature,1990,343(6257):479-482.
43.Nixon RA,Logvinenko KB.Multiple fates of newly synthesized neurofilament proteins:evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons.J Cell Biol,1986,102(2):647-659.
44.Pant HC.Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain[j].Biochem J,1988,256(2):665-668.
45.Sun F,Kanthasamy A,Anantharam V,et al.Environmental neurotoxic chemicals-induced ubiquitin proteasome system dysfunction in the pathogenesis and progression of Parkinson's disease[J]. Pharmacol Ther, 2007, 114(3): 327-344.
46. Zhai Q, Wang J, Kim A, et al. Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration[J]. Neuron, 2003, 39(2): 217-225.
47. Nixon VL, Levasseur M, McDougall A, et al. Ca~(2+) oscillations promote APC/C-dependent cyclin Bl degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs[J]. Curr Biol. 2002,12(9): 746-750.
48. Ciechanover A, Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg[J]. Neuron, 2003, 40(2): 427-446.
1.Opanashuk LA,He DK,Lehning E J,et al.Gamma-diketone peripheral neuropathy Ⅲ.Neurofilament gene expression[J].Neurotoxicology,2001,22(2):215-220.
2.Sun F,Kanthasamy A,Anantharam V,et al.Environmental neurotoxic chemicals-induced ubiquitin proteasome system dysfunction in the pathogenesis and progression of Parkinson's disease[J].Pharmacol Ther,2007,114(3):327-344.
3.Hershko A,Ciechanover A.The ubiquitin system[J].Annu Rev Biochem,1998,67:425-479.
4.Ravid T,Hochstrasser M.Diversity of degradation signals in the ubiquitin-proteasome system[J].Nat Rev Mol Cell Biol,2008,9(9):679-690.
5.DeMartino GN,Slaughter CA.The proteasome,a novel protease regulated by multiple mechanisms[J].J Biol Chem,1999,274(32):22123-22126.
6.周筠梅.蛋白质的错误折叠与疾病[J].生物化学与生物物理进展,2000,27(2):581-584.
7.McNaught KS,Jenner P.Proteasomal function is impaired in substantia nigra in Parkinson's disease[J].Neurosci Lett,2001,297(3):191 - 194.
8.Sherman MY,Goldberg AL.Cellular defenses against unfolded proteins:a cell biologist thinks about neurodegenerative diseases[J].Neuron,2001,29(1):15-32.
9.Golab J,Bauer TM,Daniel V,et al.Role of the ubiquitin-proteasome pathway in the diagnosis of human diseases[J].Clin Chim Acta,2004,340(1-2):27-40.
10.Petrucelli L,Dickson D,Kehoe K,et al.CHIP and Hsp70 regulate tau ubiquitination,degradation and aggregation[J].Hum Mol Genet,2004,13(7):703-714.
11.Dickey CA,Kamal A,Lundgren K,et al.The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins[J].J Clin Invest,2007,117(3):648-658.
12.Kumar P,Ambasta RK,Veereshwarayya V,et al.CHIP and HSPs interact with beta-APP in a proteasome-dependent manner and influence Abeta metabolism[J].Hum Mol Genet,2007,16(7):848-864.
13.Figueiredo-Pereira ME,Cohen G.The ubiquitin/proteasome pathway:friend or foe in zinc-,cadmium-,and H202-induced neuronal oxidative stress[J].Mol Biol Rep,1999,26(1-2):65-69.
14.Zhai Q,Wang J,Kim A,et al._Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration[J].Neuron,2003,39(2):217-225.
15.Graham DG,Szakal-Quin G,Priest JW,et al.In vitro evidence that covalent crosslinking of neurofilaments occurs in gamma-diketone neuropathy[J].Proc Natl Acad Sci USA,1984,81(15):4979-4982.
16.Nixon VL,Levasseur M,McDougall A,et al.Ca(2+) oscillations promote APC/C-dependent cyclin B 1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs[J].Curr Biol,2002,12(9):746-750.
17.Ardley HC,Robinson PA.The role of ubiquitin-protein ligases in neurodegenerative disease[J].Neurodegener Dis.2004,1(2-3):71-87.
