Humanin拮抗NMDA诱导的兴奋性神经毒的作用观察
|
摘要
Humanin(HN)是一个由24个氨基酸残基组成的短肽,2001年首先由日本学者在阿尔采末病(Alzhemer's disease,AD)患者的脑内发现。由于AD患者的枕叶在发病过程极少受累,他们据此推测,枕叶的神经元一定启动了某种基因的表达,并由此形成了特殊的保护机制,使神经元免受AD病变过程的毒性作用而死亡。最终他们发现了一种由24个氨基酸残基组成的新型多肽,并将其命名为HN。研究发现,在离体培养的细胞中,HN能有效抑制多种FAD基因(APP、PS-1和PS一2)、Aβ完整肽链(Aβ1-43或Aβ1-42)及其衍生物(Aβ25-35、Aβ31-35)诱发的神经毒作用;而且在大鼠在体AD模型中也发现了HN的神经保护作用。但他们的实验发现,HN对AD以外的损伤似乎不发挥保护作用。因此,HN被认为是AD特异或AD相关毒性(AD-related insults)的神经保护肽。
然而,有关HN神经保护作用的细胞机制提示,HN的神经保护作用可能不只限于AD相关的神经毒。(1)HN分布于脑组织以外的其他组织及不同的动物种属,即HN的作用不只局限于神经组织;(2)HN可拮抗线粒体功能障碍;(3)HN可抑制不同途径的凋亡;(4)HN可拮抗Aβ引起的细胞内钙超载。HN的这些作用特点强烈提示,HN极有可能具有广泛的神经保护作用,而不只是针对某种特定损伤(如AD相关毒性)而产生保护作用的神经保护肽。因此,有必要在AD以外的损伤模型中进行验证并分析其机制,而谷氨酸(NMDA)造成的兴奋性神经毒由于具有使用普遍,毒性作用强,损伤机制复杂,临床意义重要等特点,因此成为验证HN具有广泛的神经保护作用的最理想的模型。基于上述假设,我们拟进行如下观察:1)采用培养的大鼠大脑皮层神经元,通过MTT、LDH及胞内Ca~(2+)测定,建立NMDA诱导的兴奋性神经毒的细胞模型,通过氧自由基(reactive oxygenspecies,ROS)、NO检测,评价NMDA引起的线粒体损伤,通过电镜观察、TUNEL染色、caspase-3活性测定评价NMDA引起的神经元凋亡,并通过检测caspase-8和caspase-9的活性,初步分析外源性及内源性凋亡途径在神经元凋亡中发挥的作用;2)通过MTT、LDH、Calcein染色及胞内Ca~(2+)测定,观察HN对NMDA引起的兴奋性神经毒的拮抗作用;3)通过激光共聚焦、流式细胞术及生化方法检测琥珀酸脱氢酶的活性、ROS水平、NO水平及线粒体膜电位的改变,观察HN对NMDA引起的线粒体损伤的拮抗作用;4)通过电镜观察、TUNEL染色、流式细胞术、激光共聚焦及生化检测,观察HN对NMDA诱发的凋亡的拮抗作用及机制;并通过检测LDH、MTT,结合各种Caspase抑制剂的使用,分析HN通过抑制凋亡对抑制兴奋性神经毒作用的贡献,即大致区分HN通过抑制凋亡或坏死在抑制兴奋性神经毒中发挥的作用。通过以上观察验证我们的假说,即HN不只是拮抗AD相关毒性的特异性神经保护肽,而是具有广谱保护作用的内源性神经活性多肽。由于HN主要在大脑皮层表达,又具有广谱神经保护作用,因此,HN将可能通过拮抗包括AD在内的神经退行性疾病及其他神经病理损伤发挥重要作用。
第一部分:NMDA诱导的大鼠大脑皮层神经元神经毒性作用观察及机制探讨
兴奋性神经毒是一个广泛应用的神经损伤模型。虽有相关报道,但目前兴奋性神经毒的细胞模型在应用NMDA的浓度、时间上差别很大。因此,为客观评价HN的神经保护作用,首先有必要摸索NMDA作用的浓度及时间,建立一个稳定的神经毒模型,以利于后续的一系列实验观察。
方法:大鼠大脑皮层神经元培养,纯度达90%左右时,通过倒置显微镜下形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca~(2+)的动态测定,摸索NMDA毒性诱导的适当浓度及时间,建立兴奋性神经毒的细胞模型。并通过ROS、NO检测,分析模型中线粒体的损伤情况;通过电镜观察、TUNEL染色、caspase-3测定,研究模型中神经元的凋亡情况;并进一步通过检测模型中caspase-8、caspase-9的活性,分析外源性和内源性凋亡途径在神经元凋亡中发挥的作用。所有数据均经SPSS10.0处理获得,以均数±标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:NMDA与皮层神经元共同孵育,引起了培养的皮层神经元形态学的改变,以及由NMDA造成的时间和浓度依赖性的神经元细胞活力的下降。而且,NMDA(100μmol/L,2h)造成了大鼠大脑皮层神经元细胞活力下降了47%,LDH释放增加66%,皮层神经元内Ca~(2+)的快速升高,并维持在高水平形成一平台,ROS和NO的生成明显增加,电镜下凋亡形态出现,TUNEL染色阳性,caspase-3、caspase-8和caspase-9活性明显升高。
上述结果提示:NMDA(100μmol/L)作用2h诱发了皮层神经元明显的细胞毒性,是一个比较理想的兴奋性神经毒模型。该模型观察到了细胞整体的结构及功能的损伤(MTT下降、LDH释放增加、Ca~(2+)的快速升高),也观察到了线粒体功能的损伤。此外,该剂量的NMDA也诱发了皮层神经元的凋亡,内源性和外源性凋亡途径同时介导了该凋亡过程。
第二部分:Humanin拮抗NMDA诱导的兴奋性神经毒的作用观察
虽然HN被认为是AD特异或AD相关毒性(AD-related insults)的神经保护肽,然而,目前已积累的有关HN的细胞、分子水平的作用机制强烈提示,HN极有可能具有广泛的神经保护作用。本部分内容拟在前期建立的大鼠大脑皮层神经元的兴奋性神经毒细胞模型上,观察HN是否通过拮抗兴奋性神经毒而发挥保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况;实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(各种浓度)组。用Fluro-3进行Ca~(2+)探针标记,在激光共聚焦显微镜下,按以上分组要求加入干预,对神经元内Ca~(2+)浓度进行动态检测,每660ms采集一次数据,共采集300次,即对每个细胞跟踪观察198s;NMDA毒性诱导24h后,检测培养液中LDH的活性,以反映神经元的受损情况;收集细胞进行MTT测定,评价存活神经元的生活状态;收集细胞行Calcein染色,反映神经元的存活数量,即通过LDH、MTT和calcein染色对神经毒进行评价。所有数据均经SPSS10.0处理、获得,以均数±标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:1)HN抑制了NMDA引起的神经元内Ca~(2+)浓度的升高。在跟踪观察的198s时间内,NMDA引起了神经元内Ca~(2+)的快速升高,并始终维持在高水平形成一平台;HN(0.1μmol/L)不能拮抗NMDA升高Ca~(2+)的作用;HN(1μmol/L)虽未能阻止NMDA升高神经元内Ca~(2+)的作用,但能使升高的Ca~(2+)迅速降至对照水平。2)HN抑制了NMDA引起的存活神经元的减少。Calcein染色显示,NMDA引起了存活神经元数量的明显减少,为对照组的51.3%;预先应用HN(100μmol/L)几乎完全抑制了NMDA引起的神经元减少,存活神经元恢复到98.6%。3)HN抑制了NMDA引起的神经元细胞活力的下降。MTT检测提示,NMDA引起细胞活力下降约47%,而HN浓度依赖性地抑制了NMDA引起的神经元细胞活力的下降;4)HN降低了NMDA引起的神经元LDH的释放。虽然HN不能完全阻断NMDA引起的LDH释放,但HN可剂量依赖性地使之降低。
上述结果提示:1)HN抑制了NMDA引起的神经元内Ca~(2+)浓度的升高;2)HN阻止了NMDA引起的细胞活力的下降,抑制了NMDA所致的LDH释放,拮抗了NMDA所致存活神经元的减少。即HN可通过拮抗NMDA引起的兴奋性神经毒而发挥神经保护作用。
第三部分:Humanin拮抗NMDA诱导的线粒体损伤的作用观察
谷氨酸(NMDA)诱导的兴奋性神经毒通过过度激活NMDA受体,引起细胞内Ca~(2+)超载,造成神经元损伤,直至凋亡或坏死。线粒体既是细胞的能量工厂,又是重要的Ca~(2+)库,在缓解细胞Ca~(2+)超载方面发挥重要作用。然而,当线粒体缓解Ca~(2+)的能力超过极限时,线粒体功能也遭到破坏,因此,兴奋性神经毒可造成线粒体功能的损伤。由于前期的实验观察已证实,HN可拮抗NMDA诱导的兴奋性神经毒,包括抑制NMDA诱导的细胞内Ca~(2+)的升高,本部分内容拟观察HN通过拮抗NMDA造成的线粒体损伤而发挥的神经保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况。实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(各种浓度)组。NMDA毒性作用2h后,收集培养介质进行NO含量的定量分析;收集细胞,用JC-1标记线粒体膜电位,用DCFH-DA标记ROS,通过流式细胞术分析标记荧光的荧光强度,通过激光共聚焦显微镜检测提供形态学依据,从而对ROS含量和线粒体膜电位进行检测;NMDA毒性作用24h后,收集细胞,通过MTT对琥珀酸脱氢酶活性进行分析。所有数据均经SPSS10.0处理、获得,以均数土标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:1)HN抑制了NMDA引起的琥珀酸脱氢酶活性的降低。MTT检测提示,NMDA使琥珀酸脱氢酶活性下降约47%,而HN浓度依赖性地抑制了NMDA的作用;2)HN阻止了NMDA引起的线粒体膜电位(Δψm)的降低。用JC-1标记线粒体膜电位,通过激光共聚焦显微镜和流式细胞术检测JC-1的红色荧光强度。JC-1在具有正常膜电位的线粒体中发出红色荧光,因此,细胞红色荧光越强,表示具有的正常线粒体越多,反之,细胞中的线粒体膜电位下降越多。激光共聚焦显微镜下照片显示,与对照组相比,NMDA引起了线粒体膜电位下降,表明HN抑制了NMDA对线粒体膜电位的作用。用流式细胞术对JC-1的红色荧光强度进行了定量分析,结果与激光共聚焦显微镜检测一致;3)HN抑制了NMDA引起的ROS的生成。用DCFH-DA标记细胞,通过激光共聚焦显微镜和流式细胞术检测DCF的荧光强度。激光共聚焦显微镜下照片显示,与对照组相比,NMDA引起了ROS的生成增加;HN阻止了NMDA引起的ROS的生成增加,表明HN抑制了NMDA对ROS生成的影响。用流式细胞术对DCF荧光强度进行了定量分析,结果与激光共聚焦显微镜检测一致;4)HN降低了NMDA引起的NO生成。与对照组相比,NMDA引起NO生成明显升高;而提前应用HN则可明显抑制NMDA引起的NO生成,与对照组相似。
上述结果提示:1)HN抑制了NMDA引起的琥珀酸脱氢酶活性的降低;2)HN抑制了NMDA引起的线粒体膜电位(Δψm)的降低;3)HN抑制了NMDA引起的ROS和NO的生成。即HN可能通过拮抗线粒体功能损伤在抑制兴奋性神经毒中发挥神经保护作用。
第四部分:Humanin拮抗NMDA诱导的神经元凋亡的作用观察
兴奋性神经毒使神经元以凋亡或坏死两种形式死亡。由于细胞凋亡是迟发性的、程序性的细胞死亡,因此被认为是可控制和调节的,凋亡也因此成为研究的热点。线粒体介导的内源性凋亡途径是重要的凋亡途径之一,而且还可以借助于Bid与外源性途径发生联系。因此,线粒体在凋亡的发生中处于无可替代的重要地位。我们的前期研究发现,HN可拮抗NMDA诱导的兴奋性神经毒,而且可拮抗NMDA诱导的线粒体损伤。那么,HN是否可抑制NMDA诱导的神经元凋亡呢?本部分内容拟观察HN通过拮抗NMDA诱导的神经元凋亡而发挥的神经保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况;实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(1μmol/L)组。在毒性作用6h后,收集细胞进行电镜观察、TUNEL染色、caspase-3活性测定,证实NMDA诱导神经元凋亡的发生;通过PI染色、流式细胞术定量分析及caspase-3活性测定,评价HN对凋亡的拮抗作用。通过caspase-8、caspase-9活性测定,评价外源性及内源性凋亡途径在NMDA诱导的兴奋性神经毒中的作用,以及HN对外源性及内源性凋亡途径的抑制作用。通过使用caspase-8和总caspases抑制剂,结合MTT和LDH检测,初步分析HN通过抑制凋亡和(或)坏死在抑制兴奋性神经毒发生中的作用。通过使用caspase-8抑制剂,结合NO检测,分析兴奋性神经毒发生时外源性凋亡途径对线粒体功能的影响。所有数据均经SPSS10.0处理、获得,以均数士标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.35。
结果:1)电镜观察、TUNEL染色、caspase-3活性测定一致表示,NMDA诱导了神经元凋亡,而PI的流式细胞术定量分析及caspase-3活性测定显示,HN可抑制NMDA诱导的神经元凋亡。2)caspase-8和caspase-9活性测定显示,外源性及内源性凋亡途径共同参与了NMDA诱导的兴奋性神经毒中神经元的凋亡,而HN对外源性凋亡途径所致的神经元凋亡有一定的抑制作用。3)通过使用caspase-8的抑制剂(IETD)并结合MTT和LDH检测,发现外源性凋亡途径在NMDA诱导的兴奋性神经毒中的贡献不大。因此,HN通过抑制外源性凋亡途径对NMDA诱导的兴奋性神经毒发挥的神经保护作用也是有限的。4)通过使用总caspases抑制剂(VAD)并结合MTT和LDH检测发现,caspase依赖的凋亡在NMDA诱导的兴奋性神经毒中的贡献不大。因此,HN通过抑制caspase依赖的凋亡对NMDA诱导的兴奋性神经毒而发挥的神经保护作用也是有限的。5)通过使用caspase-8抑制剂,结合NO检测,发现caspase-8被抑制后NO水平降低,因此,HN可能通过抑制外源性凋亡途径影响NO的生成。
上述结果提示:1)HN对NMDA诱发的皮层神经元的凋亡具有一定的抑制作用;2)HN可能通过抑制外源性途径抑制NMDA诱导的凋亡;3)HN通过抑制caspases依赖的凋亡在抑制兴奋性神经毒中的作用并不明显;4)HN可能通过抑制caspase-8的激活而抑制NO的生成,从而抑制NMDA通过NO造成的神经损伤。
结论:1)NMDA(100μmol/L)作用2h诱发了皮层神经元明显的细胞毒性,造成了线粒体损伤和神经元凋亡,是一个比较理想的兴奋性神经毒模型;2)HN可通过拮抗NMDA引起的兴奋性神经毒而发挥神经保护作用;3)HN可能通过拮抗线粒体功能损伤在抑制兴奋性神经毒中发挥神经保护作用;4)HN可抑制NMDA引起的外源性凋亡途径介导的神经元凋亡,并可抑制caspase-8介导生成的NO造成的神经损伤。
Humanin(HN)is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzhermer's disease(AD)in 2001.Studies showed that HN protected neurons from insults of various familial Alzheimer's disease mutations(APP、PS-1 and PS-2),anti-APP(amyloid precussor protein,APP)antibodies, amyloid peptide(Aβ)(Aβ1-43 or Aβ1-42)and its segments(Aβ25-35、Aβ31-35)with efficiency.It seemed that HN didn't inhibit other toxic insults to neurons,such as Fas,etoposide, glutamate and NMDA triggered neurotoxicity.HN was,therefore,considered as a selectively neuroprotective factor rescuing neurons from Alzheimer's disease-related insults.
