磷酸化C—JUN不参与谷氨酸诱导的小脑颗粒神经元凋亡

观察磷酸化 C- JUN与谷氨酸诱导的小脑颗粒神经元凋亡的关系。在培养的小脑颗粒神经元建立谷氨酸凋亡模型 ;采用 MTT法分析细胞存活率 ,相差显微镜观察形态学 ,DNA凝胶电泳法分析细胞凋亡和原位细胞荧光免疫组织化学法检测磷酸化 C- JUN。结果显示 ,谷氨酸诱导大鼠小脑颗粒神经元细胞体积缩小 ,突触断裂、消失 ,DNA电泳呈典型的“梯状”条带 ;谷氨酸处理 2 4 h后细胞存活率为 2 8.6%± 5.2 %。神经元在谷氨酸处理 5,30 min及 1 ,2 ,4,8,1 6和 2 4 h后均未检测到有磷酸化 C- JUN阳性细胞 ,与去极化组 ( 2 5mmol/L KCl)相同。而复极化组 ( 5mmol/L KCl)则在 30 min检测到大量的磷酸化 C- JUN阳性细胞 ,4h荧光最强并持续。处理 4h后 ,40 0倍荧光显微镜下 ,复极化组、去极化组和谷氨酸组的磷酸化 C- JUN阳性细胞数分别为 1 2 4± 1 7,8± 3,5± 3。上述结果提示 ,谷氨酸诱导小脑颗粒神经元凋亡 ,磷酸化 C- JUN不参与谷氨酸诱导的大鼠小脑颗粒神经元凋亡。     

高钾诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst33258 染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现：①高钾诱导神经元死亡呈剂量（50－100mmol/L）和时间依赖性;②神经元死亡呈现明显的凋亡特征：胞体缩小,染色质浓缩,DNA“梯形”条带形成和蛋白质合成抑制剂（cycloheximide,1.0mg/L)可阻断其毒性等;③MK－801（2μmol/L)、尼莫地平（10μmol/L）、硫酸镁（20μmol/L）可阻断高钾的大部分毒性作用。结果提示：高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗料神经元凋亡。     

高钾可诱导培养的小脑颗粒神经元凋亡

采用四唑盐比色、琼脂糖凝胶电泳、乙二酸荧光素染色和Hoechst332 58染色等方法研究高钾对原代培养的大鼠小脑颗粒神经元的毒性作用及其机制。结果发现 :①高钾诱导神经元死亡呈剂量 ( 50～ 10 0mmol/L)和时间依赖性 ;②神经元死亡呈现明显的凋亡特征 :胞体缩小 ,染色质浓缩 ,DNA“梯形”条带形成和蛋白质合成抑制剂 (cycloheximide ,1.0mg/L)可阻断其毒性等 ;③MK 80 1( 2 μmol/L)、尼莫地平 ( 10 μmol/L)、硫酸镁 ( 2 0mmol/L)可阻断高钾的大部分毒性作用。结果提示 :高钾可能通过刺激内源性谷氨酸释放从而诱导小脑颗粒神经元凋亡     

尼莫地平诱导小脑颗粒神经元凋亡及机制研究

目的探讨尼莫地平能否诱导小脑颗粒神经元凋亡及可能机制。方法取Sprague-Dawley(SD)大鼠小脑颗粒神经元,体外培养7d,用含10μM尼莫地平培养基处理神经元24h,对照组为含1‰二甲亚砜(DMSO)培养基处理细胞。Hoechst33258染胞核检测凋亡率。Westernblot检测c-Fos和c-Jun蛋白的表达水平;腺病毒作载体过表达c-Fos和负显性c-Jun突变体阻断c-Jun功能,检测能否抑制尼莫地平诱导的神经元凋亡,免疫细胞化学方法检测腺病毒感染率。结果对照组神经元的凋亡率为(10±4)%,尼莫地平处理6、12、24h后神经元的凋亡率分别为(13±4)%、(28±5)%、(45±3)%。尼莫地平处理使c-Fos表达下调,但c-Jun表达水平上调。腺病毒Ad-c-Fos和Ad-c-JunDN(负显性c-Jun突变体)对神经元感染率为85%以上,过表达c-Fos或负显性c-Jun突变体使尼莫地平诱导的神经元凋亡率从(42±6)%减少到(28±6)%或(20±3)%。结论尼莫地平通过下调c-Fos和上调c-Jun表达诱导小脑颗粒神经元凋亡。     