18.杨东叶,刘凯于,余泽华.泛素连接酶E3[J].细胞生物学杂志,2005,27(3):281-285.
19.Wang G,Yang J,Huibregtse JM.Functional domains of the Rsp5ubiquitin-protein ligase[J].Mol Cell Biol,1999,19(1):342-352.
20.Skowyra D,Koepp DM,Kamura T,et al.Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrrl and Rbxl[J].Science,1999,284(5414):662-665.
21.Koegl M,Hoppe T,Schlenker S,et al.A novel ubiquitination factor,E4,is involved in multiubiquitin chain assembly[J].Cell,1999,96(5):635-644.
22.Jiang J,Ballinger CA,Wu Y,et al.CHIP is a U-box-dependent E3 ubiquitin ligase:identification of Hsc70 as a target for ubiquitylation[J].J Biol Chem,2001,276(46):42938-42944.
23.Connell P,Ballinger CA,Jiang J,et al.The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins[J].Nat Cell Biol,2001,3(1):93-96.
24.Dai Q,Zhang C,Wu Y,et al.CHIP activates HSF1 and confers protection against apoptosis and cellular stress[J].EMBO J,2003,22(20):5446-5458.
25.Dai Q,Qian SB,Li HH,et al.Regulation of the cytoplasmic quality control protein degradation pathway by BAG2[J].J Biol Chem,2005,280(46):38673-38681.
26.Urushitani M,Kurisu J,Tateno M,et al.CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70[J].J Neurochem,2004,90(1):231-244.
27.Ballinger CA,Connell P,Wu Y,et al.Identification of CHIP,a novel tetratricopeptide repeatcontaining protein that interacts with heat shock proteins and negatively regulates chaperone functions[J].Mol.Cell.Biol,1999,19(6):4535-4545.
28.Hatakeyama S,Yada M,Matsumoto M,et al.U box proteins as a new family of ubiquitin-protein ligases[J].J Biol Chem,2001,276(35):33111-33120.
29.Zheng N,Schulman BA,Song L,et al.Structure of the Cull-Rbxl-Skpl-F boxSkp2 SCF ubiquitin ligase complex[J].Nature,2002,416(6882):703-709.
30.Connell P,Ballinger CA,Jiang J,et al.The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins[J].Nat.Cell Biol,2001,3(1):93-96.
31.Shimura H,Schwart D,Gygi SP,et al.CHIP-Hsc70 complex uiquitinates phosphorylated tau and enhances cell survival[J].J Biol Chem.2004,279(6): 4869-4876.
32. Figueiredo-Pereira ME, Cohen G. Disruption of the intracelular sulthydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquination in neuronal cells[J]. J Biol Chem, 1998,273(21): 12703-12709.
33. Lee MH, Hyun DH, Jenner P. Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production[J]. J Neurochem, 2001, 78(1): 32-41.
34. Mizushima N. Collaboration of proteolytic systems[J]. Autophagy, 2007, 3(3): 179-80.
35. Zhang JY, Peng C, Shi H, et al. Inhibition of autophagy causes tau proteolysis by activating calpain in rat brain[J]. J Alzheimers Dis, 2009,16(1):39-47.
36. Ding WX, Ni HM, Gao W, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability[J]. Am J Pathol, 2007,171 (2):513-524.
37. Nedelsky NB, Todd PK, Taylor JP. Autophagy and the ubiquitin-proteasome system: collaborators in neuroprotection[J]. Biochim Biophys Acta, 2008, 1782(12): 691-699.
38. Komatsu M, Wang QJ, Holstein GR, et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration[J]. Proc Natl Acad Sci USA, 2007,104(36): 14489-14494.
39. Levine B, Kroemer G Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132(1): 27-42.
40. Posmantur RM, Zhao X, Kampfl A, et al. Immunoblot analyses of the relative contributions of cysteine and aspartic proteases to neurofilament breakdown products following experimental brain injury in rats[J]. Neurochem Res, 1998, 23(10): 1265-1276.
