SOD2在姜黄素减轻氧糖剥夺所致神经元损伤中的作用

目的:探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了姜黄素(Curcumin,Cur)对氧糖剥夺模型(Oxygen-Glucose Deprivation,OGD)损伤神经元的保护作用。方法:本研究采用HT22神经元细胞暴露于OGD环境中3 h模拟神经元缺血缺氧损伤,SOD2-si RNA抑制神经元SOD2蛋白表达后,通过噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,比色法测量培养基乳酸脱氢酶(Lactic Dehydrogenase LDH)水平,流式细胞仪计算细胞凋亡率,Western blot测定凋亡蛋白Cleaved Caspase-3表达,并观察细胞形态和线粒体功能。结果:与正常培养的Control组相比,OGD组细胞活力显著降低,LDH释放明显增加,细胞凋亡率和Cleaved Caspase-3表达显著上升,细胞形态破坏并降低线粒体膜电位(MMP)和线粒体复合物1(Mitochondrial Complex 1 Activity)的活力(P0.05),100 ng/ml的Cur可显著减轻OGD诱导的神经元细胞的上述损伤性改变(P0.05)。而SOD2-si RNA显著逆转Cur对OGD诱导的神经元细胞损伤的保护作用(P0.05),SC-si RNA则未对Cur产生的神经保护作用造成显著干扰(P0.05)。结论:Cur可能通过上调SOD2的表达,减轻OGD对神经元细胞的损伤。     

SIRT3在氧糖剥夺再灌注损伤小鼠神经元中的表达及意义

目的:通过建立体外脑缺血模型,探讨沉默信息因子3(SIRT3)在小鼠皮层神经元氧糖剥夺再灌注(OGD/R)损伤后的表达和意义。方法:C57BL/6J小鼠皮层神经元原代培养7天后,以氧糖剥夺不同时长(2 h、4 h、6 h、8 h)再灌注24 h作为观察时间点,利用细胞增殖-毒性检测试剂盒(Cell Counting Kit-8,CCK-8)检测细胞活力;小鼠乳酸脱氢酶(LDH)试剂盒检测LDH释放;蛋白印迹法(Western blot WB)观察微管相关蛋白1轻链3(LC3-Ⅱ)、活化凋亡蛋白3(Cleaved caspase-3)、以及SIRT3的表达变化;免疫荧光下进一步观察LC3-II、SIRT3表达。结果:与正常组比,随着氧糖剥夺时间的延长,LDH释放量呈台阶式升高(P0.01),而神经元活性进展性下降(P0.01);蛋白印迹结果发现在缺血损伤后LC3-Ⅱ整体上调,并于OGD 4h达峰值,SIRT3分子表达趋势与LC3-Ⅱ相似均呈抛物线状,而Cleaved caspase-3整体上调;相应的,细胞免疫荧光结果显示缺血损伤后神经元胞体和突起中LC3呈点状高表达,与此同时SIRT3荧光强度亦增高。结论:神经元缺血时间越长损伤越重;LC3-Ⅱ和SIRT3表达呈现相似性;SIRT3可能通过调控线粒体自噬参与了拮抗神经元缺血损伤的作用。     