Although HN was firstly identified from human brain with Alzheimer's disease,more and more its homologues have been discovered in other species of animals and its location is also far beyond brain,suggesting that the role of HN be versatile other than just attenuating Alzheimer's disease-related insults.In support of this notion,other investigators including our lab have done some works to decipher the neuroprotective mechanism of HN at celluar and molecular level:1)to elevate ATP level,which is necessary for any cells to survive stresses;2) to block apoptosis mediated by extracelullar signals as well as intrinsic signals;3)to interfere with intracellular Ca~(2+)accumulation induced by amyloid beta peptide(Aβ).
The mechanisms underling the protective activity of HN mentioned above imply that it may play versatile protective roles against various insults other than as an AD- selective neuroprotective peptide.To verify the hypothesis,we employed NMDA induced neurotoxicity, an in vitro paradigm of neurodegeneration representative of pathologically neurotoxic insults of excitotoxicity which brings heavy damage to neurons by complicated mechanisms and is implicated in most of the neurodegenerative processes,to confirm the versatile neuroprotective role of HN in this in vitro insult model.The program of this research are as followings:1) Build an excitatory neurotoxic model in cultured cortical neurons of rat by measurement MTT, LDH in the medium and Ca~(2+)concentration in the cytoplasm,which reflect general NMDA-induced neurotoxicity;evaluate the mitochondrial damage in the model by detecting ROS and NO;and assay the neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,and analysis the apoptotic pathways involved in by the measurement of the activities of caspase-8 and caspase-9;2)Evaluate the neuroprotective effects of HN on NMDA induced neurotoxicity by detecting Ca~(2+)concentration in cytoplasm,MTT,Calcein staining and LDH release in the medium;3)Evaluate the protective effect of HN on NMDA-induced mitochondrial damage by determining the activity of fumaric reductase(MTT),the level of ROS and NO,and mitochondrial membrane potential;4)Evaluate the protective effects of HN on NMDA-induced neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,the measurement of the activity of caspase-3,and sub-summit of PI,analysis the potential protective pathways of HN against apoptosis by the measurement of the activities of caspase-8 and caspase-9,and evaluate the contribution of HN in attenuating NMDA-induced neurotoxicity by blocking neuronal apoptosis with the aid of caspase inhibitors.Our results showed that:1)HN blocked NMDA induced neurotoxicity by attenuating LDH release and Ca~(2+)overloading,and rescue cortical neurons from excitatory neurotoxicity with the increase of MTT and Calcein staining cells;2)HN antagonized mitochondrial dysfunction by suppressing ROS and NO with the increase of mitochondrial membrane potential and the activity of fumaric reductase;3)HN inhibited neuronal apoptosis by interfering extrinsic apoptotic signals involved in caspase-dependent pathway as well as neurotoxicity mediated by NO.
Part One:NMDA-Induced Neurotoxicity in Cortical Neurons and Potential Mechanisms Underlying
To evaluate the neuroprotective effect of HN on excitatory neurotoxicity,an appropriate cellular model must be employed which have definite and moderate neuronal damage and loss.The degree of neuronal loss triggered by NMDA largely depends on the concentration of NMDA in the medium and the time it works.But the concentration of NMDA and the time it works varied dramatically in reported models currently.To find a reasonable concentration of NMDA and the time it works is therefore a key factor in this part.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.An appropriate concentration of NMDA and the time it works were explored by monitoring morphological changes under contrast phase microscope,MTT,LDH release in the medium,and Ca~(2+) concentration in the cytoplasm so as to build an appropriate cellular model in cortical neurons in rat.ROS and NO were detected to evaluate the damage of mitochondria in the model.Electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,caspase-8 and caspase-9 were employed to evaluate neuronal apoptosis and analysis the pathways involved in the model.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:The administration of NMDA in the cultures caused pathological changes of morphology and cell viability decrease dose-dependently and time-dependently.NMDA (100μmol/L,2h)caused cell viability decrease by about 47%and LDH release increase by about 66%compared with control.It triggered the rise of Ca~(2+)in cytoplasm and the maintenance of the highest level during the observation.Excessive ROS and NO were detected in the model.NMDA also caused the appearance of neuronal apoptosis suggested by electron microscopy,TUNEL staining.Caspase-3,caspase-8 and caspase-9 were activated in the model.
Conclusions:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate concentration and duration to cause excitatory neurotoxicity;2)Mitochondria are damaged in the model;3)NMDA(100μmol/L,2h)triggers neuronal apoptosis mediated both by extrinsic and intrinsic apoptotic signal pathways.
Part Two:Humanin Attenuates NMDA-Induced Excitatory Neurotoxicity
Although HN was considered as a selectively neuroprotective factor against AD-related insults, the mechanism underlying its neuroprotective functions at molecular and cellular level strongly suggests that HN plays protective roles for neurons at various stresses other than AD-related insults.The current research was to investigate the protective role of HN against excitatory neurotoxicity.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L) and Gly 10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L) was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected,labeled with Fluo-3 and subjected to laser scanning confocl microscopy.Dynamic changes of cytoplasmic Ca~(2+)were captured and recorded every 660ms and 300 records(for about 198s)were needed for each neuron.24h after NMDA treatment,the medium was collected for the measurement of LDH. Neurons were subjected to MTT detection and Calcein staining.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:1)HN inhibits the increase of cytoplasmic Ca~(2+)concentration induced by NMDA. NMDA triggered the rapid increase of cytoplasmic Ca~(2+)concentration and maintenance of the highest level during the observation.From the control level to the highest one,it took only 2s. HN(0.1μmol/L)has no effect on interfering with NMDA-induced increase of cytoplasmic Ca~(2+) concentration.Although HN(1μmol/L)didn't block the increase of cytoplasmic Ca~(2+) concentration induced by NMDA,it caused dropping of the Ca~(2+)concentration;2)HN inhibited the loss of living neurons caused by NMDA.NMDA triggered the loss of neurons by 51.3%compared with control showed by Calcein staining.Pretreated with HN(100μmol/L), the loss of neurons was almost blocked completely.The rate of living cells reached 98.6%of control;3)HN blocked the decrease of cell viability caused by NMDA.NMDA caused cell viability dropping by 47%showed by MTT.HN blocked the toxic effect of NMDA and attenuated the decrease dose-dependently;4)HN neutralized the LDH release triggered by NMDA.Compared with control,additional LDH release(about 66%of control)was detected when neurons were treated with NMDA.HN inhibited the additional release of LDH does-dependently although it didn't stop it completely.
Conclusions:1)HN inhibits the increase of cytoplasmic Ca~(2+)concentration induced by NMDA; 2)HN inhibits the loss of living nerurons,blocks the decrease of cell viability,and neutralizes the additional release of LDH when neurons confront with NMDA-induced extitatory stress. HN,therefore,rescues cortical neurons from NMDA-triggered excitatory neurotoxicity effectively.
Part Three:Humanin Attenuates NMDA-Induced Mitochondria Dysfunction
Excitatory neurotoxicity is triggered by the overloading of Ca~(2+)for the overactivation of NMDA receptor.Since mitochondria is an important Ca~(2+)pool beside producing ATP,it plays an important role in soothing excessive Ca~(2+)in the situation.Once more Ca~(2+)is uptaken into mitochondria than its capacity,mitochondria itself is damaged and loss normal function of producing ATP.In the part,the protective role of HN against mitochondria dysfunction caused by NMDA were to investigate since we have confirmed its neuroprotective effect on NMDA-induced excitatory neurotoxicity and its inhibitory effect on Ca~(2+)increase triggered by NMDA.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected and labeled with JC-1 for mitochondria membrane potential or DCFH-DA for ROS.Neurons were subjected to laser scanning confocl microscopy and FCM.The medium was collected for the detection of NO.24h later,neurons were collected for the determination of fumaric reductase by MTT.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D. Means of different groups were compared by ANOVA,followed by the Dunnett's test. Significance level was 0.05.
Results:1)HN blocked the decrease of the activity of fumaric reductase.NMDA triggered the decrease of the activity of fumaric reductase by about 47%of control.HN inhibited the effect of NMDA dose-dependently showed by MTT;2)HN attenuated the drop of mitochondrial membrane potential caused by NMDA.The red fluorescent intensity of JC-1 labeled on neurons for mitochondrial membrane potential was detected and analyzed both by laser scanning confocal microscopy and FCM.The fluorescent intensity decreases when treated with NMDA. When HN(1μmol/L)and NMDA coincubated with the neurons,the decrease of the fluorescent intensity of JC-1 was inhibited;3)HN inhibited the excessive production of ROS triggered by NMDA.The measurement of fluorescent intensity of DCFH-DA for ROS by laser scanning confocal microscopy and FCM showed that NMDA induces excessive production of ROS and HN(1μmol/L)inhibited the production of ROS caused by NMDA.4)HN neutralized the excessive production of NO.Compared with control,NMDA triggered more NO and HN attenuated the activity of NMDA.
