Received 2002-04-09  Accepted 2002-04-29

  This work was supported by grants from National Natural Science Foundation of China (No.39825109) and National Key Project of Basic Science Research (No.G1999054007). 

  *Corresponding author. Tel:  +86-21-64041900-2774; E-mail: fysun@shmu.edu.cn生理学报, Aug. 2002, 54  (4): 287～293 

Acta Physiologica Sinica

 研究论文 

岗田酸诱导大鼠脑神经细胞表达谷氨酸转运体EAAT1

魏建设, 张玲妹, 黄娅琳, 朱粹青, 孙凤艳*

复旦大学上海医学院医学神经生物学国家重点实验室,  上海 200032

 

摘 要:   为研究tau蛋白高度磷酸化与谷氨酸转运体功能之间的关系, 实验采用免疫组织化学、荧光双标记技术及大鼠额叶皮质定位注射的方法, 观察了蛋白磷酸酶抑制剂岗田酸(okadaic acid, OA)所致神经细胞退化对谷氨酸转运体亚型EAAT1表达的影响。结果如下: (1) 在OA注射中心区神经元早期出现胞体固缩、肿胀、核移位, 在注射3 d时细胞破碎, 发生坏死, 并有大量炎性细胞浸润等病理现象; 边周区细胞呈AT8(微管相关蛋白tau磷酸化指标)免疫阳性反应; (2) OA首先诱导神经细胞突起远端tau蛋白磷酸化, 并逐渐向胞体发展, 形成营养不良的神经细胞突起和神经纤维缠结样病理改变; (3) AT8免疫阳性反应脑区的神经细胞高表达谷氨酸转运体EAAT1, 在12 h阳性表达细胞数显著增多(P<0.01), 1 d时达峰值(P<0.001), 3 d时明显减少。在OA作用下EAAT1表达于星形胶质细胞和神经元。结果提示, OA致微管相关蛋白tau高度磷酸化时可诱导该区星形胶质细胞和神经元高表达谷氨酸转运体EAAT1。EAAT1高表达的病理生理意义有待进一步的阐明。

 

关键词: 岗田酸; tau蛋白; 磷酸化; 神经纤维缠结; 谷氨酸转运体EAAT1

学科分类号:  Q421; R338; R741.02

 

Okadaic acid induces the expression of glutamate transporter EAAT1 in the neurons of rat brain

WEI Jian-She, ZHANG Ling-Mei, HUANG Ya-Lin, ZHU Cui-Qing, SUN Feng-Yan*State Key Laboratory of Medical Neurobiology,  Shanghai Medical College,  Fudan University,  Shanghai  200032

 

Abstract:   To study the relationship between tau hyperphosphorylation and the function of glutamate transporter  okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5  μl volume,  was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression  of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling,   nuclear pyknosis, and dislocation at the early stage, and  necrosis appeared  3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters   with immuno-positive reation for  AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the  cell body at 1 d,  which brought about   dystrophic neurites and neurofibrillary tangle  (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the   mechanism and significance of the induction of glutamate transporter EAAT1 expression remain  to be further elucidated.

 

Key words: okadaic acid; tau protein; phosphorylation; neurofibrillary tangle (NFT); excitatory amino acid transporter 1 (EAAT1)

 

谷氨酸是中枢神经系统内重要的兴奋性神经递质, 其在突触快速传递, 学习记忆以及神经系统发育过程中起着十分重要的作用。正常情况下胞外谷氨酸含量维持在1 μmol/L以下, 浓度过高则通过激活谷氨酸受体, 介导兴奋毒性作用, 参与中枢神经系统损伤和神经退行性疾病的发病机制。谷氨酸能神经传递的终止依赖于谷氨酸与其转运体结合并被摄入胞体。在神经系统中存在5种Na+/K+依赖性高亲和谷氨酸转运体EAAT1-EAAT5, 它们具有不同的脑区分布, 细胞定位, 机能特性和发育特征[1]。谷氨酸转运体的功能和表达异常可见于中风、 脑缺血[2-5]、 癫痫以及阿尔茨海默病(AD)[6,7]、Huntinton舞蹈病(HD)和肌萎缩性脊髓侧索硬化(ALS)等。根据谷氨酸转运体的Km值大小推测, 胶质细胞型EAAT1(GLAST)可能在限制胞外谷氨酸浓度过度升高, 抑制病理条件下神经损伤等方面具有重要的作用[8]。