41. Weissman AM. Themes and variations on ubiquitylation[J]. Nat. Rev. Mol. Cell Biol, 2001,2(3): 169-178.
42. Gao M, Karin M. Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli[J]. Mol Cell, 2005, 19(5): 581-593.
2.Spencer PS,Bischoff MC,Schaumburg HH.On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central--peripheral distal axonopathy[J].Toxicol Appl Pharmacol,1978,44(1):17-28.
3.Abbritti G,Siracusa A,Cianchetti C,et al.Shoe-makers' polyneuropathy in Italy:the aetiological problem[J].Br J Ind Med,1976,33(2):92-99.
4.Echaniz AM,Castro A,de Miguel J,et al.Toxic polyneuropathy caused by occupational use of glues[J].Med Clin(Bare),1985,85(16):685-685.
5.Wang JD,Chang YC,Kao KP,et al.An outbreak of n-hexane induced polyneuropathy among press proofing workers in Taipei[J].Am J Ind Med,1986,10(2):111-118.
6.林斯星,陈嘉斌.一起严重的正己烷职业中毒事故报告[J].中国工业医学杂志,1997,10(3):172-173.
7.杨玉珍.14例正己烷中毒患者的观察及护理[J].中国职业医学,1995,22(2):63-64.
8.张永汉,周奇,黄俊超.慢性正己烷中毒9例[J].中华劳动卫生职业病杂志,1999,17(2):118-120.
9.邝守仁,黄汉林,刘惠芳,等.慢性正己烷中毒102例临床分析[J].中华内科杂志,2001,40(5):329-331.
10.Graham DG,Amamath V,Valentine WM,et al.Pathogenetic studies of hexane and carbon disulfide neurotoxicity[J].Crit Rev Toxicol,1995,25(2):91-112.
11.Carden MJ,Lee VM,Schlaepfer WW.2,5-Hexanedione neuropathy is associated with the covalent crosslinking of neurofilament proteins[J].Neurochem.Pathol,1986,5:25-35.
12.Spencer PS,Sabri MI,Schaumburg HH,et al.Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies[J]?Ann.Neurol,1979,5:501-507.
13.Sahenk Z,Mendell JR.Acrylamide and 2,5-hexanedione neuropathies:abnormal bidirectional transport rate in distal axons[J].Brain Res,1981,219(2):397-405.
14.Monaco S,Autilio-Gambetti L,Zabel D,et al.Giant axonal neuropathy: acceleration of neurofilament transport in optic axons[J].Proe.Natl Acad.Sci.USA,1985,82:920-924.
15.Monaco S,Jacob J,Jenich H,et al.Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication[J].Brain Res,1989,491:328-334.
16.Monaco S,Wongrnongkolrit T,Shearson CM,et al.Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport[J].Brain Res,1990,519:73-81.
17.Pyle S J,Amarnath V,Graham DG,et al.Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure[J].Cell Motil Cytoskeleton,1993,26(2):133-143.
18.Genter MB,Szakal-Quin G,Anderson CW,et al.Evidence that pyrrole formation is a pathogenetic step in gamma-diketone neuropathy[J].Toxicol Appl Pharmacol,1987,87(2):351-362.
19.Genter St,Clair MB,Amamath V,et al.Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy[J].Chem Res Toxicol,1988,1(3):179-185.
20.Sayre LM,Shearson CM,Wongmongkolrit T,et al.Structural basis of gamma-diketone neurotoxicity:non-neurotoxicity of 3,3-dimethyl-2,5-hexanedione,a gamma-diketone incapable of pyrrole formation[J].Toxicol Appl Pharmacol,1986,84(1):36-44.
21.Sickles DW.Toxic neurofilamentous axonopathies and fast anterograde axonal transport.Ⅲ.Recovery from single injections and multiple dosing effects of acrylamide and 2,5-hexanedione[J].Toxicol.Appl.Pharmacol,1991,108(3):390-396.
22.Sickles DW.Toxic neurofilamentous axonopathies and fast anterograde axonal transport.Ⅳ.In vitro analysis of transport following acrylamide and 2,5-hexanedione[J].Toxicol.Lett,1992,61(2-3):199-204.