Conclusions:1)HN blocks the decrease of the activity of fumaric reductase;2)HN attenuates the drop of mitochondrial membrane potential caused by NMDA.3)HN inhibits the excessive production of ROS triggered by NMDA;4)HN neutralizes the excessive production of NO. HN,therefore,protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.
Part Four:Humanin Attenuates NMDA-Induced Neuronal Apoptosis
Excitatory neurotoxicity causes neuronal loss by necrosis and apoptosis.Apoptosis draws more and more attention nowadays because it is a delayed cell death and can be controlled and regulated to some extent.Mitochondria play a key role in apoptosis.For one reason,it is in the centre of intrinsic apoptotic signal system.For another,it is involved in extrinsic apoptotic signals via Bid.In the part,the protective role of HN against neuronal apoptosis was to explore since it protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(1μmol/L),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.6h later,neurons were collected and subjected to electron microscopy,TUNEL staining,the measurement of the activation of caspase-3,caspase-8 and caspase-9,and the detection of sub-summit of PI by FCM.By electron microscopy,TUNEL staining and the measurement of the activity of caspase-3,NMDA-induced neuronal apoptosis were to confirm.By the quantitative analysis of the activity of caspase-3 and the sub-summit of PI,the protective effect of HN on NMDA-induced neuronal apoptosis were to evaluate.By comparing the activity of caspase-8 and caspase-9 in different groups,the mechanism underlying HN's protective role against neuronal apoptosis were to investigate.By two caspase inhibitors, caspase-8 inhibitor(IETD)and general caspase inhibitor(VAD),and the detection of MTT and LDH release,the share of HN's inhibiting apoptosis in its protective effect against excitatory neurotoxicity were to evaluate.By caspase-8 inhibitor(IETD)and the detection of NO,the effect of extrinsic apoptosis signals on the function of mitochondria was to explore.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:1)Electron microscopy,TUNEL staining and the measurement of caspase-3 suggested that NMDA induce neuronal apoptosis.The quantitative analysis of sub-summit of PI and caspase-3 showed that HN rescued cortical neurons from NMDA-induced apoptosis;2)The detection of caspase-8 and caspase-9 indicated that NMDA triggered neuronal apoptosis by both extrinsic and intrinsic apoptosis signals.HN attenuated neuronal apoptosis induced by NMDA via extrinsic apoptotic pathway;3)With caspase-8 inhibitor(IETD)and the detection of MTT and LDH release,it was believed that the contribution of neuronal apoptosis mediated by extrinsic apoptotic signal was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was,therefore,limited;4)With general caspase inhibitor(VAD) and the detection of MTT and LDH release,it was believed that the share of caspase-dependent neuronal apoptosis was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was,therefore,limited;5)With caspase-8 inhibitor(IETD)and the detection of NO,excessive NO triggered by NMDA was found to be inhibited suggesting that HN attenuates the excessive production of NO induced by NMDA by interfering with extrinsic apoptotic signal.
Conclusions:1)HN attenuates NMDA-induced neuronal apoptosis;2)HN attenuates NMDA-induced neuronal apoptosis by interfering with extrinsic apoptotic signal pathway;3) The contribution of HN to inhibition of extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was limited;4)the contribution of HN to inhibition of caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was limited;5)HN attenuates NO mediated neurotoxicity induced by NMDA at least in part via interfering with extrinsic apoptotic signal.
The conclusion in all:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate cortical neuronal model of excitatory neurotoxicity with mitochondrial dysfunction and neuronal apoptosis;2)HN rescues cortical neurons from NMDA induced excitatory neurotoxicity with efficiency;3)HN attenuates NMDA-triggered mitochondrial dysfunction;4)HN inhibits NMDA-induced neuronal apoptosis by interfering with the activity of extrinsic apoptotic signals;5)HN inhibits NO mediated neurotoxicity triggered by NMDA at least in part by interfering with the activity of extrinsic apoptotic signals.
然而,有关HN神经保护作用的细胞机制提示,HN的神经保护作用可能不只限于AD相关的神经毒。(1)HN分布于脑组织以外的其他组织及不同的动物种属,即HN的作用不只局限于神经组织;(2)HN可拮抗线粒体功能障碍;(3)HN可抑制不同途径的凋亡;(4)HN可拮抗Aβ引起的细胞内钙超载。HN的这些作用特点强烈提示,HN极有可能具有广泛的神经保护作用,而不只是针对某种特定损伤(如AD相关毒性)而产生保护作用的神经保护肽。因此,有必要在AD以外的损伤模型中进行验证并分析其机制,而谷氨酸(NMDA)造成的兴奋性神经毒由于具有使用普遍,毒性作用强,损伤机制复杂,临床意义重要等特点,因此成为验证HN具有广泛的神经保护作用的最理想的模型。基于上述假设,我们拟进行如下观察:1)采用培养的大鼠大脑皮层神经元,通过MTT、LDH及胞内Ca~(2+)测定,建立NMDA诱导的兴奋性神经毒的细胞模型,通过氧自由基(reactive oxygenspecies,ROS)、NO检测,评价NMDA引起的线粒体损伤,通过电镜观察、TUNEL染色、caspase-3活性测定评价NMDA引起的神经元凋亡,并通过检测caspase-8和caspase-9的活性,初步分析外源性及内源性凋亡途径在神经元凋亡中发挥的作用;2)通过MTT、LDH、Calcein染色及胞内Ca~(2+)测定,观察HN对NMDA引起的兴奋性神经毒的拮抗作用;3)通过激光共聚焦、流式细胞术及生化方法检测琥珀酸脱氢酶的活性、ROS水平、NO水平及线粒体膜电位的改变,观察HN对NMDA引起的线粒体损伤的拮抗作用;4)通过电镜观察、TUNEL染色、流式细胞术、激光共聚焦及生化检测,观察HN对NMDA诱发的凋亡的拮抗作用及机制;并通过检测LDH、MTT,结合各种Caspase抑制剂的使用,分析HN通过抑制凋亡对抑制兴奋性神经毒作用的贡献,即大致区分HN通过抑制凋亡或坏死在抑制兴奋性神经毒中发挥的作用。通过以上观察验证我们的假说,即HN不只是拮抗AD相关毒性的特异性神经保护肽,而是具有广谱保护作用的内源性神经活性多肽。由于HN主要在大脑皮层表达,又具有广谱神经保护作用,因此,HN将可能通过拮抗包括AD在内的神经退行性疾病及其他神经病理损伤发挥重要作用。
第一部分:NMDA诱导的大鼠大脑皮层神经元神经毒性作用观察及机制探讨
兴奋性神经毒是一个广泛应用的神经损伤模型。虽有相关报道,但目前兴奋性神经毒的细胞模型在应用NMDA的浓度、时间上差别很大。因此,为客观评价HN的神经保护作用,首先有必要摸索NMDA作用的浓度及时间,建立一个稳定的神经毒模型,以利于后续的一系列实验观察。
方法:大鼠大脑皮层神经元培养,纯度达90%左右时,通过倒置显微镜下形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca~(2+)的动态测定,摸索NMDA毒性诱导的适当浓度及时间,建立兴奋性神经毒的细胞模型。并通过ROS、NO检测,分析模型中线粒体的损伤情况;通过电镜观察、TUNEL染色、caspase-3测定,研究模型中神经元的凋亡情况;并进一步通过检测模型中caspase-8、caspase-9的活性,分析外源性和内源性凋亡途径在神经元凋亡中发挥的作用。所有数据均经SPSS10.0处理获得,以均数±标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:NMDA与皮层神经元共同孵育,引起了培养的皮层神经元形态学的改变,以及由NMDA造成的时间和浓度依赖性的神经元细胞活力的下降。而且,NMDA(100μmol/L,2h)造成了大鼠大脑皮层神经元细胞活力下降了47%,LDH释放增加66%,皮层神经元内Ca~(2+)的快速升高,并维持在高水平形成一平台,ROS和NO的生成明显增加,电镜下凋亡形态出现,TUNEL染色阳性,caspase-3、caspase-8和caspase-9活性明显升高。
上述结果提示:NMDA(100μmol/L)作用2h诱发了皮层神经元明显的细胞毒性,是一个比较理想的兴奋性神经毒模型。该模型观察到了细胞整体的结构及功能的损伤(MTT下降、LDH释放增加、Ca~(2+)的快速升高),也观察到了线粒体功能的损伤。此外,该剂量的NMDA也诱发了皮层神经元的凋亡,内源性和外源性凋亡途径同时介导了该凋亡过程。
第二部分:Humanin拮抗NMDA诱导的兴奋性神经毒的作用观察
虽然HN被认为是AD特异或AD相关毒性(AD-related insults)的神经保护肽,然而,目前已积累的有关HN的细胞、分子水平的作用机制强烈提示,HN极有可能具有广泛的神经保护作用。本部分内容拟在前期建立的大鼠大脑皮层神经元的兴奋性神经毒细胞模型上,观察HN是否通过拮抗兴奋性神经毒而发挥保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况;实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(各种浓度)组。用Fluro-3进行Ca~(2+)探针标记,在激光共聚焦显微镜下,按以上分组要求加入干预,对神经元内Ca~(2+)浓度进行动态检测,每660ms采集一次数据,共采集300次,即对每个细胞跟踪观察198s;NMDA毒性诱导24h后,检测培养液中LDH的活性,以反映神经元的受损情况;收集细胞进行MTT测定,评价存活神经元的生活状态;收集细胞行Calcein染色,反映神经元的存活数量,即通过LDH、MTT和calcein染色对神经毒进行评价。所有数据均经SPSS10.0处理、获得,以均数±标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:1)HN抑制了NMDA引起的神经元内Ca~(2+)浓度的升高。在跟踪观察的198s时间内,NMDA引起了神经元内Ca~(2+)的快速升高,并始终维持在高水平形成一平台;HN(0.1μmol/L)不能拮抗NMDA升高Ca~(2+)的作用;HN(1μmol/L)虽未能阻止NMDA升高神经元内Ca~(2+)的作用,但能使升高的Ca~(2+)迅速降至对照水平。2)HN抑制了NMDA引起的存活神经元的减少。Calcein染色显示,NMDA引起了存活神经元数量的明显减少,为对照组的51.3%;预先应用HN(100μmol/L)几乎完全抑制了NMDA引起的神经元减少,存活神经元恢复到98.6%。3)HN抑制了NMDA引起的神经元细胞活力的下降。MTT检测提示,NMDA引起细胞活力下降约47%,而HN浓度依赖性地抑制了NMDA引起的神经元细胞活力的下降;4)HN降低了NMDA引起的神经元LDH的释放。虽然HN不能完全阻断NMDA引起的LDH释放,但HN可剂量依赖性地使之降低。
上述结果提示:1)HN抑制了NMDA引起的神经元内Ca~(2+)浓度的升高;2)HN阻止了NMDA引起的细胞活力的下降,抑制了NMDA所致的LDH释放,拮抗了NMDA所致存活神经元的减少。即HN可通过拮抗NMDA引起的兴奋性神经毒而发挥神经保护作用。
第三部分:Humanin拮抗NMDA诱导的线粒体损伤的作用观察
谷氨酸(NMDA)诱导的兴奋性神经毒通过过度激活NMDA受体,引起细胞内Ca~(2+)超载,造成神经元损伤,直至凋亡或坏死。线粒体既是细胞的能量工厂,又是重要的Ca~(2+)库,在缓解细胞Ca~(2+)超载方面发挥重要作用。然而,当线粒体缓解Ca~(2+)的能力超过极限时,线粒体功能也遭到破坏,因此,兴奋性神经毒可造成线粒体功能的损伤。由于前期的实验观察已证实,HN可拮抗NMDA诱导的兴奋性神经毒,包括抑制NMDA诱导的细胞内Ca~(2+)的升高,本部分内容拟观察HN通过拮抗NMDA造成的线粒体损伤而发挥的神经保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况。实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(各种浓度)组。NMDA毒性作用2h后,收集培养介质进行NO含量的定量分析;收集细胞,用JC-1标记线粒体膜电位,用DCFH-DA标记ROS,通过流式细胞术分析标记荧光的荧光强度,通过激光共聚焦显微镜检测提供形态学依据,从而对ROS含量和线粒体膜电位进行检测;NMDA毒性作用24h后,收集细胞,通过MTT对琥珀酸脱氢酶活性进行分析。所有数据均经SPSS10.0处理、获得,以均数土标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.05。