岗田酸(okadaic acid, OA)是从Halichondria okadai提取的聚醚类单羧酸化合物(C44H66O13, Mr 802)[9]。实验表明, OA是蛋白磷酸酶(protein phosphatase, PP)1和2A的抑制剂[11], 能使神经元微管相关蛋白(MAP) tau磷酸化[12-15], 产生自由基造成氧化损伤[13], 此外也可提高神经细胞突触体谷氨酸基础释放量[10]; 在整体还能造成Aβ聚积, 记忆障碍, 产生类AD样病理特征[16,17], 从而使神经细胞退化。我们在脑缺血模型上观察到缺血致神经细胞损伤时谷氨酸转运体EAAT1高表达, 其活性呈代偿性增加反应[3-5], 然而在OA致神经元类AD样tau高度磷酸化的情况下是否也有相应的反应目前尚不清楚。本课题拟用免疫组织化学和荧光双标记技术, 在大鼠额叶皮质定位注射OA, 探讨其对大鼠神经细胞退化和谷氨酸转运体亚型EAAT1表达的影响。

 

1 材料和方法

1.1  额叶皮质定位注射OA  SD雄性大鼠, 200-250 g, 由中国科学院上海实验动物中心提供。大鼠在水合氯醛(360 mg/kg, i.p.)麻醉下固定于脑立体定位仪(Narishige, Japan)。颅顶正中切开, 根据大鼠颅脑定位图谱, 对右侧额叶皮质立体定位(A 1.20 mm, L 1.90 mm, H 2.20 mm), 用牙科钻钻开颅骨, 用1 μl微量注射器取溶于0.9%生理盐水的OA (Sigma, USA; pH  7.4) 0.5 μl (浓度40 ng/μl)于10 min内缓注在注射位点, 留针5 min。对照组施以等剂量生理盐水。

1.2  脑组织切片的制备  不同组别大鼠在OA注射后3、6、12 h及1、3、7 d (n=4)灌注取脑。在水合氯醛(360 mg/kg, i.p.)麻醉下, 经左心室快速灌流150 ml 0.9%生理盐水, 缓慢滴注300 ml 4%中性多聚甲醛(0.1 mol/L PB, pH 7.4); 后固定脑组织8 h, 梯度蔗糖液脱水至下沉。冠状冰冻切片, 厚度30 μm, 入保护液－20℃保存。

1.3  AT8 和EAAT1免疫组织化学  脑片(Bregma 1.20 mm)经常规固定, 漂洗后用10%羊血清孵育30 min, 再用鼠抗人AT8 (PHFs-tau)抗体(Innogenetics, Belgium; 1:60) 37℃孵育2 h, 4℃为48 h。漂洗后用生物素化羊抗鼠IgG(Vector, USA; 1:200) 37℃孵育1 h,按常规ABC法(ABC kit, Vector, USA; 1:200)反应检测, 行DAB (Sigma, USA)显色反应, 常规封片。阴性对照则用抗体稀释液替代一抗。EAAT1 (Novocastra, UK; 1:80)免疫组化染色方法同上。用光学显微镜观察免疫阳性反应细胞, 在放大40倍下进行EAAT1阳性细胞计数, 记录每张切片上额叶皮质阳性细胞胞体数目的总和, 对所有计数的阳性神经细胞均不考虑其染色深浅度。