一氧化氮及谷氨酸在内皮素-1诱导神经元凋亡中的作用

目的　观察一氧化氮 (NO)和谷氨酸在内皮素 (ET) 1诱导培养神经元凋亡中的作用。方法　神经元培养取自新生SD大鼠大脑皮质。培养 5天后分 4组 :对照组、ET 1组 (2 0nM)、ET 1 L NAME(N 硝基左旋精氨酸甲酯 ,NO合酶抑制剂 ,1 0 0mM)组和ET 1 APV组 (N 甲基 D 天冬氨酸型受体拮抗剂 ,1 0 0 μM)。培养 2 4h后 ,收集细胞用流式细胞仪定量检测凋亡率。上清液中NO水平通过硝酸还原酶法检测亚硝酸盐浓度反映 ,谷氨酸浓度测定用高压液相法。结果　 2 0nMET 1处理后 2 4h,培养神经元凋亡率较对照组显著增高 (P <0 0 0 1 )。L NAME和APV分别明显阻断ET 1诱导神经元凋亡的作用 ,与ET 1组比较 ,凋亡率降幅分别为 40 % (P<0 0 5)和 80 % (P <0 0 0 1 )。ET 1作用 2 4h后。神经元培养液中NO和谷氨酸浓度较对照组显著增高 (P <0 0 0 1 ) ,L NAME完全抑制了ET 1引起的培养液中NO的升高。结论　NO和谷氨酸参与了ET 1诱导培养大鼠大脑神经元凋亡过程 ,其中谷氨酸更为重要     

刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用

目的观察神经元在谷氨酸毒性损伤时一氧化氮(NO)的动态变化及其与凋亡的关系,探讨刺五加皂甙(ASS)的有效保护浓度。方法采用谷氨酸(Glu)诱导的皮质神经元凋亡模型。随机分成Glu组、正常对照组及ASS3组;用流式细胞仪检测神经元凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT法测定神经元存活率并在电镜下观察细胞形态学变化。结果(1)Glu呈剂量和时间依赖性增加神经元培养液中NO含量,ASS能不同程度地减少NO含量;(2)与Glu共培养的神经元,其存活率呈剂量和时间依赖性下降,ASS能增加神经元存活率;(3)经Glu处理的神经元发生凋亡,细胞超微结构呈现凋亡样改变,其凋亡率与正常对照组比较有显著性差异(P<0.01)。ASS能减少Glu毒性神经元凋亡。结论NO介导了Glu毒性神经元凋亡,ASS可能通过抑制NO的释放及其神经毒性作用,拮抗Glu引起的神经元凋亡。     

糖氧剥夺诱导神经元磷酸化Rb蛋白表达特点及其与凋亡关系研究

目的观察糖氧剥夺(OGD)诱导体外培养的皮层神经元细胞内磷酸化Rb蛋白(p-Rb)的表达特点并探讨其与神经元凋亡的关系。方法将体外培养的皮层神经元随机分为对照组、OGD1 h后恢复糖氧供给(OGD/R)6 h、12 h和24 h组。应用免疫荧光细胞化学染色观察神经元p-Rb表达特点及其与TUNEL染色的关系。结果 p-Rb在OGD/R各组神经元的表达率较对照组明显增高,主要在神经元细胞浆内强表达,OGD/R6h组p-Rb与TUNEL染色共定位细胞比例开始增高,12 h达最高,与对照组比较差异明显(P<0.01)。OGD/R24 h组很少观察到两者共定位染色。结论 Rb磷酸化介导的神经元细胞周期紊乱可能是缺血缺氧诱导的神经元早期凋亡机制之一。     