结果:1)HN抑制了NMDA引起的琥珀酸脱氢酶活性的降低。MTT检测提示,NMDA使琥珀酸脱氢酶活性下降约47%,而HN浓度依赖性地抑制了NMDA的作用;2)HN阻止了NMDA引起的线粒体膜电位(Δψm)的降低。用JC-1标记线粒体膜电位,通过激光共聚焦显微镜和流式细胞术检测JC-1的红色荧光强度。JC-1在具有正常膜电位的线粒体中发出红色荧光,因此,细胞红色荧光越强,表示具有的正常线粒体越多,反之,细胞中的线粒体膜电位下降越多。激光共聚焦显微镜下照片显示,与对照组相比,NMDA引起了线粒体膜电位下降,表明HN抑制了NMDA对线粒体膜电位的作用。用流式细胞术对JC-1的红色荧光强度进行了定量分析,结果与激光共聚焦显微镜检测一致;3)HN抑制了NMDA引起的ROS的生成。用DCFH-DA标记细胞,通过激光共聚焦显微镜和流式细胞术检测DCF的荧光强度。激光共聚焦显微镜下照片显示,与对照组相比,NMDA引起了ROS的生成增加;HN阻止了NMDA引起的ROS的生成增加,表明HN抑制了NMDA对ROS生成的影响。用流式细胞术对DCF荧光强度进行了定量分析,结果与激光共聚焦显微镜检测一致;4)HN降低了NMDA引起的NO生成。与对照组相比,NMDA引起NO生成明显升高;而提前应用HN则可明显抑制NMDA引起的NO生成,与对照组相似。
上述结果提示:1)HN抑制了NMDA引起的琥珀酸脱氢酶活性的降低;2)HN抑制了NMDA引起的线粒体膜电位(Δψm)的降低;3)HN抑制了NMDA引起的ROS和NO的生成。即HN可能通过拮抗线粒体功能损伤在抑制兴奋性神经毒中发挥神经保护作用。
第四部分:Humanin拮抗NMDA诱导的神经元凋亡的作用观察
兴奋性神经毒使神经元以凋亡或坏死两种形式死亡。由于细胞凋亡是迟发性的、程序性的细胞死亡,因此被认为是可控制和调节的,凋亡也因此成为研究的热点。线粒体介导的内源性凋亡途径是重要的凋亡途径之一,而且还可以借助于Bid与外源性途径发生联系。因此,线粒体在凋亡的发生中处于无可替代的重要地位。我们的前期研究发现,HN可拮抗NMDA诱导的兴奋性神经毒,而且可拮抗NMDA诱导的线粒体损伤。那么,HN是否可抑制NMDA诱导的神经元凋亡呢?本部分内容拟观察HN通过拮抗NMDA诱导的神经元凋亡而发挥的神经保护作用。
方法:原代培养大鼠大脑皮层神经元,通过倒置显微镜对培养细胞进行观察并确认NMDA毒性诱导的情况;实验共分5大组:1)对照组,正常培养的细胞;2)HN组,在培养的细胞中添加HN(10μmol/L);3)NMDA组,亦即兴奋性神经毒的模型组,在培养液中加入NMDA(100μmol/L)和Gly(10μmol/L),作用2h,观察NMDA引起的毒性作用;4)NMDA+MK-801(10μmol/L)组;5)NMDA+HN(1μmol/L)组。在毒性作用6h后,收集细胞进行电镜观察、TUNEL染色、caspase-3活性测定,证实NMDA诱导神经元凋亡的发生;通过PI染色、流式细胞术定量分析及caspase-3活性测定,评价HN对凋亡的拮抗作用。通过caspase-8、caspase-9活性测定,评价外源性及内源性凋亡途径在NMDA诱导的兴奋性神经毒中的作用,以及HN对外源性及内源性凋亡途径的抑制作用。通过使用caspase-8和总caspases抑制剂,结合MTT和LDH检测,初步分析HN通过抑制凋亡和(或)坏死在抑制兴奋性神经毒发生中的作用。通过使用caspase-8抑制剂,结合NO检测,分析兴奋性神经毒发生时外源性凋亡途径对线粒体功能的影响。所有数据均经SPSS10.0处理、获得,以均数士标准差((?)±s)表示,组间均数的比较用one-way ANOVA分析和Dunnett多重比较,显著性水平设为0.35。
结果:1)电镜观察、TUNEL染色、caspase-3活性测定一致表示,NMDA诱导了神经元凋亡,而PI的流式细胞术定量分析及caspase-3活性测定显示,HN可抑制NMDA诱导的神经元凋亡。2)caspase-8和caspase-9活性测定显示,外源性及内源性凋亡途径共同参与了NMDA诱导的兴奋性神经毒中神经元的凋亡,而HN对外源性凋亡途径所致的神经元凋亡有一定的抑制作用。3)通过使用caspase-8的抑制剂(IETD)并结合MTT和LDH检测,发现外源性凋亡途径在NMDA诱导的兴奋性神经毒中的贡献不大。因此,HN通过抑制外源性凋亡途径对NMDA诱导的兴奋性神经毒发挥的神经保护作用也是有限的。4)通过使用总caspases抑制剂(VAD)并结合MTT和LDH检测发现,caspase依赖的凋亡在NMDA诱导的兴奋性神经毒中的贡献不大。因此,HN通过抑制caspase依赖的凋亡对NMDA诱导的兴奋性神经毒而发挥的神经保护作用也是有限的。5)通过使用caspase-8抑制剂,结合NO检测,发现caspase-8被抑制后NO水平降低,因此,HN可能通过抑制外源性凋亡途径影响NO的生成。
上述结果提示:1)HN对NMDA诱发的皮层神经元的凋亡具有一定的抑制作用;2)HN可能通过抑制外源性途径抑制NMDA诱导的凋亡;3)HN通过抑制caspases依赖的凋亡在抑制兴奋性神经毒中的作用并不明显;4)HN可能通过抑制caspase-8的激活而抑制NO的生成,从而抑制NMDA通过NO造成的神经损伤。
结论:1)NMDA(100μmol/L)作用2h诱发了皮层神经元明显的细胞毒性,造成了线粒体损伤和神经元凋亡,是一个比较理想的兴奋性神经毒模型;2)HN可通过拮抗NMDA引起的兴奋性神经毒而发挥神经保护作用;3)HN可能通过拮抗线粒体功能损伤在抑制兴奋性神经毒中发挥神经保护作用;4)HN可抑制NMDA引起的外源性凋亡途径介导的神经元凋亡,并可抑制caspase-8介导生成的NO造成的神经损伤。
Humanin(HN)is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzhermer's disease(AD)in 2001.Studies showed that HN protected neurons from insults of various familial Alzheimer's disease mutations(APP、PS-1 and PS-2),anti-APP(amyloid precussor protein,APP)antibodies, amyloid peptide(Aβ)(Aβ1-43 or Aβ1-42)and its segments(Aβ25-35、Aβ31-35)with efficiency.It seemed that HN didn't inhibit other toxic insults to neurons,such as Fas,etoposide, glutamate and NMDA triggered neurotoxicity.HN was,therefore,considered as a selectively neuroprotective factor rescuing neurons from Alzheimer's disease-related insults.
Although HN was firstly identified from human brain with Alzheimer's disease,more and more its homologues have been discovered in other species of animals and its location is also far beyond brain,suggesting that the role of HN be versatile other than just attenuating Alzheimer's disease-related insults.In support of this notion,other investigators including our lab have done some works to decipher the neuroprotective mechanism of HN at celluar and molecular level:1)to elevate ATP level,which is necessary for any cells to survive stresses;2) to block apoptosis mediated by extracelullar signals as well as intrinsic signals;3)to interfere with intracellular Ca~(2+)accumulation induced by amyloid beta peptide(Aβ).
The mechanisms underling the protective activity of HN mentioned above imply that it may play versatile protective roles against various insults other than as an AD- selective neuroprotective peptide.To verify the hypothesis,we employed NMDA induced neurotoxicity, an in vitro paradigm of neurodegeneration representative of pathologically neurotoxic insults of excitotoxicity which brings heavy damage to neurons by complicated mechanisms and is implicated in most of the neurodegenerative processes,to confirm the versatile neuroprotective role of HN in this in vitro insult model.The program of this research are as followings:1) Build an excitatory neurotoxic model in cultured cortical neurons of rat by measurement MTT, LDH in the medium and Ca~(2+)concentration in the cytoplasm,which reflect general NMDA-induced neurotoxicity;evaluate the mitochondrial damage in the model by detecting ROS and NO;and assay the neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,and analysis the apoptotic pathways involved in by the measurement of the activities of caspase-8 and caspase-9;2)Evaluate the neuroprotective effects of HN on NMDA induced neurotoxicity by detecting Ca~(2+)concentration in cytoplasm,MTT,Calcein staining and LDH release in the medium;3)Evaluate the protective effect of HN on NMDA-induced mitochondrial damage by determining the activity of fumaric reductase(MTT),the level of ROS and NO,and mitochondrial membrane potential;4)Evaluate the protective effects of HN on NMDA-induced neuronal apoptosis by morphological observation of neurons with electron microscopy,TUNEL staining,the measurement of the activity of caspase-3,and sub-summit of PI,analysis the potential protective pathways of HN against apoptosis by the measurement of the activities of caspase-8 and caspase-9,and evaluate the contribution of HN in attenuating NMDA-induced neurotoxicity by blocking neuronal apoptosis with the aid of caspase inhibitors.Our results showed that:1)HN blocked NMDA induced neurotoxicity by attenuating LDH release and Ca~(2+)overloading,and rescue cortical neurons from excitatory neurotoxicity with the increase of MTT and Calcein staining cells;2)HN antagonized mitochondrial dysfunction by suppressing ROS and NO with the increase of mitochondrial membrane potential and the activity of fumaric reductase;3)HN inhibited neuronal apoptosis by interfering extrinsic apoptotic signals involved in caspase-dependent pathway as well as neurotoxicity mediated by NO.
Part One:NMDA-Induced Neurotoxicity in Cortical Neurons and Potential Mechanisms Underlying
To evaluate the neuroprotective effect of HN on excitatory neurotoxicity,an appropriate cellular model must be employed which have definite and moderate neuronal damage and loss.The degree of neuronal loss triggered by NMDA largely depends on the concentration of NMDA in the medium and the time it works.But the concentration of NMDA and the time it works varied dramatically in reported models currently.To find a reasonable concentration of NMDA and the time it works is therefore a key factor in this part.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.An appropriate concentration of NMDA and the time it works were explored by monitoring morphological changes under contrast phase microscope,MTT,LDH release in the medium,and Ca~(2+) concentration in the cytoplasm so as to build an appropriate cellular model in cortical neurons in rat.ROS and NO were detected to evaluate the damage of mitochondria in the model.Electron microscopy,TUNEL staining,and the measurement of the activation of caspase-3,caspase-8 and caspase-9 were employed to evaluate neuronal apoptosis and analysis the pathways involved in the model.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:The administration of NMDA in the cultures caused pathological changes of morphology and cell viability decrease dose-dependently and time-dependently.NMDA (100μmol/L,2h)caused cell viability decrease by about 47%and LDH release increase by about 66%compared with control.It triggered the rise of Ca~(2+)in cytoplasm and the maintenance of the highest level during the observation.Excessive ROS and NO were detected in the model.NMDA also caused the appearance of neuronal apoptosis suggested by electron microscopy,TUNEL staining.Caspase-3,caspase-8 and caspase-9 were activated in the model.
Conclusions:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate concentration and duration to cause excitatory neurotoxicity;2)Mitochondria are damaged in the model;3)NMDA(100μmol/L,2h)triggers neuronal apoptosis mediated both by extrinsic and intrinsic apoptotic signal pathways.
