17β雌二醇对氯胺酮诱导发育期大鼠皮质神经细胞凋亡的影响

目的探讨17β雌二醇对氯胺酮诱导发育期大鼠额叶皮质区神经细胞凋亡的影响以及机制。方法30只7日龄雄性SD幼鼠,随机分为对照组(C组)、氯胺酮组(K组)、氯胺酮+17β雌二醇组(K+E组),每组10只。C组连续3 d腹腔注射等容量生理盐水;K组连续3 d腹腔注射75 mg/kg氯胺酮;K+E组连续3 d腹腔注射75mg/kg氯胺酮同时皮下注射600μg/kg 17β雌二醇。末次注药后24 h,取额叶皮质应用TUNEL法检测神经细胞凋亡,同时应用Western-blot法检测bcl-2,Bax以及cleaved-caspase-3蛋白的水平。结果与对照组比较,氯胺酮组皮质区凋亡细胞显著性增加(P<0.01),Bcl-2蛋白水平显著性下降(P<0.01),Bax蛋白水平显著性增加(P<0.01),Bcl-2/Bax显著性降低(P<0.01),cleaved-caspase-3蛋白水平显著性增加(P<0.01)。与氯胺酮组比较,氯胺酮+17β雌二醇组皮质区凋亡细胞显著性下降(P<0.01),Bcl-2蛋白水平显著性增加(P<0.01),Bax蛋白水平显著下降(P<0.01),Bcl-2/Bax显著性增加(P<0.01),cleaved-caspase-3蛋白水平显著性下降(P<0.01)。结论 17β雌二醇可对氯胺酮诱导的发育期大鼠大脑皮质区神经细胞凋亡产生保护作用,其机制可能与影响bc1-2和Bax蛋白表达有关。     

氯胺酮对NMDA诱导大鼠脊髓背角星形胶质细胞损伤的保护作用

目的观察氯胺酮对N-甲基-D-天冬氨酸(NMDA)受体过度激活诱导大鼠脊髓背角星形胶质细胞凋亡影响,并探讨其可能的作用机制.方法取新生2～3 d wistar大鼠T11-L6脊髓背角星形胶质细胞原代纯化培养,GFAP鉴定星形胶质细胞纯度达98%后用于实验.将细胞随机分6组:对照组(C组),NMDA组(N组),氯胺酮组(K组)和三种不同浓度氯胺酮加NMDA组(0.1,0.5,1 mmol/L,标记为NK1～NK3组),再培养24 h后检测SOD活性和MDA含量,免疫组化HE复染观察Bcl-2蛋白和形态学变化,流式细胞仪检测星形胶质细胞凋亡率.结果N组细胞发生了大量凋亡,SOD活性显著降低,MDA含量明显增加,Bcl-2蛋白表达不明显;NK3组细胞凋亡被显著抑制,Bcl-2蛋白强阳性表达,SOD活性明显增加和MDA含量低.结论NMDA受体过度激活可诱导大鼠脊髓背角星形胶质细胞大量凋亡,适量氯胺酮显著抑制了细胞凋亡,其机制可能是增强了星形胶质细胞Bcl-2蛋白表达,同时抑制了自由基的产生和增强了SOD活性.     

Aβ诱导骨髓间充质干细胞对其分化的神经细胞Tau蛋白过度磷酸化的影响

目的研究β-淀粉样蛋白25-35(Aβ25-35)对大鼠骨髓间充质干细胞(BMSCs)分化的神经细胞Tau蛋白磷酸化的影响。方法体外分离纯化大鼠骨髓MSCs,将P4 MSCs分为两组:(1)Aβ25-35实验组中加入预诱导培养基(含10μg/L bFGF,10%FBS的DMEM)和20μmol/L Aβ25-35,24h后用含2%DMSO和200μmol/L BHA的DMEM诱导其向神经细胞分化,5h后收取细胞;(2)对照组诱导方法同Aβ实验组,但在诱导过程中未加入Aβ25-35。光镜下观察细胞形态变化,免疫细胞化学法检测神经神经元特异性烯醇化酶(NSE),western blotting检测GSK-3β、Tau[pSer262]和Tau[pSer396]。结果光镜下可见纺锤状的MSCs诱导后出现长突起,呈现神经细胞样形态,但Aβ25-35实验组突起数量和长度均少于对照组;免疫细胞化学法检测两组诱导后的神经元样细胞为NSE阳性;免疫蛋白印记法证明Aβ25-35实验组的GSK-3β、Tau[pSer262]和Tau[pSer396]表达均显著高于对照组。结论 Aβ25-35能够通过GSK-3β途径诱导骨髓间充质...     