23.Papasozomenos SC,Yoon M,Crane R,et al.Redistribution of proteins of fast axonal transport following administration of beta, beta'-iminodipropionitrile: a quantitative autoradiographic study[J]. J. Cell. Biol, 1982,95(2Pt1): 672-675.
24. Xu Z, Cork LC, Griffin JW, et al. Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease[J]. Cell, 1993,73(1): 23-33.
25. Lehning EJ, Jortner BS, Fox JH, et al. y-diketone peripheral neuropathy. I. Quality morphometric analyses of axonal atrophy and swelling[J]. Toxicol Appl Pharmacol, 2000,165(2): 127-140.
26. Lapadula DM, Suwita E, Abou-Donia MB. Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy [J]. Brain Res, 1988, 458(1): 123-131.
27. Lehning EJ, Dyer KS, Jortner BS, et al. Axonal atrophy is a specific component of 2,5-hexanedione peripheral neuropathy [J]. Toxicol Appl Pharmacol, 1995, 135(1): 58-66.
28. Stone JD, Peterson AP, Eyer J, et al. Neurofilaments are nonessential to pathogenesis of toxicant-induced axonal degeneration[J]. J Neurosci, 2001, 21(7): 2278-2287.
29. Elder GA, Friedrich VL Jr, Bosco P, et al. Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content[J]. J Cell Biol, 1998,141(3): 727-39.
30. Xu Z, Marszalek JR, Lee MK, et al. Subunit composition of neurofilaments specifies axonal diameter[J]. J Cell Biol, 1996,133(5): 1061-9.
31. Rao MV, Houseweart MK,Williamson TL, et al. Neurofilament -dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation[J]. J Cell Biol, 1998,143(1): 171-81.
1.Lapadula DM,Suwita E,Abou-Donia MB.Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy[J].Brain Res,1988,458(1):123-131.
2.Chiu FC,Opanashuk LA,He DK,et al.gamma-diketone peripheral neuropathy.Ⅱ.Neurofilament subunit content[J].Toxicol Appl Pharmacol.2000,165(2):141-147.
3.LoPachin RM,He D,Reid ML.2,5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve[J].NeuroToxicology.2005,26(2):229-240.
4.Opanashuk LA,He DK,Lehning E J,et al.Gamma-diketone peripheral neuropathy Ⅲ.Neurofilament gene expression[J].Neurotoxicology.2001,22(2):215-220.
5.Vosler PS,Brennan CS,Chen J.Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration[J].Mol Neurobiol.2008,38(1):78-100.
6.Seet BT,Dikic I,Zhou MM,et al.Reading protein modifications with interaction domains[J].Nat Rev Mol Cell Biol.2006,7(7):473-483.
7.Hunter T.The age of crosstalk:phosphorylation,ubiquitination,and beyond[J].Mol Cell.2007,28(5):730-738.
8.温萍.蛋白质的泛素/蛋白酶体降解[J].宜宾学院学报.2004,6(3):168-170.
9.Wilkinson KD.Ubiquitination and deubiquitination:tar2 geting of proteins for degradation by the proteasome[J].Semin.Cell Dev.Biol.2000,11(3):141-148.
10.Hershko A,Ciechanover A.The ubiquitin system[J].Annu.Rev.Biochem.1998,67:425-479.
11.Haglund K,Dikic I.Ubiquitylation and cell signaling[J].EMBO J.2005,24(19): 3353-3359.
12.Sun L,Chen ZJ.The novel functions of ubiquitination in signaling[J].Curr Opin Cell Biol.2004,16(2):119-126.
13.佟向军,陈建国,翟中和.神经丝的结构和功能[J].细胞生物学杂志,1998,20(2):63-67.
14.Elhanany E,Jaffe H,Link WT,et al.Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein[J].J Neurochem.1994,63(6):2324-2335.
15.米瑞发,范明,周长满.神经丝磷酸化及其生理病理作用[J].神经解剖学杂志,1998,14(2):199-202.