20008/神经生长因子通过PTEN保护谷氨酸诱导的海马神经元凋亡

目的：探讨NGF保护谷氨酸（Glu）诱导海马神经元凋亡中PTEN的作用。方法：新生（< 24 hr）大鼠海马神经元培养7天后,随机分为对照组、谷氨酸组（0.2 mM）和NGF组（10、50、100、200μg•L -1）采用LDH和MTT法检测NGF对Glu损伤细胞活力的影响,采用DAPI染色法检测海马神经元凋亡,采用RT-PCR法和Western blot法测定PTEN mRNA及蛋白表达。结果：NGF可明显提高Glu损伤的海马神经元的细胞活力,减少海马神经元的凋亡和PTEN的表达。结论：NGF对Glu诱导的海马神经元损伤有保护作用,其机制可能与减少PTEN表达,抑制神经元凋亡有关。     

磷酸化蛋白酪氨酸激酶及信号转导子和转录激活子在谷氨酸诱导的PC12细胞凋亡中的表达研究

目的探讨谷氨酸诱导PC12细胞凋亡后磷酸化蛋白酪氨酸激酶2（P-JAK2）、磷酸化信号转导子与转录激活子3（P-STAT3）表达的变化及意义。方法采用谷氨酸诱导PC12细胞发生凋亡，实验分为6组，分别为正常对照组、500μmol／L谷氨酸作用5、10、20、30、60min组，应用流式细胞仪观察PC12细胞凋亡．Western blot定量观测PC12细胞P-JAK2与P-STAT3蛋白表达的变化。结果对照组PC12细胞的凋亡率为（2．71±0．32）％；经500μmol／L谷氨酸作用PC12细胞20min，凋亡率增加到（61．20±4．60）％，与对照组相比差异有显著性意义（P=0．000）；Westem blot检测结果表明P-JAK 25 min在胞浆中开始表达，20min达最高峰，是5min组（3．52±0．20）倍（P=0．002）；P-STAT35min表达增强，30min达高峰，为5min组的（4．76±0．17）倍（P=0．000）。结论细胞损伤激活了JAK／STAT信号转导通路，该通路参与了神经细胞凋亡的过程。     

Neurotrophin-elicited short-term glutamate release from cultured cerebellar granule neurons

Brain-derived neurotrophic factor (BDNF) has been suggested to play an important role in neuronal plasticity. In this study, we investigated the effects of BDNF on short-term transmitter release from cultured CNS neurons. Rapid and transient glutamate and aspartate releases induced by BDNF were observed from cultured cortical, hippocampal, striatal and cerebellar neurons. We furthermore investigated the mechanism of release induced by neurotrophins from cerebellar granule cells, since granule cells represent a large homogeneous glutamatergic population. NGF and NT-3 elicited neurotrophin-induced release of glutamate as well as BDNF from the cerebellar granule neurons. The release was dependent on intracellular Ca²⁺ mobilization. Pretreatment with K252a and also TrkB-IgG completely blocked the glutamate and aspartate release elicited by BDNF, but not by NGF. The cerebellar granule neurons expressed trkB and p75 mRNAs at high levels, but not trkA mRNA. These results suggested that while BDNF induced release via TrkB, NGF-elicited release was not mediated by Trks. Furthermore, in the experiment using the styryl dye FM1–43, which selectively labels synaptic vesicles, neither BDNF nor NGF evoked dye loss, suggesting that neurotrophin-induced excitatory amino acid release occurs through a non-exocytotic pathway.     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells

Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and by reduced extracellular Cl- ion concentration. In addition, DIDS and low extracellular Cl- completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl(-)-regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Cesium chloride protects cerebellar granule neurons from apoptosis induced by low potassium