异甘草素对SHG44人脑胶质瘤干细胞增殖和分化的影响

目的探讨不同浓度异甘草素对SHG44人脑胶质瘤干细胞增殖和分化的影响及机制。方法实验分为二甲基亚砜(dimethyl sulfoxide,DMSO)对照组,异甘草素(10~160μmol/L)诱导组,氮-[氮-(3,5-二氟苯乙酰)-L-丙氨酰]-S-苯基甘氨酸丁酯(N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-ph,DAPT)(2.0μmol/L)阻断剂组,异甘草素+阻断剂组(10~160μmol/L+2.0μmol/L DAPT),采用CCK-8法、免疫荧光染色、Western blot及Real-time PCR分别检测细胞抑制率、相关分化蛋白及Notch1通路相关基因表达情况。结果异甘草素在12~48 h,随着浓度增加,细胞抑制率减弱(P0.05),且分化细胞越多,干细胞减少;72 h后随着浓度增加,细胞抑制率增强(P0.05),分化细胞及干细胞同时减少;隔日加药至第7 d时,经统计分析胶质瘤干细胞球数目减少、直径减小(与对照组比),且P0.05。异甘草素作用72 h后:与对照组比较,随着异甘草素浓度的增加Nestin蛋白表达量逐渐下调(P0.05);与对照组比较,10、40、160μmol/L组GFAP蛋白表达水平均上调(P0.05),且40μmol/L组GFAP蛋白表达量较其他浓度组均较高,(P0.05);与对照组比较,10、40、160μmol/L组β-TubulinⅢ蛋白表达水平均上调(P0.05),且10μmol/L组β-TubulinⅢ蛋白表达量较其他浓度组均较高(P0.05)。Notch1通路阻断剂作用后,与对照组比较,各异甘草素组和阻断剂组Notch1、RBP-JK及Hes1基因表达均显著下调(P0.05);与异甘草素组比较,Notch1、RBP-JK及Hes1基因表达在异甘草素加DAPT组及阻断剂组显著下调(P0.05);与阻断剂组比较,Notch1、RBP-JK及Hes1基因表达在异甘草素加DAPT组显著下调(P0.05)。结论异甘草素能诱导SHG44人脑胶质瘤干细胞向星形胶质细胞和神经元细胞分化,且能抑制其增殖,可能与下调Notch1信号通路中的Notch1、RBP-JK及Hes1有关。     

PI3K/Akt和Wnt/β-catenin信号通路在大鼠脑缺血再灌注损伤中的相互作用

目的探究PI3K/Akt信号通路在大鼠脑缺血再灌注损伤中对Wnt/β-catenin信号通路的影响。方法采用longa改良法建立局灶性脑缺血再灌注模型,将78只健康雄性SD大鼠随机分为4组:假手术组(S组)、脑缺血再灌注组(I/R组)、脑缺血再灌注+bFGF后处理组(b FGF组)、脑缺血再灌注+bFGF后处理+LY294002(LY组)。应用HE染色检测大脑皮质组织形态变化,TUNEL法检测细胞凋亡情况。应用免疫组织化学染色法、原位杂交法分别检测在各组大鼠脑皮质在不同再灌注时间点P-Akt、GSK-3βmRNA表达变化情况。结果相较于假手术组(S组),I/R组和LY组凋亡细胞数和P-Akt、GSK-3βmRNA表达明显增高(P 0. 05); b FGF处理组凋亡细胞数和GSK-3βmRNA表达减少,但P-Akt表达量较I/R组和LY组显著增高(P 0. 05)。结论在脑缺血再灌注损伤中,PI3K/Akt信号通路可以通过调控GSK-3β进而影响Wnt/β-catenin信号通路,这为深入研究PI3K/Akt和Wnt/β-catenin信号通路在缺血性脑损伤中的协同作用机制提供重要线索。     