Part Two:Humanin Attenuates NMDA-Induced Excitatory Neurotoxicity
Although HN was considered as a selectively neuroprotective factor against AD-related insults, the mechanism underlying its neuroprotective functions at molecular and cellular level strongly suggests that HN plays protective roles for neurons at various stresses other than AD-related insults.The current research was to investigate the protective role of HN against excitatory neurotoxicity.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L) and Gly 10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L) was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected,labeled with Fluo-3 and subjected to laser scanning confocl microscopy.Dynamic changes of cytoplasmic Ca~(2+)were captured and recorded every 660ms and 300 records(for about 198s)were needed for each neuron.24h after NMDA treatment,the medium was collected for the measurement of LDH. Neurons were subjected to MTT detection and Calcein staining.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:1)HN inhibits the increase of cytoplasmic Ca~(2+)concentration induced by NMDA. NMDA triggered the rapid increase of cytoplasmic Ca~(2+)concentration and maintenance of the highest level during the observation.From the control level to the highest one,it took only 2s. HN(0.1μmol/L)has no effect on interfering with NMDA-induced increase of cytoplasmic Ca~(2+) concentration.Although HN(1μmol/L)didn't block the increase of cytoplasmic Ca~(2+) concentration induced by NMDA,it caused dropping of the Ca~(2+)concentration;2)HN inhibited the loss of living neurons caused by NMDA.NMDA triggered the loss of neurons by 51.3%compared with control showed by Calcein staining.Pretreated with HN(100μmol/L), the loss of neurons was almost blocked completely.The rate of living cells reached 98.6%of control;3)HN blocked the decrease of cell viability caused by NMDA.NMDA caused cell viability dropping by 47%showed by MTT.HN blocked the toxic effect of NMDA and attenuated the decrease dose-dependently;4)HN neutralized the LDH release triggered by NMDA.Compared with control,additional LDH release(about 66%of control)was detected when neurons were treated with NMDA.HN inhibited the additional release of LDH does-dependently although it didn't stop it completely.
Conclusions:1)HN inhibits the increase of cytoplasmic Ca~(2+)concentration induced by NMDA; 2)HN inhibits the loss of living nerurons,blocks the decrease of cell viability,and neutralizes the additional release of LDH when neurons confront with NMDA-induced extitatory stress. HN,therefore,rescues cortical neurons from NMDA-triggered excitatory neurotoxicity effectively.
Part Three:Humanin Attenuates NMDA-Induced Mitochondria Dysfunction
Excitatory neurotoxicity is triggered by the overloading of Ca~(2+)for the overactivation of NMDA receptor.Since mitochondria is an important Ca~(2+)pool beside producing ATP,it plays an important role in soothing excessive Ca~(2+)in the situation.Once more Ca~(2+)is uptaken into mitochondria than its capacity,mitochondria itself is damaged and loss normal function of producing ATP.In the part,the protective role of HN against mitochondria dysfunction caused by NMDA were to investigate since we have confirmed its neuroprotective effect on NMDA-induced excitatory neurotoxicity and its inhibitory effect on Ca~(2+)increase triggered by NMDA.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(various concentration),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.2h later,neurons were collected and labeled with JC-1 for mitochondria membrane potential or DCFH-DA for ROS.Neurons were subjected to laser scanning confocl microscopy and FCM.The medium was collected for the detection of NO.24h later,neurons were collected for the determination of fumaric reductase by MTT.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D. Means of different groups were compared by ANOVA,followed by the Dunnett's test. Significance level was 0.05.
Results:1)HN blocked the decrease of the activity of fumaric reductase.NMDA triggered the decrease of the activity of fumaric reductase by about 47%of control.HN inhibited the effect of NMDA dose-dependently showed by MTT;2)HN attenuated the drop of mitochondrial membrane potential caused by NMDA.The red fluorescent intensity of JC-1 labeled on neurons for mitochondrial membrane potential was detected and analyzed both by laser scanning confocal microscopy and FCM.The fluorescent intensity decreases when treated with NMDA. When HN(1μmol/L)and NMDA coincubated with the neurons,the decrease of the fluorescent intensity of JC-1 was inhibited;3)HN inhibited the excessive production of ROS triggered by NMDA.The measurement of fluorescent intensity of DCFH-DA for ROS by laser scanning confocal microscopy and FCM showed that NMDA induces excessive production of ROS and HN(1μmol/L)inhibited the production of ROS caused by NMDA.4)HN neutralized the excessive production of NO.Compared with control,NMDA triggered more NO and HN attenuated the activity of NMDA.
Conclusions:1)HN blocks the decrease of the activity of fumaric reductase;2)HN attenuates the drop of mitochondrial membrane potential caused by NMDA.3)HN inhibits the excessive production of ROS triggered by NMDA;4)HN neutralizes the excessive production of NO. HN,therefore,protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.
Part Four:Humanin Attenuates NMDA-Induced Neuronal Apoptosis
Excitatory neurotoxicity causes neuronal loss by necrosis and apoptosis.Apoptosis draws more and more attention nowadays because it is a delayed cell death and can be controlled and regulated to some extent.Mitochondria play a key role in apoptosis.For one reason,it is in the centre of intrinsic apoptotic signal system.For another,it is involved in extrinsic apoptotic signals via Bid.In the part,the protective role of HN against neuronal apoptosis was to explore since it protects cortical neurons from NMDA-induced excitatory neurotoxicity by attenuating mitochondrial dysfunction.
Methods:Cortical neurons from neonatal rat(P1-3)were primarily cultured.For the experiment, cortical neurons were grouped as following:1)control,cultured neurons;2)HN,neurons incubated with HN(10μmol/L);3)NMDA,neurons coincubated with NMDA(100μmol/L)and Gly(10μmol/L)for 2h;4)NMDA + MK-801(10μmol/L),additional MK-801(10μmol/L)was needed compared with NMDA group;5)NMDA + HN(1μmol/L),having extra HN compared with NMDA group.Primary cultures were treated with chemicals as grouped.HN was administrated 16h in advance.6h later,neurons were collected and subjected to electron microscopy,TUNEL staining,the measurement of the activation of caspase-3,caspase-8 and caspase-9,and the detection of sub-summit of PI by FCM.By electron microscopy,TUNEL staining and the measurement of the activity of caspase-3,NMDA-induced neuronal apoptosis were to confirm.By the quantitative analysis of the activity of caspase-3 and the sub-summit of PI,the protective effect of HN on NMDA-induced neuronal apoptosis were to evaluate.By comparing the activity of caspase-8 and caspase-9 in different groups,the mechanism underlying HN's protective role against neuronal apoptosis were to investigate.By two caspase inhibitors, caspase-8 inhibitor(IETD)and general caspase inhibitor(VAD),and the detection of MTT and LDH release,the share of HN's inhibiting apoptosis in its protective effect against excitatory neurotoxicity were to evaluate.By caspase-8 inhibitor(IETD)and the detection of NO,the effect of extrinsic apoptosis signals on the function of mitochondria was to explore.Data coming from 5-8 separate experiments were subjected to SPSS10.0 and were expressed as mean±S.D.Means of different groups were compared by ANOVA,followed by the Dunnett's test.Significance level was 0.05.
Results:1)Electron microscopy,TUNEL staining and the measurement of caspase-3 suggested that NMDA induce neuronal apoptosis.The quantitative analysis of sub-summit of PI and caspase-3 showed that HN rescued cortical neurons from NMDA-induced apoptosis;2)The detection of caspase-8 and caspase-9 indicated that NMDA triggered neuronal apoptosis by both extrinsic and intrinsic apoptosis signals.HN attenuated neuronal apoptosis induced by NMDA via extrinsic apoptotic pathway;3)With caspase-8 inhibitor(IETD)and the detection of MTT and LDH release,it was believed that the contribution of neuronal apoptosis mediated by extrinsic apoptotic signal was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was,therefore,limited;4)With general caspase inhibitor(VAD) and the detection of MTT and LDH release,it was believed that the share of caspase-dependent neuronal apoptosis was limited in NMDA-induced neurotoxicity.The contribution of HN's inhibiting caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was,therefore,limited;5)With caspase-8 inhibitor(IETD)and the detection of NO,excessive NO triggered by NMDA was found to be inhibited suggesting that HN attenuates the excessive production of NO induced by NMDA by interfering with extrinsic apoptotic signal.
Conclusions:1)HN attenuates NMDA-induced neuronal apoptosis;2)HN attenuates NMDA-induced neuronal apoptosis by interfering with extrinsic apoptotic signal pathway;3) The contribution of HN to inhibition of extrinsic apoptotic signal-mediated apoptosis to its protective effect on NMDA-induced nerotoxicity was limited;4)the contribution of HN to inhibition of caspase-dependent neuronal apoptosis to its protective effect on NMDA-induced neurotoxicity was limited;5)HN attenuates NO mediated neurotoxicity induced by NMDA at least in part via interfering with extrinsic apoptotic signal.
The conclusion in all:1)NMDA(100μmol/L,2h)triggers obvious neuronal loss and is considered as an appropriate cortical neuronal model of excitatory neurotoxicity with mitochondrial dysfunction and neuronal apoptosis;2)HN rescues cortical neurons from NMDA induced excitatory neurotoxicity with efficiency;3)HN attenuates NMDA-triggered mitochondrial dysfunction;4)HN inhibits NMDA-induced neuronal apoptosis by interfering with the activity of extrinsic apoptotic signals;5)HN inhibits NO mediated neurotoxicity triggered by NMDA at least in part by interfering with the activity of extrinsic apoptotic signals.
引文
[1] Bogaert L, Scheller D, Moonen J, et al. Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia [J]. Brain Res, 2000,887:266-275
[2] Ferrari R, Ceconi C, Curello S, et al. Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity [J]. J Mol Cell Cardiol, 1985,17: 937-945
[3] Benveniste H, Drejer J, Schousboe A, et al. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis [J]. J Neurochem, 1984,43:1369-1374.
[4] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMD A receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[5] Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol., 1992,3(9): 1261-1276
[6] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels:high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996, 16(17):5457-5465.
[7] Artal-Sanz M, Tavernarakis N. Proteolytic mechanisms in necrotic cell death and neurodegeneration [J]. FEBS Lett. 2005 Jun 13;579(15):3287-3296.
[8] Dugan LL, Sensi SL, Canzoniero LM, et al. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate [J]. J Neurosci.,1995,15(10): 6377-6388.
[9] Dawson VL, Dawson TM, London ED, et al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures [J]. Proc Natl Acad Sci U S A., 1991, 88(14): 6368-6371
[10] Lucas DR, Newhouse JP. The toxic effect of sodium L-glutamate on the inner layers of the retina [J]. AMA Arch Ophthalmol, 1957, 58(2): 193-201.
[11]Olney JW. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate [J]. Science, 1969,164(880): 719-721.
[12]Inomata Y, Nakamura H, Tanito M, et al. Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina [J]. J Neurochem, 2006,98(2): 372-385.
[13] Zhang Q, Zhang J. Effect of melatonin on the spatial and temporal changes of [Ca2+] i in single living cells of cortical neurons by laser scanning confocal microscopy [J]. Chin Med J (Engl)., 2000,113(6): 558-562
[14]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[15]Hetman M, Gozdz A. Role of extracellular signal regulated kinases 1 and 2 in neuronal survival [J]. Eur J Biochem., 2004,271(11): 2050-2055
[16]Chu C, Alapat D, Wen X, et al. Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway [J].Cell Signal. 2004,16(9): 1045-1059
[17] Wang H, Yu SW, Koh DW, et al. Apoptosis-Inducing Factor Substitutes for Caspase Executioners in NMDA-Triggered Excitotoxic Neuronal Death [J]. J. Neurosci., 2004,24(48):10963-10973
[18] Koh JY, Choi DW. Vulnerability of cultured cortical neurons to damage by excitotoxins:differential susceptibility of neurons containing NADPH-diaphorase [J]. J Neurosci., 1988,8(6): 2153-2163.
[19] Yuan J, ipinski M, Degterev A. Diversity in the mechanisms of neuronal cell death [J].Neuron, 2003,40: 401-413.
[20]Benn SC, Woolf CJ. Adult neuron survival strategies - slamming on the brakes [J]. Nat.Rev. Neurosci., 2004, 5: 686-700
[21]Nicotera P, Leist M, Manzo L. Neuronal cell death: a demise with di.erent shapes [J].Trends Pharmacol. Sci, 1999,20: 46-51.
[22]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J].Neurochemical Research, 2003,28( 8): 1193-1199
[23]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[24] Kim JB, Sig Choi J, Yu YM, et al. HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain [J]. J Neurosci.2006 Jun 14;26(24):6413-2
[25] Shamloo M, Soriano L, Wieloch T, et al. Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia [J]. J Biol Chem., 2005, 280(51):42290-42299.
[26] Shirakawa H, Katsuki H, Kume T, et al. Aminoglutethimide prevents excitotoxic and ischemic injuries in cortical neurons [J]. Br J Pharmacol, 2006,147(7): 729-736.