MSCs线粒体转移对脑缺血后损伤修复的作用研究

目的探索骨髓基质细胞（MSCs）中线粒体转移在脑缺血后的损伤保护作用。 方?法采用小鼠骨髓MSCs分离与原代培养方法,培养MSCs并通过流式细胞仪进行鉴定;取出生后第0天（P0）SD幼鼠的皮层,进行原代神经元培养,并进行氧糖剥夺（OGD）处理,将含有线粒体的MSCs培养基（MCM）组和不含有线粒体的MSCs培养基（mdMCM）组与OGD神经元进行共培养,另以未经过OGD处理的神经元（Neuron组）和经过OGD处理的神经元（OGD组）作为对照;通过MitoTracker追踪线粒体,分析线粒体从MSCs向OGD神经元的转移情况;通过检测试剂盒对神经元内ATP含量和神经元活性进行分析;通过对线粒体膜电势检测,分析线粒体的功能;采用Western Blot分析线粒体Miro1蛋白的表达水平;通过MCAO造模和计算梗死体积,分析MSCs移植对脑缺血的保护作用。采用方差分析和t检验进行统计学分析。 结?果原代培养的骨髓MSCs纯度达到99﹪以上。取原代培养MSCs的培养基分别去除线粒体（mdMCM）与不去除线粒体（MCM）,与OGD神经元共培养,在MCM组,观察到神经元中存在MSCs来源的线粒体;经过OGD处理的神经元,其胞内ATP水平降低至0.634±0.023,给予MCM处理后,神经元胞内ATP水平上升至1.623±0.039,当给予mdMCM处理后,神经元胞内ATP水平降低至0.645±0.011,ATP比率变化的差异具有统计学意义（F?= 3413.62,P?< 0.01）;经过OGD处理,神经元活性降低至（73.7±1.12）﹪;给予MCM处理后,神经元活升高到（83.3±1.57）﹪,当给予mdMCM处理后,神经元活性降低至（72.9±1.25）﹪,与MCM组相比差异具有统计学意义（F?= 654.280,P?< 0.01）。在未经过处理的对照组中,线粒体膜电势丢失1.7﹪;经过OGD处理后,膜电势丢失70.3﹪;添加MCM后的OGD神经元,线粒体膜电势丢失44.7﹪,与OGD组相比,差异具有统计学意义（P?= 0.036）;而添加mdMCM的OGD处理神经元,线粒体膜电势丢失67.7﹪,与OGD+MCM组相比,差异具有统计学意义（P?= 0.041）。给予CCCP处理后的阳性对照神经元,膜电势丢失为99.3﹪。在Miro1表达干预中,空白对照组神经元胞内的ATP平均水平记为1,神经元活性为100﹪,计算其余各组相对空白对照组的ATP水平和神经元活性。在Miro1高表达组,胞内ATP水平为2.304,与对照质粒组（ratio = 1.611）相比,差异具有统计学意义（P?= 0.034）;神经元活性检测中,Miro1高表达组相比对照质粒组（90.4﹪vs 81.7﹪）,差异具有统计学意义（P?= 0.040）。在MCAO手术后,小鼠的脑梗死体积达到38.4﹪,而给予MSCs后的小鼠,梗死面积降低到14.4﹪,差异具有统计学意义（P?= 0.004）。 结论MSCs来源的线粒体可以向损伤神经元转移,提升神经元胞内ATP水平和神经元活性,降低缺血损伤中小鼠的脑梗死体积。线粒体Miro1蛋白参与了线粒体向神经元转移保护过程。     

对羟基苯甲醛对神经元缺氧所致钙超载的抑制作用

通过复制大鼠原代皮层神经元缺糖缺氧/复糖复氧(OGD/Rep)损伤模型,采用MTT法检测细胞存活率、硝酸还原酶法检测NO释放量、流式细胞仪检测胞内钙离子浓度、Western Blot法及RT-PCR法检测神经元L型钙通道、NMDA型钙通道、TRPM7通道蛋白和基因的表达,评价天麻成分对羟基苯甲醛对OGD/Rep损伤致大鼠原代皮层神经元钙通道过度开放的影响,探讨天麻成分对羟基苯甲醛的神经保护作用是否通过抑制钙通道的过度开放减轻钙超载,结果表明天麻成分对羟基苯甲醛可提高大鼠原代皮层神经元OGD/Rep损伤的存活率、减少NO的释放、降低胞内钙离子浓度、降低L型钙通道蛋白的表达。     

粒细胞集落刺激因子对氧糖剥夺后培养神经元的影响

目的:观察G-CSF时体外模拟脑缺血损伤神经元的影响,为其临床治疗脑缺血损伤提供理论的依据.方法:应用体外培养原代大脑皮质神经元氧糖剥夺模型模拟脑缺血损伤,氧糖剥夺4h,制作模型过程中加入不同浓度的G-CSF进行干预,通过观察细胞活性、死亡率、细胞色素C释放量判断G-CSF对神经元的保护作用.结果:G-CSF可明显降低氧糖剥夺神经元的死亡率,提高其生存活性,明显降低神经元由线粒体向胞浆内释放细胞色素C,与模型组比较有显著性差异(P＜0.01).结论:G-CSF对氧糖剥夺所致的神经元损伤发挥保护作用.     