MKP1通过抑制JNK信号途径减轻淀粉样蛋白的神经毒性作用

目的研究MKP1在Aβ所致MAPK激活、神经炎症和细胞凋亡中的调控作用。方法在细胞培养基中加入不同浓度的Aβ42(0μmol/L,0.1μmol/L,1μmol/L,10μmol/L和100 v),处理24 h后CCK-8检测细胞活性。向细胞培养基中加入10μmol/L的Aβ42,在不同时间点(0 h、6 h、12 h、18 h和24 h)通过qRT-PCR和Western blot检测MKP1表达。将野生型PC12细胞分为对照组(Control)和Aβ42组,敲除MKP1的细胞为MKP1 KD+Aβ42组,过表达MKP1细胞为MKP1+Aβ组,后3组加入10μmol/L Aβ42,置于培养箱中24 h,通过线粒体膜电位、DCFH-DA以及超氧化物歧化酶活性和丙二醛检测评价细胞内氧化应激水平,通过qRT-PCR检测TNF-α和IL-1β表达,Western blot检测p-JNK水平。结果发现经Aβ刺激后,PC12细胞活性受到抑制,MAPK的重要调节因子MKP1表达下调,并呈时间依赖性。过表达MKP1后,Aβ诱导的PC12细胞中p-JNK水平降低,细胞内活性氧类水平下降,TNF-α和IL-1β表达减少。而敲除MKP1可加重Aβ诱导的PC12氧化应激和炎症反应。结论 Aβ通过下调MKP1表达激活MAPK信号途径,过表达MKP1可通过抑制JNK信号途径减轻Aβ所诱导的氧化应激和神经炎症从而发挥神经保护作用。     

毛蕊花糖苷通过下调TGF-β抑制胶质母细胞瘤上皮间质转化的实验研究

目的探讨毛蕊花糖苷(VB)对胶质母细胞瘤细胞上皮间质转化的作用及分子机制。方法(1)实验1:常规培养胶质母细胞瘤T98和U251细胞并各自分为4组,依次添加0、20、40、80μmol/L的VB处理24 h。采用CCK-8实验检测4组细胞存活率;采用Transwell实验检测4组细胞迁移、侵袭情况;采用RT-PCR实验及Western blotting实验检测4组细胞转化生长因子-β(TGF-β)、波形蛋白、Snail蛋白mRNA及蛋白表达情况。(2)实验2:T98、U251细胞各分为4组,依次为对照组、VB组、TGF-β组和TGF-β+VB组。对照组及VB组细胞转染空载质粒、TGF-β组及TGF-β+VB组细胞转染TGF-β质粒;转染24 h后VB组、TGF-β+VB组细胞均加入40μmol/L VB继续培养24 h,随后进行后续实验(迁移和侵袭实验、Western blotting实验,实验方法及检测指标同实验1)。(3)体内实验:裸鼠皮下种植U251细胞后分为2组,每组8只。实验组每天腹腔注射100 mg/kg的VB,对照组注射等量的PBS。记录小鼠肿瘤体积大小和小鼠体质量的变化;实验终点(移植后21 d)时,检测小鼠肿瘤重量并采用Western blotting实验检测肿瘤中TGF-β、波形蛋白、Snail的表达。结果(1)实验1:与0μmol/L VB组相比较,20μmol/L、40μmol/L VB组的细胞存活率差异无统计学意义(P>0.05),而80μmol/L VB组细胞存活率下降,差异有统计学意义(P<0.05);与0μmol/L VB组比较,20和40μmol/L VB组迁移和侵袭到下室的细胞随浓度的增高而减少,TGF-β、波形蛋白、Snail蛋白的mRNA和蛋白表达水平也依次降低,差异有统计学意义(P<0.05)。(2)实验2:与对照组相比较,VB组细胞迁移和侵袭的细胞数量减少,TGF-β、波形蛋白及Snail蛋白的表达下调,差异有统计学意义(P<0.05);而TGF-β组细胞迁移和侵袭的细胞数量增加,TGF-β、波形蛋白及Snail蛋白的表达上调,差异有统计学意义(P<0.05);与VB组比较,TGF-β组及TGF-β+VB组细胞迁移和侵袭的细胞数量增加,TGF-β、波形蛋白及Snail蛋白的表达上调,差异均有统计学意义(P<0.05)。(3)体内实验:与对照组相比,实验组小鼠的肿瘤体积和肿瘤重量更小,TGF-β、波形蛋白及Snail蛋白表达量更小,差异均有统计学意义(P<0.05);但2组小鼠的体质量差异无统计学意义(P>0.05)。结论VB通过下调TGF-β的表达而抑制胶质母细胞瘤的上皮间质转化。     