1.4  免疫荧光双色标记  脑组织切片按常规免疫组织化学固定、 漂洗, 与抗体EAAT1孵育后, 将脑片与rhodamine结合的抗鼠IgG (Roche Molecular Biochemical, USA; 1:20)在37℃孵育1 h; 漂洗后用兔抗牛胶质纤维酸性蛋白(GFAP)抗体(DAKO, Denmark; 1:100)在37℃孵育1 h, 4℃为24 h, 漂洗后用异硫氰酸荧光黄(FITC)结合的抗兔IgG(DAKO, Denmark; 1:20)在37℃孵育1 h, 洗脱后用1:1甘油PBS封片, 然后用TCS-NT激光共聚焦扫描显微镜(Leica, Germany)观察。EAAT1与神经元特异的烯醇化酶(NSE)免疫双标方法同上,兔抗NSE抗体(ICN, USA)浓度为1:40。

1.5  统计分析  所有数据均用mean±SE表示, 以方差分析、组间t检验作统计学处理, P<0.05为差别有显著性统计意义。

 

2 结果

2.1  OA所致神经元损伤的形态学变化

焦油紫染色结果显示于额叶皮质注射OA 20 ng后,  在注射中心区6 h时神经元出现胞体固缩; 12 h神经元固缩与肿胀并存; 1 d神经元肿胀, 核浓缩移位, 出现破碎细胞; 3 d时细胞严重受损, 出现坏死, 而边周区细胞极不规则, 且有大量炎性细胞浸润(图1, 见p.290)。

2.2  OA对tau蛋白磷酸化的影响

AT8免疫组织化学染色结果显示, 在OA注射后6 h注射区的额叶皮质出现AT8免疫阳性染色的神经细胞突起, 呈丝线状排列, 可见着色较淡的胞体与其相连; 1 d时在OA作用脑区出现大量胞体深染的神经细胞, 阳性颗粒物质成束聚集于整个胞浆, 细胞突起较为粗大, 呈现典型的tau蛋白磷酸化的细胞形态变化; 3 d时此类细胞突起缩短乃至消失, 胞浆着色变浅, 而膜周仍深染(图 2)。该结果提示神经细胞在OA作用早期即出现营养不良的细胞突起, 且该突起远端先出现tau磷酸化, 形成螺旋细丝PHFs (paired helical filaments), 并逐渐发展到胞体。

图 2. 额叶皮质注射OA对神经细胞AT8和EAAT1表达的影响

Fig. 2. Photomicrographs showing the immunostaining feature for AT8 (left) and EAAT1 (right) in the peri-injected areas of frontal cortex after 20 ng OA injection. Arrowheads in the photographs indicate that AT8 immunostaining appear in distal neurites at 6 h and accumulated in cell bodies along with single axon-like neurites at 1 d. Scale bar, 30 μm.

图 1.  额叶皮质注射OA对神经元形态学的影响

Fig. 1. Photomicrographs (cresyl violet staining) showing the morphological changes in the ipsilateral frontal cortex after 20 ng OA injection. The arrows in the low power photomicrographs (A-E) correspond to those in the high power photomicrographs (a-e). In vehicle rats (A  a) the neurons presented regular morphological characteristics. At 6 h (B b) after OA injection the neurons with injury displayed cytoplasmic shrinkage. At 12 h (C c) the neurons showed cytoplasmic shrinkage or swelling. At 1 d (D  d) the neurons displayed cytoplasmic swelling and nuclear pyknosis, dislocation. At 3 d (E  e)  more neurons showed necrosis in the center of injection region, but displayed greater morphological changes and inflammatory infiltration in the peri-injected areas. Scale bars, 200 μm (A), 30 μm (a).