[27]Kim SH, Won SJ, Mao XO, et al. Molecular mechanisms of cannabinoid protection from neuronal excitotoxicity [J]. Mol Pharmacol., 2006,69(3): 691-696.
[28]Ha HJ, Kwon YS, Park SM, et al. Quercetin attenuates oxygen-glucose deprivation- and excitotoxin-induced neurotoxicity in primary cortical cell cultures [J]. Biol Pharm Bull. 2003 Apr;26(4):544-546.
[29]Kang HJ, Choi YS, Hong SB, et al. Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity [J]. J Neurosci. 2004 Feb 11;24(6):1280-1287.
[30] Rosenberg PA, Amin S, Leitner M. Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture [J]. J Neurosci., 1992,12(1):56-61
[31]Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme [J].FEBS J., 2006,273(3): 433-450.
[1] Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. Proc Natl Acad Sci USA, 2001,98:6336-6341.
[2] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. Neurosci, 2001, 21:9235-9245.
[3] Lin-min Li, Ai-ling Cui, Jian Wang, et al. Effects of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[4] Tauima H, Kawasumi M, Chiba T, et al. A humanin derivative, SGly14-Humanin, prevents amyloid-beta-induced memory impairment in mice. J Neurosci Res, 2005,79: 714-723
[5] Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol, 2001,134(8): 1597-1599.
[6] Krejcova G, Patcoka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats. J Pept Sci, 2004,10(10): 636 - 639
[7] Niikura T, Chiba T, Aiso S, et al. Humanin: after the discovery [J]. Mol Neurobiol., 2004,30(3): 327-340
[8] Niikura T, Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults [J]. Eur J Neurosci., 2003,17(6):1150-1158.
[9] Benaki D, Zikos C, Evangelou A, et al. Solution structure of Serl4Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease [J]. Biochem Biophys Res Commun. 2006 Oct 20;349(2):634-642
[10]Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005,64(6): 816-823
[11]Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[12] Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[13] Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[14] Colon E, Strand ML, Carlsson-Skwirut C, et al. Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis [J]. J Cell Physiol., 2006,208(2): 373-385.
[15]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[16]Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem, 2003,254(1-2): 83-89.
[17]Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA [J]. Neuropeptides, 2005,39(2005):97-101
[18]Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[19] Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[20]Lin-min Li, Ai-ling Cui, Jian Wang, et al. The mechanisms underlying the protection of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[21]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1 -40. Peptides, 2003,24(5): 679-685.
[22]Bogaert L, Scheller D, Moonen J, et al. Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia [J]. Brain Res, 2000,887:266-275
[23]Ferrari R, Ceconi C, Curello S, et al. Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity [J]. J Mol CellCardiol, 1985,17: 937-945
[24]Benveniste H, Drejer J, Schousboe A, et al. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis [J]. J Neurochem, 1984,43: 1369-1374.
[25]Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[26]Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol., 1992, 3(9): 1261-1276 Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996,16(17): 5457-5465.
[27] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996,16(17): 5457-5465.
[28]Dempsey RJ, Baskaya MK, Dogan A. Attenuation of brain edema, blood-brain barrier breakdown, and injury volume by ifenprodil, a polyamine-site N-methyl-D-aspartate receptor antagonist, after experimental traumatic brain injury in rats [J]. Neurosurgery, 2000, 47: 399-404
[29]Lipton SA , Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders [J]. N Engl J Med, 1994, 330: 613-622.
[30]Olney JW. Glutamate, a neurotoxic transmitter [J]. J Child Neurol, 1989,4: 218-226
[31] Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies [J]. Curr Drug Targets, 2001,2: 241-271
[32]Montoliu C, Llansola M, Monfort P, et al. Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death [J]. Neurotox Res, 2001,3(2):179-88.
[33]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J]. Neurochemical Research, 2003,28( 8): 1193-1199
[34]Brenneman DE, Hauser J, Neale E, et al. Activity-dependent neurotrophic factor: structure-activity relationships of femtomolar-acting peptides [J]. J Pharmacol Exp Ther,1998,285(2): 619-627.
[35] Mark RJ, Keller JN, Kruman I, et al. Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons [J]. Brain Res, 1997, 756(1-2): 205-14.
[36]Dore S, Kar S, Quirion R. Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity [J]. Proc Natl Acad Sci U S A, 1997, 94(9): 4772-47777
[37]Low SJ and Roland CL..Review of NMDA antagonist-induced neurotoxicity and implications for clinical development. Int J Clin Pharmacol Ther. 2004,42(1):1-14
[38]Lees KR, Asplund K, Carolei A, et al. Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomised controlled trial. GAIN International Investigators [J]. Lancet. 2000,355(9219): 1949-54
[39] Sacco RL,DeRosa JT, Haley EC Jr, et al. Glycine antagonist in neuroprotection for patients with acute stroke: GAIN Americas: a randomized controlled trial [J]. JAMA. 2001,285(13):1719-28
[40] Caricasole A, BrunoV,CappuccioI, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002(10): 1331-1333
[41]Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke [J]. Stroke., 2006,37(10): 2613-2619
[42]Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005,64(6): 816-823.
[43] Kin T, Sugie K, Hirano M, et al. Humanin expression in skeletal muscles of patients with chronic progressive external ophthalmoplegia [J]. J Hum Genet. 2006,51(6): 555-558.
[44]Kariya S, Hirano M, Furiya Y,et al. Humanin detected in skeletal muscles of MEL AS patients: a possible new therapeutic agent [J]. Acta Neuropathol (Berl). 2005, 109(4):367-372
[45] Chiba T, Yamada M, Sasabe J, et al. Colivelin prolongs survival of an ALS model mouse [J]. Biochem Biophys Res Commun., 2006,343(3): 793-798.
[46]Kariya S, Hirano M, Nagai Y, et al. Humanin attenuates apoptosis induced by DRPLA proteins with expanded polyglutamine stretches [J]. J Mol Neurosci. 2005,25(2): 165-169
[1] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[2] Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol, 1992,3(9): 1261-1276
[3] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels:high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996, 16(17):5457-5465.
[4] Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme [J]. FEBS J, 2006,273(3): 433-450.
[51 Campanella M, Pinton P, Rizzuto R. Mitochondrial Ca2+ homeostasis in health and disease [J].Biol Res, 2004,37(4): 653-660?
[6] Bernardi P. Mitochondrial transport of cations: channels , exchangers , and permeability transition. Physiol Rev, 1999,79(4): 1127-1155
[7] Di Paola M, Lorusso M. Interaction of free fatty acids with mitochondria: coupling, uncoupling and permeability transition [J]. Biochim Biophys Acta., 2006, 1757(9-10):1330-1337.
[8] Dutra F, Bechara EJ. Aminoacetone induces iron-mediated oxidative damage to isolated rat liver mitochondria [J]. Arch Biochem Biophys., 2004,430(2): 284-289
[9] Schild L, Huppelsberg J, Kahlert S, et al. Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration [J]. J Biol Chem, 2003,278(28): 25454-25460.
[10] Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. Proc Natl Acad Sci USA,2001,98:6336-6341.
[11] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. Neurosci, 2001,21:9235-9245.
[12]Lin-min Li, Ai-ling Cui, Jian Wang, et al. Effects of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[13]Tauima H, Kawasumi M, Chiba T, et al. A humanin derivative, SGly14-Humanin, prevents amyloid-beta-induced memory impairment in mice. J Neurosci Res, 2005, 79: 714-723
[14]Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol, 2001,134(8): 1597-1599.
[15]Krejcova G, Patcoka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats. J Pept Sci, 2004,10(10): 636 - 639
[16]Niikura T, Chiba T, Aiso S, et al. Humanin: after the discovery [J]. Mol Neurobiol., 2004,30(3): 327-340
[17]Niikura T, Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults [J]. Eur J Neurosci., 2003,17(6):1150-1158.
[18]Benaki D, Zikos C, Evangelou A, et al. Solution structure of Ser14Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease [J]. Biochem Biophys Res Commun. 2006 Oct 20;349(2):634-642
[19] Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005, 64(6): 816-823
[20] Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[21]Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[22] Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[23] Colon E, Strand ML, Carlsson-Skwirut C, et al. Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis [J]. J Cell Physiol, 2006,208(2): 373-385.
[24]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[25] Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem, 2003, 254(1-2): 83-89.
[26]Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA [J]. Neuropeptides, 2005,39(2005):97-101
[27]Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[28] Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[29]Lin-min Li, Ai-ling Cui, Jian Wang, et al. The mechanisms underlying the protection of Humanin on A β 31-35-induced apoptosis in cultured cortical neurons, (in submission)
[30]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1 -40. Peptides, 2003,24(5): 679-685.
[31] Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J].Neurochemical Research, 2003,28( 8): 1193-1199.
[1] Dempsey RJ, Baskaya MK, Dogan A. Attenuation of brain edema, blood-brain barrier breakdown, and injury volume by ifenprodil, a polyamine-site N-methyl-D-aspartate receptor antagonist, after experimental traumatic brain injury in rats [J]. Neurosurgery,2000,47: 399-404
[2] Lipton SA , Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders [J]. N Engl J Med, 1994,330: 613-622.
[3] Olney JW. Glutamate, a neurotoxic transmitter [J]. J Child Neurol, 1989,4:218-226
[4] Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies [J]. Curr Drug Targets, 2001,2: 241-271
[5] Montoliu C, Llansola M, Monfort P, et al. Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death [J]. Neurotox Res, 2001, 3(2): 179-88.
[6] Nath R, Scott M, Nadimpalli R, et al. Activation of apoptosis-linked caspase(s) in NMDA-injured brains in neonatal rats [J]. Neurochem Int, 2000, 36(2): 119-26.
[7] Okamoto S, Li Z , Ju C, et al. Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis [J]. PNAS, 2002, 99(6):3974-3979
[8] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003, 285: H2592-H2598.
[9] Low SJ and Roland CL..Review of NMDA antagonist-induced neurotoxicity and implications for clinical development. Int J Clin Pharmacol Ther. 2004,42(1): 1-14
[10] Lees KR, Asplund K, Carolei A, et al. Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomised controlled trial. GAIN International Investigators [J]. Lancet. 2000, 355(9219): 1949-54
[11] Sacco RL,DeRosa JT, Haley EC Jr, et al. Glycine antagonist in neuroprotection for patients with acute stroke: GAIN Americas: a randomized controlled trial [J]. JAMA.2001,285(13):1719-28
[12]Wu J, Anwyl R, Rowan MJ. β-amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus [J]. Neuroreport, 1995, 6(17): 2409-2413
[13]Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ [J]. Proc Natl Acad Sci USA, 2001, 98(11): 6336-6341.
[14] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults [J]. Neurosci, 2001,21(23): 9235-9245.
[15]Tajima H, Kawasumi M, Chiba T, et al. A humanin derivative, S14G-HN, prevents amyloid-beta-induced memory impairment in mice [J]. J Neurosci Res., 2005, 79(5):714-723.
[16]Krejcova G, Patocka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats [J]. J Pept Sci., 2004,10(10): 636-639
[17]Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol., 2001,134(8):1597-1599
[18] Hashimoto Y, Tsuji O, Niikura T, et al. Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death. J Neurochem, 2003, 84(4): 864-877.
[19] Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[20]Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[21]Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature, 2003,423(6938): 456-461
[22]Kariya S, Hirano M, Furiya Y, et al. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent [J]. Acta Neuropathol (Berl)., 2005, 109(4):367-372
[23]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[24]Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem. 2003,254(1-2): 83-89.
[25]Nishimoto I, Matsuoka M, Niikura T. Unravelling the role of Humanin [J]. Trends Mol Med, 2004,10(3): 102-105
[26]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ_(1-40). Peptides, 2003,24(5): 679-685.
[27] Wang D, Li H, Yuan H, et al. Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase [J]. Apoptosis. 2005,10(5):963-971
[28]Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link [J]. Nat Rev Mol Cell Biol., 2003, 4(7): 552-565
[29]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J]. Neurochemical Research, 2003,28( 8): 1193-1199
[30]Inomata Y, Nakamura H, Tanito M, et al. Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina [J]. J Neurochem, 2006,98(2): 372-385.
[31] Zhang Q, Zhang J. Effect of melatonin on the spatial and temporal changes of [Ca2+] i in single living cells of cortical neurons by laser scanning confocal microscopy [J]. Chin Med J (Engl)., 2000,113(6): 558-562
[32]SuzukiY, OnoY, HirabayashiY. Rapid and specific reactive oxygen species generation via NADPH oxidase activation durong Fas mediated apoptosis [J]. FEBS Lett, 1998,425(2):209-212.