线粒体大麻素受体1在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响

目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca~(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca~(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca~(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。     

自噬在高压氧预处理预防脊髓缺血再灌注损伤中 的作用机制研究*

目的:研究自噬在高压氧预处理预防脊髓缺血再灌注损伤中的机制。方法:新生大鼠脊髓神经元原代培养,分为对照组(氧糖剥夺)和高压氧(HBO)预处理组。通过应用免疫组织化学、Western blot分析两组LC3-Ⅱ与凋亡相关分子Beclin-1,Bcl-2,Casp-ase-3的表达变化。结果:发现重复高压氧预处理对氧糖剥夺诱导原代培养的脊髓神经元损伤具有明显的保护作用。免疫组化和Western blot显示与对照组相比高压氧预处理显著增加脊髓神经元细胞Bcl-2的表达,降低Beclin-1,Caspase-3以及自噬的特异性标记蛋白LC3-Ⅱ的表达。氧糖剥夺后对照组与高压氧组相比,LDH释放量明显增多(P<0.05)。结论:HBO预处理通过调节自噬减轻缺血再灌注损伤,为HBO预处理神经保护提供一条新的作用机制。     

血管紧张素1-7对氧糖剥夺/复氧的原代培养大白鼠海马神经元的保护作用

血管紧张素1-7(angiotensin 1-7,Ang1-7)在神经系统中发挥重要作用。已有研究发现,Ang1-7在脑缺血动物模型中发挥保护作用,但至今未见有关Ang1-7对氧糖剥夺/复氧(oxygenglucose deprivation/reoxygenation,OGD/R)损伤神经元的保护作用及其机制的研究报道。本研究以厌氧培养及不含葡萄糖的EBSS培养基培养、建立新生大白鼠原代培养的海马神经元OGD/R模型模拟脑缺血环境,实验分为3组:正常对照组、实验对照组和Ang1-7处理组。倒置显微镜观察神经元形态显示,Ang1-7处理组的神经元形态明显改善;CCK8试剂盒检测发现,Ang1-7处理组的细胞活性提高;流式细胞术研究发现,Ang1-7处理组的神经元凋亡和坏死率降低、神经元内Ca2+及NO水平降低;Western印迹结果发现,Ang1-7处理组Bax表达降低,Bcl-2表达增加。以上结果说明,Ang1-7可降低OGD/R神经元中NO和Ca2+水平,降低Bax蛋白、增加Bcl-2蛋白的表达,减少OGD/R神经元凋亡和坏死率,对OGD/R神经元发挥了保护作用。本研究为进一步在神经元水平上研究Ang-1-7的保护机制奠定基础,对中风等脑缺血疾病的防治具有重要意义。     

4-苯基丁酸钠通过抑制内质网应激减轻皮层神经元氧糖剥夺/再灌注损伤

4-苯基丁酸钠（4-phenylbutyric acid,4-PBA）是协助内质网中蛋白质转录后修饰和折叠的分子伴侣,故可减轻非折叠蛋白反应（unfolded protein response,UPR）及其介导的细胞凋亡。既往研究表明,4-PBA可以减轻脑组织的缺血性损伤,但采用原代皮层神经元构建氧糖剥夺/再灌注（oxygen glucose deprivation/reoxygenation, OGD/R）损伤模型,来研究4-PBA对神经元损伤的保护作用及其机制尚未见报道。本文采用原代培养的皮层神经元OGD/R损伤模型,同时给予4-PBA处理,探讨4-PBA对OGD/R诱导的神经元内质网应激（endoplasmic reticulum stress,ERS）的作用及其机制。分别采用MTT、LDH和Hoechst 33342染色法检测神经元存活率、细胞膜完整性和细胞凋亡情况。Western印迹检测ERS标志物葡萄糖调节蛋白78 (glucose regulated protein 78,GRP78),以及肌醇必需酶1（inositol requiring enzyme 1, IRE1）通路相关蛋白质的表达。Western印迹结果显示,在OGD/R后0～48 h,GRP78的表达较对照组明显升高。MTT、LDH漏出率和Hoechst 33342染色法检测显示,4-PBA显著改善OGD/R所导致的神经元存活率下降、LDH漏出率升高和细胞凋亡增加,且具有明显的剂量依赖性。通过Western印迹检测发现,4-PBA显著逆转OGD/R所致GRP78蛋白表达水平的上调。此外,对肌醇必需酶1通路相关蛋白质的检测显示,4-PBA下调氧糖剥夺/再灌注组神经元p IRE1和p JNK的表达,增加抗凋亡蛋白Bcl 2表达。上述研究结果表明,4-PBA在氧糖剥夺/再灌注情况下对神经元具有保护作用,该保护作用可能是通过抑制肌醇必需酶1信号通路介导的非折叠蛋白反应和内质网应激实现的。     