 

2.3  OA对神经细胞谷氨酸转运体亚型EAAT1表达的影响

EAAT1免疫组织化学染色结果显示, 对照组大鼠额叶皮质EAAT1阳性细胞表达较少。注射OA后, 在注射区周边额叶皮质尤其是锥体细胞层表达EAAT1的神经细胞逐渐增多。从形态学观察到OA作用后6 h可见EAAT1免疫阳性的神经细胞突起呈细丝状, 胞膜有点状深染; 1 d时锥体细胞层表达EAAT1的神经细胞明显增多, 胞浆淡染或不着色, 而胞膜周围着色加深。与AT8相比, 形态学有明显不同, EAAT1阳性免疫反应着色部位始终位于突起及细胞膜周围(图2)。EAAT1阳性胞体计数显示, 在OA作用下3 h (47.8±6.7/slice)、12 h (95.8±7.9/slice)和1 d (296.0±20.6/slice)时, EAAT1的表达数量与对照相比有显著性差异(P<0.01), 3 d后表达阳性细胞数明显减少(图3)。免疫荧光双标记分析显示, 在OA作用下EAAT1分别与GFAP及NSE免疫反应于同一细胞。由此提示, 在OA诱导下EAAT1不仅在星形胶质细胞表达, 也在神经元表达(图4)。

图 3. 额叶皮质注射OA后不同时刻EAAT1阳性细胞的表达

Fig. 3. Time-course of EAAT1 expression in ipsilateral frontal cortex after 20 ng OA injection. Number of EAAT1 expressed cells was shown as the sum of positive cells per slice (n=4). Values are mean±SE. *P<0.01, **P<0.001 vs vehicle; #P<0.01 vs 1 d. Note that the number of EAAT1-like immunopositive cells in frontal cortex time-dependently increased   after 20 ng OA injection.

图 4. 额叶皮质注射OA后表达EAAT1的阳性细胞

Fig. 4. Immunofluorescent double staining showed cellular localization of EAAT1 expression (red) in the peri-injected areas of frontal cortex after 20 ng OA injection at 1 d. GFAP and NSE (green) were used as markers of astrocytes and neurons respectively. Overlapping of red and green color produced yellow color. Scale bar, 20 μm.

 

3 讨论

蛋白磷酸酶(PP)分为PP1和PP2A、2B、2C, 它们在神经元内均有分布。OA主要抑制PP2A和PP1, 而不抑制PP2C[11]。 正常胞内蛋白磷酸酶和磷酸激酶处于动态平衡之中,OA通过抑制蛋白磷酸酶使激酶处于相对高活性状态, 从而使蛋白质磷酸化增加。神经元内微管相关蛋白tau正常时位于轴突内, 成熟脑极少磷酸化。编码tau蛋白的基因位于人17号染色体, 在发育过程中其mRNA翻译后修饰不同, 从而形成6种同功异构体。Tau蛋白磷酸化程度受激酶活性的影响, 其生物学活性的表达与多个位点的磷酸化作用有关。Tau异常高度磷酸化后在胞内聚积形成PHFs, 是AD神经元退化的较早病理特征之一。已有实验表明, PHFs-tau形成后能抵御蛋白水解酶对其水解, 使正常tau与微管蛋白和胞浆膜相脱离, 阻断tau与微管蛋白结合, 使其不能形成微管而丧失生物学功能。这在神经元退化过程中起重要作用[18,19]。

离体时OA使神经元tau磷酸化形成PHFs已被诸多实验所证实, 但在整体条件下OA的作用尚有疑义[20]。本实验采用能识别tau serine202和threonine205位点异常磷酸化的特异性抗体AT8 (PHF-tau)对PHFs进行检测, 结果表明额叶皮质注射OA能使神经元tau磷酸化形成PHFs, 同时观察到额叶神经细胞在OA作用下先在突起远端形成PHFs, 沿轴突向胞体发展, 形成致密聚集的早期神经纤维缠结, 诱导神经细胞退化。在OA作用3 d后AT8 阳性表达减弱可能与OA浓度降低, 有效PP活性增加, 以及早期NFTs高度聚集使抗原暴露不足有关[21]。