[33]GotohY, Cooper JA. Reactive oxygen species and dimerization induced activation of apoptosis signal regulating kinase 1 in tumour necrosis factor alpha signal transduction [J]. J Biol Chem, 1998,273(28): 17477-17482.
[34]Koppenol WH, Moreno JJ, Pryor WA, et al. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide [J]. Chem Res Toxicol, 1992, 5(6): 834-842
[35]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[36]Hetman M, Gozdz A. Role of extracellular signal regulated kinases 1 and 2 in neuronal survival [J]. Eur J Biochem., 2004,271(11): 2050-2055
[37]Chu C, Alapat D, Wen X, et al. Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway [J].Cell Signal. 2004,16(9):1045-1059
[38] Wang H, Yu SW, Koh DW, et al. Apoptosis-Inducing Factor Substitutes for Caspase Executioners in NMDA-Triggered Excitotoxic Neuronal Death [J]. J. Neurosci., 2004,24(48): 10963-10973
[39]Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke [J].Stroke., 2006,37(10): 2613-2619
[1]Selkoe DJ.The molecular pathology of Alzheimer's disease[J].Neuron,1991,6:487-498.
[2]Kawasumi M,Hashimoto Y,Chiba T,et al.Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults[J].Neurosignals,2002,11(5):236-250.
[3]Evans DA,Funkenstein HH,Albert MS,et al.Prevalance of Alzhemimer's disease in a community population of older persons.Higher than previously reported.JAMA,1989,262(18):2551-2556.
[4]Price DL,Sisodia SS.Mutant genes in familial Alzheimer's disease and transgenic models [J].Annu Rev Neurosci,1998,21:479-505.
[5]Katzman R,Kawas C.The epidemiology of dementia and Alzheimer disease.In Terry RD,Katzman R,Bick KL,editors.Alzheimer disease.New York:Raven Press,1994,P.105.
[6]罗焕敏,陈以慈.Alzheimer病病因学研究新进展及可能的发病机理[J].国外医学,老年医学分册,1996,17(1):22-28.
[7]Hardy JA,Higgins GA.Alzheimer's disease:the amyloid cascade hypothesis.Science,1992,256:184-185.
[8]Lashuel HA,Hartley D,Petre BM,et al.Neurodegenerative disease:amyloid pores from pathogenic mutations[J].Nature,2002,418(6895):291.
[9]Zou P,Ding Y,Sha Y,et al.Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ_1-40[J].Peptides,2003,24(5):679-685.
[I0]Ying G,Iribarren P,Zhou Y,et al.Humanin,a newly identified neuroprotective factor,uses the G protein-coupled Formylpeptide receptor-like-1 as a functional receptor[J].The Journal of Immunology,2004,172:7078-7085.
[11]Gearing M,Mori H,Mirra SS.Abeta-peptide length and apolipoprotein E genotype in Alzheimer's disease[J].Ann Neurol,1996,39(3):395-399.
[12]Steiner H,Capell A,Leimer U,et al.Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease[J].Eur Arch Psychiatry Clin Neurosci,1999,249(6):266-270.
[13]Xia W.Role of presenilin in gamma-secretase cleavage of amyloid precursor protein[J].Exp Gerontol,2000,35(4):453-460.
[14]Hashimoto Y,Niikura T,Ito Y,et al.Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer;s disease-relevant insults[J].Neurosci,2001,21(23):9235-9245.
[15]Fraser PE,Yu G,Levesque L,et al.Presenilin function:connections to Alzheimer's disease and signal transduction[J].Biochem Soc Syrup,2001,(67):89-100.
[16]Kulic L,Walter J,Multhaup G,et al.Separation of presenilin function in amyloid beta-peptide generation and endoproteolysis of Notch[J].Proc Natl Acad Sci U S A,2000,97(11):5913-5918.
[17]Yoshida H,Hastie C J,McLauchlan H,et al.Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase(JNK)[J].J Neurochem,2004,90(2):352-358.
[18]Loo DT,Copani A,Pike C J,et al.Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.Proc Natl Acad Sci U S A,1993,90(17):7951-7955.
[19]廖海妮,张宗玉,童坦君.Alzheimer病与细胞凋亡 生命的化学 1999,19(3):141-143.
[20]Cummings JL,Vinters HV,Cole GM,et al.Alzheimer's disease:Etiologies,pathophysiology,cognitive reserve,and treat opportunities[J].Neurology,1998,51(1Suppl 1):S2-17;discussion S65-7.
[21]Niikura T,Hashimoto Y,Tajima H,et al.Death and survival of neuronal cells exposed to Alzheimer's insults[J].J Neurosci Res,2002,70(3):380-391.
[22]Hashimoto Y,Ito Y,Niikura T,et al.Mechanism of neuropretection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein[J].Biochem Biophys Res Commun,2001,283(2):460-468.
[23]Hashimoto Y,Niikura T,Tajima H,et al.A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ[J].Proc Natl Acad Sci USA,2001,98(11):6336-6341.
[24]Mamiya T,Ukai M.[Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo[J].Br J Pharmacol,2001,134(8):1597-1599.
[25]Jung SS,Van Nostrand WE.Humanin rescues human cerebrovascular smooth muscle cells from Abeta-induced toxicity[J].J Neurochem,2003,84(2):266-272.
[26]Kariya S,Takahashi N,Hirano M,et al.Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes[J].Mol Cell Biochem.2003,254(1-2):83-89.
[27]Kariya S,Takahashi N,Ooba N,et al.Humanin inhibits cell death of serum-deprived PC12h cells[J].Neuroreport,2002,13(6):903-907.
[28]Terashita K,Hashimoto Y,Niikura T,et al.Two serine residues distinctly regulate the rescue function of Humanin,functiona;potentiation by isomerization and dimerization[J].J Neurochem,2003,85(6):1521-1538.
[29]Yamagishi Y,Hashimoto Y,Niikura T,et al.Identification of essential amino acids in Humanin,a neuroprotective factor against Alzheimer's disease-relevant insults[J].Peptide, 2003,24(4): 585-595.
[30]Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[31]Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation [J]. Nature, 2003,423(6938): 456-461.
[32]Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[33]Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-relatted insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[34] Hashimoto Y, Niikura T, Ito Y, etal. Multiple mechanisms underlie neurotoxicity by different type Alzheimer's disease mutations of amyloid precursor protein [J]. J Biol Chem,2000,275(44): 34541-34551.
[35]Ikonen M, Liu B, Hashimoto Y, et al. Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis [J].Proc Natl Acad Sci U S A,2003,100(22): 13042-13047
[36]Maximov V, Martynenko A, Hunsmann G,et al. Mitochondrial 16S rRNA gene encodes a functional peptide, a potential drug for Alzheimer's disease and target for cancer therapy [J].Med Hypotheses, 2002, 59(6): 670-673.
[37]von Heijne G. Signal sequences. The limits of variation [J]. J Mol Biol,1985, 184(1):99-105.
[38]Hashimoto Y, Terashita K, Niikura T, et al. Humanin antagonists: mutants that interfere with dimerization inhibit neuroprotection by Humanin [J]. Eur J Neurosci, 2004, 19(9):2356-2364.35
[39]Nishimoto I, Matsuoka M, Niikura T. Unravelling the role of Humanin [J]. Trends Mol Med, 2004,10(3): 102-105.
[40]Nishimoto I. ["Death and survival of neuronal cells exposed to Alzheimer's disease-relevant insults"] [J]. Nippon Yakurigaku Zasshi, 2002,120(1): 11P-15P.
[41]Jobling MF, Huang X, Stewart LR, et al. Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide prP(106-126) [J]. Biochemistry, 2001,40(27): 8073-8084.
[42]Rottkamp CA, Raina AK, Zhu X, et al. Redox-active iron mediates amyloid-β toxicity [J]. Free Radic Biol Med, 2001, 30(4): 447-450.
[43]O'Donovan CN, Tobin D, Cotter TG. Priori protein fragment PrP-(106-126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells [J].J Biol Chem,2001,276(47):43516-43523.
[44]Sponne I, Fifre A, Koziel V, et al. Humanin rescues cortical neurons from prion-peptide-induced apoptosis [J]. Mol Cell Neurosci, 2004,25(1): 95-102.
[45]Pillot T, Drouet B, Pincon-Raymond M,et al. A nonfibrillar form of the fusogenic prion protein fragment [118-135] induces apoptotic cell death in rat cortical neurons [J]. J Neurochem, 2000,75(6):2298-308.
[46]Granata R, Trovato L, Garbarino G, et al. Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways [J]. FASEB J.2004 Jul 9 [Epub ahead of print]
[47] Liu B, Lee HY, Weinzimer SA, et al. Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis [J]. J Biol Chem, 2000,275(43):33607-33613.
[48] Kim HS, Ingermann AR, Tsubaki J, et al. Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells [J]. Cancer Res, 2004, 64(6):2229-2237.
[49] Rajah R, Valentinis B, Cohen P. Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGF-independent mechanism [J]. J Biol Chem, 1997, 272(18):12181-12188.
[50]Rensink AA, Gellekink H, Otte-Holler I, et al. Expression of the cytokine leukemia inhibitory factor and pro-apoptotic insulin-like growth factor binding protein-3 in Alzheimer's disease [J]. Acta Neuropathol (Berl), 2002,104(5): 525-533.
[51] Zheng H, Jiang M, Trumbauer ME, et al. beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity [J]. Cell, 1995, 81(4):525-531.
[52] Heber S, Herms J, Gajic V, et al. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members [J]. J Neurosci, 2000, 20(21):7951-7963.
[53]Rohn TT, Ivins KJ, Bahr BA,et al. A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis [J]. J Neurochem, 2000, 74(6): 2331-2342.
[54]Sudo H, Jiang H, Yasukawa T, et al. Antibody-regulated neurotoxic function of cell-surface beta-amyloid precursor protein [J]. Mol Cell Neurosci, 2000, 16(6): 708-723.
[55] Sudo H, Hashimoto Y, Niikura T, et al. Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein [J]. Biochem Biophys Res Commun, 2001, 282(2):548-556.
[56]Hashimoto Y,Tsuji O,Niikura T,et al.Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death[J].J Neurochem,2003,84(4):864-877.
[57]Kawasumi M,Hashimoto Y,Chiba T,et al.Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults[J].Neurosignals,2002,11(5):236-250.
[58]Hashimoto Y,Niikura T,Chiba T,et al.The cytoplasmic domain of Alzheimer's amyloid-beta protein precursor causes sustained apoptosis signal-regulating kinase 1/c-Jun NH2-terminal kinase-mediated neurotoxic signal via dimerization[J].J Pharmacol Exp Ther,2003,306(3):889-902.
[59]Scheinfeld MH,Roncarati R,Vito P,et al.Jun NH2-terminal kinase(JNK)interacting protein 1(JIP1)binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein(APP)[J].J Biol Chem,2002,277(5):3767-3775.
[60]Matsuda S,Yasukawa T,Homma Y,et al.c-Jun N-terminal kinase(JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK[J].J Neurosci,2001,21(17):6597-6607.
[61]Niikura T,Hashimoto Y,Tajima H,et al.A tripartite motif protein TRIM11 binds and destabilizes Humanin,a neuroprotective peptide against Alzheimer's disease-relevant insults[J].Eur J Neurosci,2003,17(6):1150-1158.
[62]Brenneman DE,Hauser J,Neale E,et al.Activity-dependent neurotrophic factor:structure-activity relationships of femtomolar-acting peptides[J].J Pharmacol Exp Ther,1998,285(2):619-627.
[63]Mark R J,Keller JN,Kruman I,et al.Basic FGF attenuates amyloid beta-peptide-induced oxidative stress,mitochondrial dysfunction,and impairment of Na+/K+-ATPase activity in hippocampal neurons[J].Brain Res,1997,756(1-2):205-14.
[64]Dore S,Kar S,Quirion R.Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity[J].Proc Natl Acad Sci U S A,1997,94(9):4772-47777.
[65]Adams JM,Cory S.Life-or-death decisions by the Bcl-2 protein family[J].Trends Riochem Sci,2001.26(1):61-66
[2] Ferrari R, Ceconi C, Curello S, et al. Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity [J]. J Mol Cell Cardiol, 1985,17: 937-945
[3] Benveniste H, Drejer J, Schousboe A, et al. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis [J]. J Neurochem, 1984,43:1369-1374.
[4] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMD A receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[5] Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol., 1992,3(9): 1261-1276
[6] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels:high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996, 16(17):5457-5465.