蛋白激酶D1对Aβ诱导的SH-SY5Y神经细胞损伤的调节作用

目的探究蛋白激酶D1(PKD1)对β淀粉样蛋白(Aβ)诱导的阿尔茨海默病(AD细胞模型的调节作用和分子机制。方法分别以0、10、20、30、40、50μmol·L~(-1)的Aβ25-35处理SHSY5Y细胞24 h,流式细胞术检测细胞凋亡,MTT检测细胞活力,蛋白印迹法(Western blotting)检测PKD1蛋白水平变化。在AD细胞模型中分别过表达和干扰PKD1,流式细胞术检测细胞凋亡和活性氧(ROS),Western blotting检测凋亡相关蛋白(B细胞淋巴瘤/白血病-2(Bcl-2)和B细胞淋巴瘤/白血病-xl(Bcl-x L))、磷酸化c-Jun氨基末端激酶(P-JNK)、磷酸化细胞外调节蛋白激酶(p-ERK 1/2)、磷酸化JAK激酶2(p-JAK2)、磷酸化信号传导及转录激活蛋白3(p-STAT3)及其下游靶基因c-Myc的蛋白水平变化。过表达PKD1同时添加10μmol·L~(-1) FLLL32(JAK2/STAT3通路抑制剂)处理AD细胞模型,流式细胞术检测细胞凋亡和ROS,Western blotting检测Bcl-2、Bcl-x L、P-JNK和P-ERK 1/2蛋白水平变化。结果 30μmol·L~(-1) Aβ25-35处理SH-SY5Y细胞,细胞存活率和凋亡率分别约为70%和20%(P0.05),为构建AD模型的最适浓度。过表达PKD1抑制Aβ25-35诱导的细胞凋亡增加、ROS升高、Bcl-2和Bcl-x L蛋白表达下调、P-JNK和P-ERK1/2蛋白表达上调;干扰PKD1,加剧Aβ25-35对细胞的毒性作用。过表达PKD1,P-JAK2、P-STAT3和C-Myc蛋白表达上调;干扰PKD1,p-JAK2、p-STAT3和c-Myc蛋白表达下调。FLLL32处理抑制PKD1对AD细胞模型的作用。结论 Aβ诱导SH-SY5Y神经细胞中P-JAK2、P-STAT3表达下调和P-JNK和P-ERK 1/2表达上调。过表达PKD1可通过上调p-JAK2、p-STAT3的表达抑制P-JNK和P-ERK 1/2表达,缓解Aβ对细胞的毒性作用。     

胰岛素信号通路磷脂酰肌醇-3激酶/丝氨酸苏氨酸蛋白激酶对海马神经元β-淀粉样前体蛋白裂解酶1表达的影响

目的 通过胰岛素和磷脂酰肌醇-3激酶(PDK)抑制剂渥曼青霉素(wortmannin,WORT)对PI3K/丝氨酸苏氨酸蛋白激酶(PDK/Akt)信号通路的激活和抑制作用,观察PI3K/Akt信号通路对海马神经元B-淀粉样前体蛋白裂解酶1(BACE1)表达的影响.方法 40只SD大鼠随机分为空白对照组、假手术组、胰岛素组和WORT组(每组10只),海马立体定向注射胰岛素和PI3K抑制剂WORT.免疫组织化学和Western blot法检测PI3K/Akt信号传导相关蛋白以及BACE1的表达水平.结果 注射胰岛素的海马PI3K信号通路下游信号分子较对照组:Akt表达增加(0.952±0.060与0.835±0.029,t=4.9150,P=0.0001),Akt set473位点磷酸化(pAkt)水平上调(0.800±0.075与0.657±0.025,t=4.5598,P=0.0002),糖原合成激酶-3α(GSK-3α)磷酸化水平降低(0.604±0.062与0.726±0. 041,t=3.5871,P=0.0018),而成熟的BACE1及其裂解产物β分泌酶C末端(β-CTF)表达下调.WORT组的PI3K下游信号分子Akt、pAkt表达明显被抑制,磷酸化GSK-3α表达增加,同时成熟的BACE1(1.004±0.096)和β-CTF(1.031±0.048)的表达较对照组(分别0.498±0.064,0.786±0.101)上调(分别t=11.5980,P=0.0000;t=4.2194,P=0.0004).结论 胰岛素信号通路PI3K/AKt可以调节BACE1的表达和活性并参与阿尔茨海默病的发病机制.     