实验表明OA可提高神经细胞突触体谷氨酸基础释放量[10], 使胶质细胞谷氨酸转运体磷酸化增加谷氨酸的转运效率[22], 同时在诱导神经元tau磷酸化时也能产生过氧亚硝基阴离子(ONOO－)造成氧化损伤[13],而谷氨酸转运体极易受其影响使转运机能降低[23,24], 提示在OA诱导神经细胞退化过程中tau高度磷酸化和神经细胞氧化损伤均存在, 而谷氨酸转运体的功能活动在其中相当复杂。我们的结果表明额叶皮质注射20 ng OA后其药理作用范围和EAAT1阳性表达细胞仅局限于额叶皮质, 细胞计数则说明在OA作用下表达EAAT1的神经细胞数量随时间而变化, 12 h后显著增加, 1 d时达峰值, 3 d后减少。EAAT1随时间依赖性地上调表达与神经细胞tau磷酸化严重程度相关联, 且两者的分布区域也同位于注射区周边, 由此间接提示OA使神经细胞tau高度磷酸化时能诱导EAAT1表达上调, 细胞的退化可能与谷氨酸介导的兴奋毒性有关。免疫荧光双标结果发现在OA诱导的tau磷酸化脑区EAAT1分别与NSE和GFAP共存于同一细胞, 提示在该区域EAAT1被诱导表达于神经元和星形胶质细胞。由于EAAT1正常表达于胶质细胞, OA能诱导神经元表达EAAT1的原因可能与tau蛋白主要存在于神经元[25], OA通过抑制PP2A造成tau高度磷酸化诱导神经元损伤, 继而使神经元内EAAT1表达增加有关。这一现象与我们以往的缺血损伤诱导谷氨酸转运体在脑内神经细胞重分布的报道相一致[3-5]。然而, OA使tau高度磷酸化诱导EAAT1高表达后, 谷氨酸转运体EAAT1是促进递质摄取进入细胞参与再循环, 减少细胞兴奋毒性, 还是使更多谷氨酸反向转运入细胞间隙, 进一步加重神经细胞的损伤, 目前尚不清楚; 此外其高表达机制也有待进一步研究。综上所述, 本文结果提示OA能诱导大鼠额叶皮质神经细胞微管相关蛋白tau高度磷酸化, 同时使该脑区星形胶质细胞和神经元高表达谷氨酸转运体EAAT1; 谷氨酸转运体EAAT1的高表达参与OA诱导的神经细胞退化, 其病理生理机制及意义则有待进一步阐明。

*  Acknowledgements:  We wish to thank Dr. LI Xue-Jun and WEI Wen-Xiang for valuable discussions and suggestions.

 

参 考 文 献

[1] Danbolt NC. Glutamate uptake. Prog Neurobiol, 2001,65:1~105.

[2] Rossi DJ, Oshima T, Attwell D. Glutamate release in severe brain ischemia is mainly by reversed uptake. Nature, 2000, 403(6767):316-321.

[3] Yan YP, Yin KJ, Sun FY. Effect of glutamate transporter on neuronal damage induced by photochemical thrombotic brain ischemia. NeuroReport, 1998,9:441-446.

[4] Yin KJ, Yan YP, Sun FY. Altered expression of glutamate transporter GLAST mRNA in rat brain after photochemically induced focal ischemia. Anat Rec, 1998,251:9-14.

[5] Tao F, Lu SD, Zhang LM, Huang YL, Sun FY. Role of excitatory amino acid transporter 1 in neonatal rat neuronal damage induced by hypoxia-ischemia. Neuroscience, 2001,102(3):503-513.

[6] Li S, Mallory M, Alford M, Tanaka S, Masliah E. Glutamate transporter alterations in Alzheimer's disease are possibly associated with abnormal APP expression. J Neuropath Exp Neurol, 1997,56:901-911.

[7] Masliah E. Mechanisms of synaptic pathology in Alzheimer's disease. J Neurol Transm-Suppl, 1998,53:147-158.

[8] Kanai Y, Smith CP, Hediger MA. A new family of neurotransmitter transporters: the high affinity glutamate transporters. FASEB J, 1993,7:1450-1459.