16.Elhanany E.Identification of endogenously phosphorylated KSP sites in rat neurofilament protein[J].J Neurochem.1992,58(4):1975-1984.
17.Balastik M,Ferraguti F,Pires-da Silva A,et al.Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration[J].Proc Natl Acad Sci USA,2008,105(33):12016-12021.
1.Simpson PB,Russell JT.Role of mitochondrial Ca~(2+) regulation in neuronal and glial cell signalling[J].Brain Res Brain Res Rev,1998,26(1):72-81.
2.Bofill-Cardona E,Kudlacek O,Yang Q,et al.Binding of calmodulin to the D2-dopamine receptor reduces receptor signaling by arresting the G protein activation switch[J].J Biol Chem,2000,275(42):32672-32680.
3.Drum CL,Yan SZ,Sarae R,et al.An extended conformation of ealmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis[J].J Biol Chem,2000,275(46):36334-36340.
4.Shen Y,Zhukovskaya NL,Guo Q,et al.Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.EMBO J,2005,24(5):929-941.
5.Hollander BA,Bennett GS,Shaw G.Localization of sites in the tail domain of the middle molecular mass neurofilament subunit phosphorylated by a neurofilament-associated kinase and by casein kinase Ⅰ[J].J Neurochem,1996,66(1):412-420.
6.Zhang GY,Wang JH,Seharma RK.Purification and characterization of bovine brain calmodulin-dependent protein kinase.Ⅱ.The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme[J].Mol Cell Bioehem,1993,122(2):159-169.
7.Darios F,Muriel MP,Khondiker ME,et al.Neurotoxic calcium transfer from endoplasmic reticulum to mitoehondria is regulated by cyclin-dependent kinase 5-dependent phosphorylation oftau[J].J Neurosci,2005,25(16):4159-4168.
8.鲁吉波,黄金祥,张寿林,等.二硫化碳对大鼠神经细胞内游离钙离子、蛋白激酶C和细胞骨架的影响[J].职业卫生与应急救援,1996,14(4):4-6.
9.孙鹂.铅对细胞内钙离子及钙调蛋白影响的研究进展[J].国外医学卫生学分册,2002,29(6):328-330.
10.王英鹏,宋俊峰,饶志仁.CDK5与神经退行性疾病[J].生理科学进展,2004,350):45-48.
11.Borshi R,Giliberto L,Assim A,et al.Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclearpalsy[J].Neurology,2002,58(4):589-592.
12.Thomas CP,Dunn MJ,Mattera R.Ca~(2+) signalling in K562 human erythroleukaemia cells:effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways[J].Biochem J,1995,312(Ptl),151-158.
13.Mahadev K,Chetty CS,Vemuri MC.Effect of prenatal and postnatal ethanol exposure on Ca~(2+) /calmodulin-dependent protein kinase Ⅱ in rat cerebral cortex[J].Alcohol,2001,23(3):183-188.
14.Chin D,Means AR.Calmodulin:a prototypical calcium sensor.Trends Cell Biol,2000,10(8):322-8.
15.Fukui Y,De Lozanne A,Spudich JA.Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant[J].J Cell Biol,1990,110(2):367-378.
16.Hahn K,DeBiasio R,Taylor DL.Patterns of elevated free calcium and calmodulin activation in living cells[J].Nature,1992,359(6397):736-738.
17.Craske M,Takeo T,Gerasimenko O,et al.Hormone-induced secretory and nuclear translocation of calmodulin:oscillations of calmodulin concentration with the nucleus as an integrator[J].Proc Natl Acad Sci USA,1999,96(8):4426-4431.
18.张艳淑,刘强,刘清君,等.2,5一己二酮对运动神经细胞钙稳态的影响[J].中化劳动卫生职业病杂志,2006,24(5):270-272.
19.Gupta RP,Abou-Donia MB.Tau proteins-enhanced Ca2+/calmodulin (CaM)-dependent phosphorylation by the brain supematant of diisopropyl phosphorofluoridate(DFP)-treated hen:tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II.Brain Res,1998,813(1):32-43.