益智仁对谷氨酸损伤的大鼠离体培养皮层神经元的保护作用研究

目的:通过观察单味中药(益智仁)对离体培养的大鼠皮层神经元谷氨酸损伤模型的保护作用,探讨中药益智仁益智作用的机理,以初步论证中药益智仁对人体的益智效果.方法:利用原代离体培养的大鼠皮层神经元,分为6组:空白对照组,损伤模型组,阳性对照组,益智仁高剂量组,益智仁中剂量组,益智仁低剂量组,制备离体培养皮层神经元的谷氨酸损伤模型,采用MTT法测定细胞存活率,并测定LDH漏出率.结果:MTT法检测结果显示,与空白对照组相比,损伤模型组皮层神经元OD值显著降低(P＜0.05);与损伤模型组相比,各治疗组皮层神经元OD值显著升高(P＜0.05).LDH漏出率检测结果显示,与空白对照组相比,损伤模型组皮层神经元LDH漏出率显著升高(P＜o.05);与损伤模型组相比,益智仁中、高剂量组皮层神经元LDH漏出率显著降低(P＜0.05);其他组皮层神经元与损伤模型组相比,LDH漏出率无显著性差异(P＞0.05).结论:益智仁能够有效的降低细胞的死亡,提示其对线粒体功能具有保护作用,并对初期和后期损伤细胞均有保护作用,但具体作用靶点还需后续实验进一步研究.     

Enhancement of oxidative damage to cultured cells and Caenorhabditis elegans by mitochondrial electron transport inhibitors

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans (C. elegans) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans. In addition the cell damage is different dependent on cell type and organism.     

Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca²⁺) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca²⁺ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca²⁺ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.     

Nrf2对缺氧/复氧诱导的SH-SY5Y细胞线粒体分裂的影响

目的:通过体外模拟脑缺血再灌注损伤的细胞模型,探究核转录相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)与线粒体分裂是否存在调控关系。方法:通过三气培养箱和无糖培养基模拟氧糖剥夺/复氧(Oxygen and glucose deprivation/reperfusion,OGD/R)的条件,将OGD4 h、6h、8h和10h与R0h、6h、12h、18h、24h多个时间点组合,通过CCK8试剂盒(Cell Counting Kit-8,CCK8)检测细胞的存活率,最终以细胞凋亡明显但仍有半数存活的OGD4 h/恢复R18 h为条件建立细胞OGD/R模型;用Nrf2激动剂叔丁基对苯二酚(Tert-butylhydroquinone,t BHQ)和抑制剂鸦胆子苦醇(Brusatol,Bru)对细胞进行干预处理;蛋白免疫印迹(Western blot,WB)法检测Nrf2、动力相关蛋白1(Dynamin-related protein 1,Drp1)的表达量;制备细胞沉淀的切片,通过电镜观察细胞内线粒体的形态。结果:OGD/R+t BHQ组Nrf2蛋白的表达明显高于OGD/R组,且OGD/R+t BHQ组Drp1蛋白的表达则低于OGD/R组(P0.05),而OGD/R+Bru组Nrf2蛋白的表达低于OGD/R组,且OGD/R+Bru组Drp1蛋白的表达高于OGD/R组(P0.05);电镜观察结果显示OGD/R+t BHQ组线粒体分裂的程度和比例较OGD/R组有所减少,而在OGD/R+Bru组有增多(P0.05)。结论:Nrf2对线粒体分裂有负向调控作用,可能机制是经由Drp1蛋白发挥作用。     

Role of exercise intensities in oxidized low-density lipoprotein-mediated redox status of monocyte in men.