[9] Tachibana K, Scheuer PJ, Tsuitani Y, Kikuchi H, Van Engen D, Clardy J, Gopichand Y, Schmitz FJ. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Halichondria. J Am Chem Soc, 1981,103:2469-2471.

[10] Sim AT, Lloyd HG, Jarvie PE, Morrison M, Rostas JA, Dunkley PR. Synaptosomal amino acid release: effect of inhibiting protein phosphatases with okadaic acid. Neurosci Lett, 1993,160:181-184.

[11] Bialojan C, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J, 1988,256:283-290.

[12] Kim D, Su J, Cotman CW. Sequence of neurodegeneration and accumulation of phosphorylated tau in cultured neurons after okadaic acid treatment. Brain Res, 1999,839:253-262.

[13] Kim D, Koh WK, Kim JU, Lee JH, Hong HN. Okadaic acid-induced upregulation of nitrotyrosine and heme oxygenase-1 in rat cortical neuron cultures. Neurosci Lett, 2001,297:33-36.

[14] Lee JH, Hong HN, Im JO, Byun H, Kim D. The formation of PHF-1 and SMI-31 positive dystrophic neurites in rat hippocampus following acute injection of okadaic acid. Neurosci Lett, 2000,282:49-52.

[15] Gong CX, Lidsky T, Wegiel J, Zuck L, Grundke-Iqbal I, Iqbal K. Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain-implications for neurofibrillary degeneration in Alzheimer's disease. J Biol Chem, 2000,275:5535-5544.

[16] Arendt T, Holzer M, Fruth R, Bruckner MK, Gartner U. Paired helical filament-like phosphorylation of tau, deposition of β/A4-amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A. Neuroscience, 1995,69:691-698.

[17] Arendt T, Holzer M, Fruth R, Bruckner MK, Gartner U. Phosphorylation of tau, Aβ-formation, and apoptosis after in vivo inhibition of PP-1 and PP-2A. Neurobiol Aging, 1998,19:3-13.

[18] Iqbal K, Alonso A del C, Gong CX, Khatoon S, Pei JJ, Wang JZ, Grundke-Iqbal I. Mechanisms of neurofibrillary degeneration and the formation of neurofibrillary tangles. J Neural Transm-Suppl, 1998,53:169-180.

[19] Ekinci FJ, Shea TB. Phosphorylation of tau alters its association with the plasm membrane. Cell Mol Neurobiol, 2000,20:497-508.

[20] Van Dam AM, Bol JG, Binnekade R, van-Muiswinkel FL. Acute or chronic administration of okadaic acid to rats induces brain damage rather than Alzheimer-like neuropathology. Neuroscience, 1998,85:1333-1335.

[21] Bancher C, Brunner C, Lassmann H, Budka H, Jellinger K, Wiche G, Seitelberger F, Grundke-Iqbal I, Iqbal K, Wisniewski HM. Accumulation of abnormally phosphorylated τ precedes the formation of neurofibrillary tangles in Alzheimer's disease. Brain Res, 1989,477:90-99.

[22] Daniels KK, Vickroy TW. Reversible activation of glutamate transport in rat brain glia by protein kinase C and an okadaic acid-sensitive phosphoprotein phosphatase. Neurochem Res, 1999,24:1017-1025.

[23] Trotti D, Danbolt NC, Volterra A. Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci, 1998,19:328-334.

[24] Lauderback CM, Hackett JM, Huang FF, Keller JN, Szweda LI, Markesbery WR, Butterfield DA. The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer's disease brain: the role of Aβ1-42. J Neurochem, 2001,78:413-416.

[25] Schultz C, Dehghani F, Hubbard GB, Thal DR, Struckhoff G, Braak E, Braak H. Filamentous tau pathology in nerve cells, astrocytes and oligodendrocytes of aged baboons. J Neuropathol Exp Neurol, 2000,59:39-52.