20.Gupta RP,Bing G,Hong JS,et al.cDNA cloning and sequencing of Ca~(2+) /calmodulin-dependent protein kinase subunit and its mRNA expression in diisopropyl phosphorofluoridate(DFP)-treated hen central nervous system.Mol Cell Biochem,1998,181(1-2):29-39.
21.宋力,黄巧冰,赵克森.蛋白激酶C与内皮细胞骨架蛋白和血管通透性研究进展[J].中国微循环.2003,7(3):184-187.
22.Kishimoto A,Mikawa K,Hashimoto K,et al.Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease(calpaln).J Bio Chem,1989,264(7):4088-4092.
23.Gonda Y,Nishizawa K,Ando S,et al.Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro[J].Biochem Biophys Res Commun,1990,167(3):1316-1325.
24.Bressler J,Kim KA,Chakraborti T,et al.Molecular mechanisms of lead neurotoxicity[J].Neurochem Res.1999,24(4):595-600.
25.Ase K.Limited proteolysis and differential down-regulation of protein kinase C subspecies in KM3 cells[J].Kobe J Med Sci,1989,35(3):137-149.
26.Wieloch T,Cardell M,Bingren H,et al.Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia[J].J Neurochem,1991,56(4):1227-1235.
27.陈康宁,董为伟.蛋白激酶C和脑缺血[J].国外医学·内科学分册,1996,23(4):164-167.
28.Hisanaga S,Gonda Y,Inagaki M,et al.Effects of phosphorylation of the neurofilament L protein on filamentous structures[J].Cell Regul,1990,1(2):237-248.
29.孙莉,刘声远,王建枝,等.蛋白激酶A与阿尔茨海默病[J].生命的化学,2002,22(1):45-47.
30.Kaur A,Gill KD.Disruption of neuronal calcium homeostasis after chronic aluminium toxicity in rats[J].Basic Clin Pharmacol Toxicol.2005,96(2):118-122.
31.Connell-Crowley L,Le Gall M,Vo DJ,et al.The cyclin-dependent kinase CDK5control multiple aspects of axon patterning in vivo[J].Curr Biol,2000,10(10):599-602.
32.Tsai LH,Delalle I,Caviness VS Jr,et al.p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5[J].Nature,1994,371(6496):419-423.
33.Humbert S,Dhavan R,Tsai L.p39 activates cdk5 in neurons,and is associated with the actin cytoskeleton[J].J Cell Sci,2000,113(Pt 6):975-983.
34.Ko J,Humbert S,Bronson RT,et al.p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment[J].J Neurosci,2001,21(17):6758-6771.
35.Cruz JC,Tseng HC,Goldman JA,et al.Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles[J].Neuron,2003,40(3):471-483.
36.Sharma M,Sharma P,Pant HC.CDK-5-mediated neurofilament phosphorylation in SHSY5Y human neuroblastoma cells[J].J Neurochem,1999,73(1):79-86.
37.Elhanany E,Jaffe H,Link WT,et al.Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein[J].J Neurochem,1994,63(6):2324-2335.
38.Borshi R,Giliberto L,Assim A,et al.Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy[J].Neurology,2002,58(4):589-592.
39.Nguyen MD,Lariviere RC,Julien JP.Deregulation of CDK5 in a monse model of ALS.Toxicity alleviated by perikaryal neurofilament inclusions[J].Neuron,2001,30(1):135-147.
40.Wang YP,Mou DL,Song JF,et al.Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN[J].J Neurochem,2006,99(1):186-197.
41.Otth C,Concha Ⅱ,Arendt T,et al.AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576[J].J Alzheimers Dis,2002,4(5):417-430.
42.Okabe S,Hirokawa N.Turnover of fluorescently labelled tubulin and actin in the axon[J].Nature,1990,343(6257):479-482.
43.Nixon RA,Logvinenko KB.Multiple fates of newly synthesized neurofilament proteins:evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons.J Cell Biol,1986,102(2):647-659.
44.Pant HC.Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain[j].Biochem J,1988,256(2):665-668.