Neuronal apoptosis plays a critical role in the pathogenesis of neurodegenerative disorders, and neuroprotective agents targeting apoptotic signaling could have therapeutic use. Here we report that cesium chloride, an alternative medicine in treating radiological poison and cancer, has neuroprotective actions. Serum and potassium deprivation induced cerebellar granule neurons to undergo apoptosis, which correlated with the activation of caspase-3. Cesium prevented both the activation of caspase-3 and neuronal apoptosis in a dose-dependent manner. Cesium at 8 mM increased the survival of neurons from 45 +/- 3% to 91 +/- 5% of control. Cesium's neuroprotection was not mediated by PI3/Akt or MAPK signaling pathways, since it was unable to activate either Akt or MAPK by phosphorylation. In addition, specific inhibitors of PI3 kinase and MAP kinase did not block cesium's neuroprotective effects. On the other hand, cesium inactivated GSK3beta by phosphorylation of serine-9 and GSK3beta-specific inhibitor SB415286 prevented neuronal apoptosis. These data indicate that cesium's neuroprotection is likely via inactivating GSK3beta. Furthermore, cesium also prevented H(2)O(2)-induced neuronal death (increased the survival of neurons from 72 +/- 4% to 89 +/- 3% of control). Given its relative safety and good penetration of the brain blood barrier, our findings support the potential therapeutic use of cesium in neurodegenerative diseases.     

AMPA prevents glutamate-induced neurotoxicity and apoptosis in cultured cerebellar granule cell neurons

Exposure of cultured cerebellar neurons to ±-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the presence of aniracetam protects all of the vulnerable neurons against the excitotoxic actions of glutamate acting on N-methyl-D-aspartate receptors. The protective effect of AMPA was both time- and concentration-dependent. Aniracetam alone did not protect the neurons against the excitotoxic effects of glutamate. Pretreatment of cerebellar neurons with the AMPA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione blocked the neuroprotective effect mediated by AMPA indicating that the neuroprotective effect is mediated specifically by AMPA receptors. An excitotoxic concentration of glutamate, which killed between 60–80% of granule cell neurons on day 8in vitro, mediated its toxic effect via a time-dependent apoptotic pathway. Pretreatment of cerebellar granule cell neurons with AMPA (500 μM) completely blocked glutamate-mediated apoptosis. Our results suggest that AMPA receptors may play an important role in neuronal survival.     

Depolarization-dependent survival of cultured mouse cerebellar granule neurons is strain-restrained

Cerebellar granule neurons (CGNs) isolated from the rat are often used as a model system for the analysis of activity-dependent survival of neurons. These cells do not survive in culture without addition of a depolarizing agent (KCl or glutamate) to the medium. However, it has been reported that mouse CGN behave differently. Here we found that the requirement for depolarization for the survival of the mouse CGN was strain-dependent, which may be important for future analyses using transgenic animals. CGNs from the Balb/C mouse could survive without KCl addition, whereas CGNs from the C57Bl/6 mouse could not (similar to the rat CGN). The survival-promoting activity of the Balb/C mouse CGNs was transferable by coculturing. However, the medium of the Balb/C mouse CGN culture was ineffective, suggesting that the neurotrophic substance that is supposed to be released is labile. Although we are yet to specify the substance, it might be independent of neurotrophins. It is necessary to select the strain of mouse in the production of transgenic animals for the analyses of activity-dependent neuronal survival.     

Cell volume regulation in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons exposed to solutions of reduced osmolarity, responded initially by swelling followed by a regulatory volume decrease (RVD) which is completed within 15 min. Increasing external osmolarity lead to cell shrinking but no evidence of volume regulation was observed within 1 hr. Replacing Na⁺ by choline did not affect RVD whereas N-methyl-D-glucamine accelerated the volume recovery and K⁺ suppressed it completely. The blockade of RVD in high extracellular K⁺ was only observed when chloride and nitrate but not sulfate or gluconate were the accompanying anions. Replacing intracellular Cl^?, by long incubations with gluconate, markedly inhibited RVD. Removal of extracellular Ca²⁺ or addition of dantrolene which blocks Ca²⁺ released from intracellular stores had no effect on RVD. Increasing extracellular taurine prevented RVD. These results indicate that membrane permeability to K⁺, Cl^?, and taurine is increased by hyposmolarity and suggest the involvement of these molecules in RVD in granule neurons. © 1993 Wiley-Liss, Inc.