苁蓉精对MPP~+诱导多巴胺能神经细胞损伤后GSK-3β表达的影响

目的观察苁蓉精水提物及苁蓉精纳米微粉对1-甲基-4-苯基吡啶离子(MPP+)诱导损伤的多巴胺能神经细胞MES23.5细胞中帕金森病相关蛋白α-突触核蛋白(α-synuclein)、糖原合成酶激酶-3β(GSK-3β)、Tau蛋白表达的影响,探讨苁蓉精治疗帕金森病的作用机制。方法分别将苁蓉精水提物及纳米微粉经PBS溶解后加入培养基使终浓度为100、200、250μg/mL,孵育MPP+诱导的PD模型细胞24h后,并设模型对照(MPP+诱导的PD模型细胞,不加干预)及空白对照组(未经任何处理的MES23.5细胞),以MTT法检测细胞的存活率,Western Blot法测定各组细胞内α-synuclein、GSK-3β、Tau蛋白表达。结果同空白对照组比较,模型组及苁蓉精水提物及纳米微粉100、200、250μg/mL组α-synuclein、GSK-3β及Tau蛋白的磷酸化水平均升高(分别P0.01、P0.05);与模型组比较,苁蓉精水提物及纳米微粉100、200、250μg/mL组α-synuclein、GSK-3β及Tau蛋白的磷酸化水平均显著降低(分别P0.01、P0.05)。除苁蓉精纳米微粉100μg/mL组GSK-3β磷酸化水平与苁蓉精水提物100μg/mL组比较差异无统计学意义(P0.05)外,其余各苁蓉精纳米微粉组α-synuclein、GSK-3β及Tau蛋白的磷酸化水平均低于相同浓度的苁蓉精水提物组(分别P0.01、P0.05)。结论苁蓉精水提物及苁蓉精纳米微粉均能够减轻MPP+诱导的PD模型细胞的损伤,且苁蓉精纳米微粉的效果相对较好。其机制可能与苁蓉精可以减少α-synuclein的聚集,降低GSK-3β的活性,抑制Tau蛋白的过度磷酸化有关。     

氯胺酮对抑郁大鼠前额皮层及海马IL-1β和IL-6表达的影响

目的 检测抑郁大鼠给予氯胺酮后,前额皮层及海马区组织内IL-1β和IL-6表达的变化.方法 Wistar大鼠雄性20只按照随机方式分为2组,各10只,给予生理盐水的大鼠入对照组（C组）,给予10 mg/kg氯胺酮的大鼠为K组.应用行强迫游泳实验15 min的方法建立大鼠抑郁模型.次日,腹腔注射氯胺酮或等体积生理盐水,注射30 min后再次进行强迫游泳实验5 min,记录不动时间.并分别采用Western Blot法和双抗体夹心ABC-ELISA法检测大鼠前额皮层及海马组织中IL-1β和IL 6的表达情况.结果 与对照组比较,应用氯胺酮后大鼠强迫游泳不动时间明显减少（P＜0.01）,大鼠前额皮层及海马区的IL-1β和IIL-6表达均明显下调（P＜0.05）.结论 氯胺酮对抑郁大鼠的抗抑郁作用可能与前额皮层及海马IL-1和IL-6的表达下调有关.     