45.Sun F,Kanthasamy A,Anantharam V,et al.Environmental neurotoxic chemicals-induced ubiquitin proteasome system dysfunction in the pathogenesis and progression of Parkinson's disease[J]. Pharmacol Ther, 2007, 114(3): 327-344.
46. Zhai Q, Wang J, Kim A, et al. Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration[J]. Neuron, 2003, 39(2): 217-225.
47. Nixon VL, Levasseur M, McDougall A, et al. Ca~(2+) oscillations promote APC/C-dependent cyclin Bl degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs[J]. Curr Biol. 2002,12(9): 746-750.
48. Ciechanover A, Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg[J]. Neuron, 2003, 40(2): 427-446.
1.Opanashuk LA,He DK,Lehning E J,et al.Gamma-diketone peripheral neuropathy Ⅲ.Neurofilament gene expression[J].Neurotoxicology,2001,22(2):215-220.
2.Sun F,Kanthasamy A,Anantharam V,et al.Environmental neurotoxic chemicals-induced ubiquitin proteasome system dysfunction in the pathogenesis and progression of Parkinson's disease[J].Pharmacol Ther,2007,114(3):327-344.
3.Hershko A,Ciechanover A.The ubiquitin system[J].Annu Rev Biochem,1998,67:425-479.
4.Ravid T,Hochstrasser M.Diversity of degradation signals in the ubiquitin-proteasome system[J].Nat Rev Mol Cell Biol,2008,9(9):679-690.
5.DeMartino GN,Slaughter CA.The proteasome,a novel protease regulated by multiple mechanisms[J].J Biol Chem,1999,274(32):22123-22126.
6.周筠梅.蛋白质的错误折叠与疾病[J].生物化学与生物物理进展,2000,27(2):581-584.
7.McNaught KS,Jenner P.Proteasomal function is impaired in substantia nigra in Parkinson's disease[J].Neurosci Lett,2001,297(3):191 - 194.
8.Sherman MY,Goldberg AL.Cellular defenses against unfolded proteins:a cell biologist thinks about neurodegenerative diseases[J].Neuron,2001,29(1):15-32.
9.Golab J,Bauer TM,Daniel V,et al.Role of the ubiquitin-proteasome pathway in the diagnosis of human diseases[J].Clin Chim Acta,2004,340(1-2):27-40.
10.Petrucelli L,Dickson D,Kehoe K,et al.CHIP and Hsp70 regulate tau ubiquitination,degradation and aggregation[J].Hum Mol Genet,2004,13(7):703-714.
11.Dickey CA,Kamal A,Lundgren K,et al.The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins[J].J Clin Invest,2007,117(3):648-658.
12.Kumar P,Ambasta RK,Veereshwarayya V,et al.CHIP and HSPs interact with beta-APP in a proteasome-dependent manner and influence Abeta metabolism[J].Hum Mol Genet,2007,16(7):848-864.
13.Figueiredo-Pereira ME,Cohen G.The ubiquitin/proteasome pathway:friend or foe in zinc-,cadmium-,and H202-induced neuronal oxidative stress[J].Mol Biol Rep,1999,26(1-2):65-69.
14.Zhai Q,Wang J,Kim A,et al._Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration[J].Neuron,2003,39(2):217-225.
15.Graham DG,Szakal-Quin G,Priest JW,et al.In vitro evidence that covalent crosslinking of neurofilaments occurs in gamma-diketone neuropathy[J].Proc Natl Acad Sci USA,1984,81(15):4979-4982.
16.Nixon VL,Levasseur M,McDougall A,et al.Ca(2+) oscillations promote APC/C-dependent cyclin B 1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs[J].Curr Biol,2002,12(9):746-750.
17.Ardley HC,Robinson PA.The role of ubiquitin-protein ligases in neurodegenerative disease[J].Neurodegener Dis.2004,1(2-3):71-87.
18.杨东叶,刘凯于,余泽华.泛素连接酶E3[J].细胞生物学杂志,2005,27(3):281-285.
19.Wang G,Yang J,Huibregtse JM.Functional domains of the Rsp5ubiquitin-protein ligase[J].Mol Cell Biol,1999,19(1):342-352.