Exercise significantly influences the progression of atherosclerosis. Oxidized LDL (ox-LDL), as a stimulator of oxidative stress, facilitates monocyte-related atherogenesis. This study investigates how exercise intensity impacts ox-LDL-mediated redox status of monocytes. Twenty-five sedentary healthy men exercised mildly, moderately, and heavily (i.e., 40, 60, and 80% maximal oxygen consumption, respectively) on a bicycle ergometer. Reactive oxygen species (ROS) production, cytosolic and mitochondrial superoxide dismutase (c-SOD and m-SOD, respectively) activities, and total and reduced-form gamma-glutamylcysteinyl glycine (t-GSH and r-GSH, respectively) contents in monocytes mediated by ox-LDL were measured. This experiment obtained the following findings: 1) ox-LDL increased monocyte ROS production and was accompanied by decreased c-SOD and m-SOD activities, as well as t-GSH and r-GSH contents, whereas treating monocytes with diphenyleneiodonium (DPI) (a NADPH oxidase inhibitor) or rotenone/2-thenoyltrifluoroacetone (TTFA) (mitochondrial complex I/II inhibitors) hindered ox-LDL-induced monocyte ROS production; 2) production of ROS and reduction of m-SOD activity and r-GSH content in monocyte by ox-LDL were enhanced by heavy exercise and depressed by mild and moderate exercise; and 3) heavy exercise augmented the inhibition of ox-LDL-induced monocyte ROS production by DPI and rotenone/TTFA, whereas these DPI- and rotenone/TTFA-mediated monocyte ROS productions were unchanged in response to mild and moderate exercise. We conclude that heavy exercise increases ox-LDL-induced monocyte ROS production, possibly by decreasing m-SOD activity and r-GSH content in monocytes. However, mild and moderate exercise likely protects individuals against suppression of anti-oxidative capacity of monocyte by ox-LDL.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Alpha-synuclein protects naive but not dbcAMP-treated dopaminergic cell types from 1-methyl-4-phenylpyridinium toxicity

The pre-synaptic protein, alpha-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that alpha-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP+), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the beta-amyloid peptide, A-beta. Protection from MPP+ was directly correlated with the preservation of mitochondrial function. Specifically, alpha-synuclein rescued cells from MPP+ mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, alpha-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wild-type alpha-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for alpha-synuclein may change during development.     

Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson's disease model

Mitochondrial complex I dysfunction is regarded as underlying dopamine neuron death in Parkinson's disease models. However, inactivation of the Ndufs4 gene, which compromises complex I activity, does not affect the survival of dopamine neurons in culture or in the substantia nigra pars compacta of 5-wk-old mice. Treatment with piericidin A, a complex I inhibitor, does not induce selective dopamine neuron death in either Ndufs4(+/+) or Ndufs4(-/-) mesencephalic cultures. In contrast, rotenone, another complex I inhibitor, causes selective toxicity to dopamine neurons, and Ndufs4 inactivation potentiates this toxicity. We identify microtubule depolymerization and the accumulation of cytosolic dopamine and reactive oxygen species as alternative mechanisms underlying rotenone-induced dopamine neuron death. Enhanced rotenone toxicity to dopamine neurons from Ndufs4 knockout mice may involve enhanced dopamine synthesis caused by the accumulation of nicotinamide adenine dinucleotide reduced. Our results suggest that the combination of disrupting microtubule dynamics and inhibiting complex I, either by mutations or exposure to toxicants, may be a risk factor for Parkinson's disease.     

Rosuvastatin induces delayed preconditioning against oxygen-glucose deprivation in cultured cortical neurons

We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.