Involvement of PI3K/Akt/GSK-3β and mTOR in the antidepressant-like effect of atorvastatin in mice

Atorvastatin is a cholesterol-lowering statin that has been shown to exert several pleiotropic effects in the nervous system as a neuroprotective and antidepressant-like agent. Antidepressant-like effect of atorvastatin in mice is mediated by glutamatergic and serotoninergic receptors, although the precise intracellular signaling pathways involved are unknown. PI3K/Akt/GSK-3β/mTOR signaling pathway has been associated to neurobiology of depression and seems to be modulated by some pharmacological antidepressant strategies. The present study investigated the participation of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of an acute atorvastatin treatment in mice. Atorvastatin sub-effective (0.01 mg/kg) or effective (0.1 mg/kg) doses in the tail suspension test (TST) was administered orally alone or in combination with PI3K, GSK-3β or mTOR inhibitors. The administration of PI3K inhibitor, LY294002 (10 nmol/site, i.c.v) completely prevented the antidepressant-like effect of atorvastatin (0.1 mg/kg, p.o.). The participation of GSK-3β in the antidepressant-like effect of atorvastatin was demonstrated by co-administration of a sub-effective dose of atorvastatin (0.01 mg/kg, p.o.) with AR-A014418 (0.01 μg/site, i.c.v., a selective GSK-3β inhibitor) or with lithium chloride (10 mg/kg, p.o., a non-selective GSK-3β inhibitor). The mTOR inhibitor, rapamycin (0.2 nmol/site, i.c.v.) was also able to prevent atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. These behavioral findings were supported by neurochemical observations, as atorvastatin treatment increased the immunocontent of the phosphorylated isoforms of Akt, GSK-3β and mTOR in the hippocampus of mice. Taken together, our results suggest an involvement of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of atorvastatin in mice.     

α-Lipoic Acid Interaction with Dopamine D2 Receptor-Dependent Activation of the Akt/GSK-3β Signaling Pathway Induced by Antipsychotics: Potential Relevance for the Treatment of Schizophrenia

Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.     

Chronic stress affects PERIOD2 expression through glycogen synthase kinase-3β phosphorylation in the central clock

Mood disorders are considered to be associated with altered circadian rhythms, but the correlation between them has remained obscure. The mood stabilizer, lithium, is an inhibitor of glycogen synthase kinase-3β (GSK-3β), which is a modulator of the circadian clock system. Here, we show that chronic restraint (CR) stress diminishes behavioral activity and rhythmicity in mice. CR stress elevated GSK-3β phosphorylation and blunted the rhythmic expression of PERIOD2 (PER2) in the brain. Moreover, lithium, when administered to the stress-imposed mice, reduced GSK-3β phosphorylation and restored PER2 expression in the suprachiasmatic nucleus in a nighttime-specific manner. These data suggest that CR stress altered the circadian behavioral rhythm through a change in circadian gene expression of PER2 and GSK-3β phosphorylation in the suprachiasmatic nucleus.     

Inhibition of GSK-3beta Signaling Pathway Rescues Ketamine-Induced Neurotoxicity in Neural Stem Cell-Derived Neurons

Clinical application of anesthetic reagent, ketamine (Keta), may induce irreversible neurotoxicity in central nervous system. In this work, we utilized an in vitro model of neural stem cells-derived neurons (nSCNs) to evaluate the role of GSK-3 signaling pathway in Keta-induced neurotoxicity. Embryonic mouse-brain neural stem cells were differentiated into neurons in vitro. Keta (50 μM)-induced neurotoxicity in cultured nSCNs was monitored by apoptosis, immunohistochemical and western blot assays, respectively. GSK-3 signaling pathways, including GSK-3α and GSK-3β, were inhibited by siRNA in the culture. The subsequent effects of GSK-3α or GSK-3β downregulation on Keta-induced neurotoxicity, including apoptosis and neurite loss, were then evaluated in nSCNs. Finally, caspase and Akt/ERK signal pathways were further examined by western blot to evaluate the regulatory effect of GSK-3 signaling pathways on Keta-induced neural injury. Keta (50 μM) caused markedly nSCN apoptosis and neurite degeneration in vitro. Keta decreased GSK-3β phosphorylation, but had no effect on GSK-3α phosphorylation. SiRNA-induced GSK-3β downregulation rescued Keta-induced neurotoxicity in nSCNs by reducing neuronal apoptosis and preventing neurite degeneration. On the other hand, GSK-3α downregulation had no effect on Keta-induced neurotoxicity. Western blot showed that, in Keta-injured nSCNs, GSK-3β downregulation reduced Caspase-1/3 proteins, but left phosphorylated Akt/ERK unchanged. GSK-3β, not GSK-3α, was specifically involved in the process of Keta-induced neurotoxicity in nSCNs. Inhibiting GSK-3β may be an effective approach to counter toxic effect of ketamine on central neurons in clinical and experimental applications.     