20.Skowyra D,Koepp DM,Kamura T,et al.Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrrl and Rbxl[J].Science,1999,284(5414):662-665.
21.Koegl M,Hoppe T,Schlenker S,et al.A novel ubiquitination factor,E4,is involved in multiubiquitin chain assembly[J].Cell,1999,96(5):635-644.
22.Jiang J,Ballinger CA,Wu Y,et al.CHIP is a U-box-dependent E3 ubiquitin ligase:identification of Hsc70 as a target for ubiquitylation[J].J Biol Chem,2001,276(46):42938-42944.
23.Connell P,Ballinger CA,Jiang J,et al.The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins[J].Nat Cell Biol,2001,3(1):93-96.
24.Dai Q,Zhang C,Wu Y,et al.CHIP activates HSF1 and confers protection against apoptosis and cellular stress[J].EMBO J,2003,22(20):5446-5458.
25.Dai Q,Qian SB,Li HH,et al.Regulation of the cytoplasmic quality control protein degradation pathway by BAG2[J].J Biol Chem,2005,280(46):38673-38681.
26.Urushitani M,Kurisu J,Tateno M,et al.CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70[J].J Neurochem,2004,90(1):231-244.
27.Ballinger CA,Connell P,Wu Y,et al.Identification of CHIP,a novel tetratricopeptide repeatcontaining protein that interacts with heat shock proteins and negatively regulates chaperone functions[J].Mol.Cell.Biol,1999,19(6):4535-4545.
28.Hatakeyama S,Yada M,Matsumoto M,et al.U box proteins as a new family of ubiquitin-protein ligases[J].J Biol Chem,2001,276(35):33111-33120.
29.Zheng N,Schulman BA,Song L,et al.Structure of the Cull-Rbxl-Skpl-F boxSkp2 SCF ubiquitin ligase complex[J].Nature,2002,416(6882):703-709.
30.Connell P,Ballinger CA,Jiang J,et al.The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins[J].Nat.Cell Biol,2001,3(1):93-96.
31.Shimura H,Schwart D,Gygi SP,et al.CHIP-Hsc70 complex uiquitinates phosphorylated tau and enhances cell survival[J].J Biol Chem.2004,279(6): 4869-4876.
32. Figueiredo-Pereira ME, Cohen G. Disruption of the intracelular sulthydryl homeostasis by cadmium-induced oxidative stress leads to protein thiolation and ubiquination in neuronal cells[J]. J Biol Chem, 1998,273(21): 12703-12709.
33. Lee MH, Hyun DH, Jenner P. Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production[J]. J Neurochem, 2001, 78(1): 32-41.
34. Mizushima N. Collaboration of proteolytic systems[J]. Autophagy, 2007, 3(3): 179-80.
35. Zhang JY, Peng C, Shi H, et al. Inhibition of autophagy causes tau proteolysis by activating calpain in rat brain[J]. J Alzheimers Dis, 2009,16(1):39-47.
36. Ding WX, Ni HM, Gao W, et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability[J]. Am J Pathol, 2007,171 (2):513-524.
37. Nedelsky NB, Todd PK, Taylor JP. Autophagy and the ubiquitin-proteasome system: collaborators in neuroprotection[J]. Biochim Biophys Acta, 2008, 1782(12): 691-699.
38. Komatsu M, Wang QJ, Holstein GR, et al. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration[J]. Proc Natl Acad Sci USA, 2007,104(36): 14489-14494.
39. Levine B, Kroemer G Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132(1): 27-42.
40. Posmantur RM, Zhao X, Kampfl A, et al. Immunoblot analyses of the relative contributions of cysteine and aspartic proteases to neurofilament breakdown products following experimental brain injury in rats[J]. Neurochem Res, 1998, 23(10): 1265-1276.
41. Weissman AM. Themes and variations on ubiquitylation[J]. Nat. Rev. Mol. Cell Biol, 2001,2(3): 169-178.
42. Gao M, Karin M. Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli[J]. Mol Cell, 2005, 19(5): 581-593.