Parkinson's Disease-Associated Dj-1 Mutations Increase Abnormal Phosphorylation of Tau Protein through Akt/Gsk-3β Pathways

Hyperphosphorylated tau protein is the main component of neurofibrillary tangles found in Alzheimer's disease and Parkinson's disease (PD). Mutations in DJ-1 have been identified as the causative gene for Parkinson's disease 7 (PARK7)-linked PD. DJ-1_L166P and DJ-1_D149A, two types of DJ-1 mutations, are most commonly studied as the loss-of-function mutations responsible for early-onset familial PD. Whether mutations in DJ-1 result in tauopathy is as yet unknown. In this study, we found that the L166P and D149A mutant isoforms of DJ-1 associated with familial PD cause tau phosphorylation at Ser202, Ser262, and PHF1 (396/404) sites in neuroblastoma 2a cells. Glycogen synthase kinase (GSK)-3β phosphorylation at serine 9 (Ser9) decreases around 50 % in DJ-1_L166P- or DJ-1_D149A-transfected cells, while there is no change in total levels of GSK-3β. Our results also indicate that overexpression of DJ-1_L166P or DJ-1_D149A leads to a significant decrease in the level of phosphorylation of Akt at Thr308, which plays a critical role in phosphorylating GSK-3β at Ser9 and inhibiting its kinase activity. Importantly, insulin, the activator for Akt, effectively attenuates the reduced phosphorylation level of GSK-3β at Ser9 induced by DJ-1_L166P. Neither the expression of cyclin-dependent kinase 5 nor the level of PP2A activity was found to have changed, suggesting that the familial PD-associated DJ-1_L166P and DJ-1_D149A mutations increase tau phosphorylation by increasing the activity of GSK-3β. Finally, we found that administration of lithium chloride, a well-known GSK-3β inhibitor, resulted in decreased levels of phosphorylated tau in DJ-1_L166P-transfected cells.     

Inactivation of glycogen synthase kinase-3beta protects against kainic acid-induced neurotoxicity in vivo

Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3β (GSK-3β), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3β phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3β inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3β using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3β in cell death in response to excitotoxins in vivo and further confirms that GSK-3β plays a role in cell death/cell survival pathways.     

Inhibition of glycogen synthase kinase-3β by Angelica sinensis extract decreases β-amyloid-induced neurotoxicity and tau phosphorylation in cultured cortical neurons

Increasing evidence has shown that β-amyloid (Aβ) induces hyperphosphorylation of tau and contributes to Aβ toxicity. Recently, tau hyperphosphorylation by glycogen synthase kinase-3β (GSK-3β) activation has been emphasized as one of the pathogenic mechanisms of Alzheimer's disease (AD). The phosphoinositide 3 kinase (PI3K)/Akt pathway is known as an upstream element of GSK-3β. The inhibitory control of GSK-3β, via the PI3K/Akt pathway, is an important mechanism of cell survival. In the present study, we investigated the neuroprotective effects of Angelica sinensis (AS), a traditional Chinese herbal medicine, against Aβ(1-42) toxicity in cultured cortical neurons and also the potential involvement of PI3K/Akt/GSK-3β signal pathway. We revealed that AS extract significantly attenuated Aβ(1-42) -induced neurotoxicity and tau hyperphosphorylation at multiple AD-related sites in a dose-dependent manner. Simultaneously, it increased the levels of phospho-Ser(473) -Akt and down-regulated GSK-3β activity by PI3K activation. The neuroprotective effects of AS extract against Aβ(1-42) -induced neurotoxicity and tau hyperphosphorylation were blocked by LY294002 (10 μM), a PI3K inhibitor. In addition, AS extract reversed the Aβ(1-42) -induced decrease in phosphorylation cyclic AMP response element binding protein (CREB), which could be blocked by the PI3K inhibitor. These results suggest that AS-mediated neuroprotection against Aβ toxicity is likely mediated by the PI3K/Akt/GSK-3β signal pathway.