[7] Artal-Sanz M, Tavernarakis N. Proteolytic mechanisms in necrotic cell death and neurodegeneration [J]. FEBS Lett. 2005 Jun 13;579(15):3287-3296.
[8] Dugan LL, Sensi SL, Canzoniero LM, et al. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate [J]. J Neurosci.,1995,15(10): 6377-6388.
[9] Dawson VL, Dawson TM, London ED, et al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures [J]. Proc Natl Acad Sci U S A., 1991, 88(14): 6368-6371
[10] Lucas DR, Newhouse JP. The toxic effect of sodium L-glutamate on the inner layers of the retina [J]. AMA Arch Ophthalmol, 1957, 58(2): 193-201.
[11]Olney JW. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate [J]. Science, 1969,164(880): 719-721.
[12]Inomata Y, Nakamura H, Tanito M, et al. Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina [J]. J Neurochem, 2006,98(2): 372-385.
[13] Zhang Q, Zhang J. Effect of melatonin on the spatial and temporal changes of [Ca2+] i in single living cells of cortical neurons by laser scanning confocal microscopy [J]. Chin Med J (Engl)., 2000,113(6): 558-562
[14]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[15]Hetman M, Gozdz A. Role of extracellular signal regulated kinases 1 and 2 in neuronal survival [J]. Eur J Biochem., 2004,271(11): 2050-2055
[16]Chu C, Alapat D, Wen X, et al. Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway [J].Cell Signal. 2004,16(9): 1045-1059
[17] Wang H, Yu SW, Koh DW, et al. Apoptosis-Inducing Factor Substitutes for Caspase Executioners in NMDA-Triggered Excitotoxic Neuronal Death [J]. J. Neurosci., 2004,24(48):10963-10973
[18] Koh JY, Choi DW. Vulnerability of cultured cortical neurons to damage by excitotoxins:differential susceptibility of neurons containing NADPH-diaphorase [J]. J Neurosci., 1988,8(6): 2153-2163.
[19] Yuan J, ipinski M, Degterev A. Diversity in the mechanisms of neuronal cell death [J].Neuron, 2003,40: 401-413.
[20]Benn SC, Woolf CJ. Adult neuron survival strategies - slamming on the brakes [J]. Nat.Rev. Neurosci., 2004, 5: 686-700
[21]Nicotera P, Leist M, Manzo L. Neuronal cell death: a demise with di.erent shapes [J].Trends Pharmacol. Sci, 1999,20: 46-51.
[22]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J].Neurochemical Research, 2003,28( 8): 1193-1199
[23]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[24] Kim JB, Sig Choi J, Yu YM, et al. HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain [J]. J Neurosci.2006 Jun 14;26(24):6413-2
[25] Shamloo M, Soriano L, Wieloch T, et al. Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia [J]. J Biol Chem., 2005, 280(51):42290-42299.
[26] Shirakawa H, Katsuki H, Kume T, et al. Aminoglutethimide prevents excitotoxic and ischemic injuries in cortical neurons [J]. Br J Pharmacol, 2006,147(7): 729-736.
[27]Kim SH, Won SJ, Mao XO, et al. Molecular mechanisms of cannabinoid protection from neuronal excitotoxicity [J]. Mol Pharmacol., 2006,69(3): 691-696.
[28]Ha HJ, Kwon YS, Park SM, et al. Quercetin attenuates oxygen-glucose deprivation- and excitotoxin-induced neurotoxicity in primary cortical cell cultures [J]. Biol Pharm Bull. 2003 Apr;26(4):544-546.
[29]Kang HJ, Choi YS, Hong SB, et al. Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity [J]. J Neurosci. 2004 Feb 11;24(6):1280-1287.
[30] Rosenberg PA, Amin S, Leitner M. Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture [J]. J Neurosci., 1992,12(1):56-61
[31]Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme [J].FEBS J., 2006,273(3): 433-450.
[1] Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. Proc Natl Acad Sci USA, 2001,98:6336-6341.
[2] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. Neurosci, 2001, 21:9235-9245.
[3] Lin-min Li, Ai-ling Cui, Jian Wang, et al. Effects of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[4] Tauima H, Kawasumi M, Chiba T, et al. A humanin derivative, SGly14-Humanin, prevents amyloid-beta-induced memory impairment in mice. J Neurosci Res, 2005,79: 714-723
[5] Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol, 2001,134(8): 1597-1599.
[6] Krejcova G, Patcoka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats. J Pept Sci, 2004,10(10): 636 - 639
[7] Niikura T, Chiba T, Aiso S, et al. Humanin: after the discovery [J]. Mol Neurobiol., 2004,30(3): 327-340
[8] Niikura T, Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults [J]. Eur J Neurosci., 2003,17(6):1150-1158.
[9] Benaki D, Zikos C, Evangelou A, et al. Solution structure of Serl4Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease [J]. Biochem Biophys Res Commun. 2006 Oct 20;349(2):634-642
[10]Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005,64(6): 816-823
[11]Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[12] Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[13] Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[14] Colon E, Strand ML, Carlsson-Skwirut C, et al. Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis [J]. J Cell Physiol., 2006,208(2): 373-385.
[15]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[16]Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem, 2003,254(1-2): 83-89.
[17]Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA [J]. Neuropeptides, 2005,39(2005):97-101
[18]Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[19] Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[20]Lin-min Li, Ai-ling Cui, Jian Wang, et al. The mechanisms underlying the protection of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[21]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1 -40. Peptides, 2003,24(5): 679-685.
[22]Bogaert L, Scheller D, Moonen J, et al. Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia [J]. Brain Res, 2000,887:266-275
[23]Ferrari R, Ceconi C, Curello S, et al. Oxygen-mediated myocardial damage during ischaemia and reperfusion: role of the cellular defences against oxygen toxicity [J]. J Mol CellCardiol, 1985,17: 937-945
[24]Benveniste H, Drejer J, Schousboe A, et al. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis [J]. J Neurochem, 1984,43: 1369-1374.
[25]Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[26]Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol., 1992, 3(9): 1261-1276 Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996,16(17): 5457-5465.
[27] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996,16(17): 5457-5465.
[28]Dempsey RJ, Baskaya MK, Dogan A. Attenuation of brain edema, blood-brain barrier breakdown, and injury volume by ifenprodil, a polyamine-site N-methyl-D-aspartate receptor antagonist, after experimental traumatic brain injury in rats [J]. Neurosurgery, 2000, 47: 399-404
[29]Lipton SA , Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders [J]. N Engl J Med, 1994, 330: 613-622.
[30]Olney JW. Glutamate, a neurotoxic transmitter [J]. J Child Neurol, 1989,4: 218-226
[31] Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies [J]. Curr Drug Targets, 2001,2: 241-271
[32]Montoliu C, Llansola M, Monfort P, et al. Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death [J]. Neurotox Res, 2001,3(2):179-88.
[33]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J]. Neurochemical Research, 2003,28( 8): 1193-1199
[34]Brenneman DE, Hauser J, Neale E, et al. Activity-dependent neurotrophic factor: structure-activity relationships of femtomolar-acting peptides [J]. J Pharmacol Exp Ther,1998,285(2): 619-627.
[35] Mark RJ, Keller JN, Kruman I, et al. Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons [J]. Brain Res, 1997, 756(1-2): 205-14.
[36]Dore S, Kar S, Quirion R. Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity [J]. Proc Natl Acad Sci U S A, 1997, 94(9): 4772-47777
[37]Low SJ and Roland CL..Review of NMDA antagonist-induced neurotoxicity and implications for clinical development. Int J Clin Pharmacol Ther. 2004,42(1):1-14
[38]Lees KR, Asplund K, Carolei A, et al. Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomised controlled trial. GAIN International Investigators [J]. Lancet. 2000,355(9219): 1949-54
[39] Sacco RL,DeRosa JT, Haley EC Jr, et al. Glycine antagonist in neuroprotection for patients with acute stroke: GAIN Americas: a randomized controlled trial [J]. JAMA. 2001,285(13):1719-28
[40] Caricasole A, BrunoV,CappuccioI, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002(10): 1331-1333
[41]Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke [J]. Stroke., 2006,37(10): 2613-2619
[42]Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005,64(6): 816-823.
[43] Kin T, Sugie K, Hirano M, et al. Humanin expression in skeletal muscles of patients with chronic progressive external ophthalmoplegia [J]. J Hum Genet. 2006,51(6): 555-558.
[44]Kariya S, Hirano M, Furiya Y,et al. Humanin detected in skeletal muscles of MEL AS patients: a possible new therapeutic agent [J]. Acta Neuropathol (Berl). 2005, 109(4):367-372
[45] Chiba T, Yamada M, Sasabe J, et al. Colivelin prolongs survival of an ALS model mouse [J]. Biochem Biophys Res Commun., 2006,343(3): 793-798.
[46]Kariya S, Hirano M, Nagai Y, et al. Humanin attenuates apoptosis induced by DRPLA proteins with expanded polyglutamine stretches [J]. J Mol Neurosci. 2005,25(2): 165-169
[1] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003., 285: H2592-H2598.
[2] Choi, D.W. Excitotoxic cell death [J]. J. Neurobiol, 1992,3(9): 1261-1276
[3] Lu YM, Yin HZ, Chiang J, et al. Ca(2+)-permeable AMPA/kainate and NMDA channels:high rate of Ca2+ influx underlies potent induction of injury [J]. J Neurosci., 1996, 16(17):5457-5465.
[4] Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme [J]. FEBS J, 2006,273(3): 433-450.
[51 Campanella M, Pinton P, Rizzuto R. Mitochondrial Ca2+ homeostasis in health and disease [J].Biol Res, 2004,37(4): 653-660?
[6] Bernardi P. Mitochondrial transport of cations: channels , exchangers , and permeability transition. Physiol Rev, 1999,79(4): 1127-1155
[7] Di Paola M, Lorusso M. Interaction of free fatty acids with mitochondria: coupling, uncoupling and permeability transition [J]. Biochim Biophys Acta., 2006, 1757(9-10):1330-1337.
[8] Dutra F, Bechara EJ. Aminoacetone induces iron-mediated oxidative damage to isolated rat liver mitochondria [J]. Arch Biochem Biophys., 2004,430(2): 284-289
[9] Schild L, Huppelsberg J, Kahlert S, et al. Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia/reoxygenation for functional breakdown and morphological disintegration [J]. J Biol Chem, 2003,278(28): 25454-25460.
[10] Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. Proc Natl Acad Sci USA,2001,98:6336-6341.
[11] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. Neurosci, 2001,21:9235-9245.
[12]Lin-min Li, Ai-ling Cui, Jian Wang, et al. Effects of Humanin on Aβ31-35-induced apoptosis in cultured cortical neurons. (in submission)
[13]Tauima H, Kawasumi M, Chiba T, et al. A humanin derivative, SGly14-Humanin, prevents amyloid-beta-induced memory impairment in mice. J Neurosci Res, 2005, 79: 714-723
[14]Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol, 2001,134(8): 1597-1599.
[15]Krejcova G, Patcoka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats. J Pept Sci, 2004,10(10): 636 - 639
[16]Niikura T, Chiba T, Aiso S, et al. Humanin: after the discovery [J]. Mol Neurobiol., 2004,30(3): 327-340
[17]Niikura T, Hashimoto Y, Tajima H, et al. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults [J]. Eur J Neurosci., 2003,17(6):1150-1158.
[18]Benaki D, Zikos C, Evangelou A, et al. Solution structure of Ser14Gly-humanin, a potent rescue factor against neuronal cell death in Alzheimer's disease [J]. Biochem Biophys Res Commun. 2006 Oct 20;349(2):634-642
[19] Ijiri K, Tsuruga H, Sakakima H, et al. Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality [J]. Ann Rheum Dis., 2005, 64(6): 816-823
[20] Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[21]Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[22] Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[23] Colon E, Strand ML, Carlsson-Skwirut C, et al. Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis [J]. J Cell Physiol, 2006,208(2): 373-385.
[24]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[25] Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem, 2003, 254(1-2): 83-89.
[26]Kariya S, Hirano M, Furiya Y, et al. Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA [J]. Neuropeptides, 2005,39(2005):97-101
[27]Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[28] Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[29]Lin-min Li, Ai-ling Cui, Jian Wang, et al. The mechanisms underlying the protection of Humanin on A β 31-35-induced apoptosis in cultured cortical neurons, (in submission)
[30]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ1 -40. Peptides, 2003,24(5): 679-685.
[31] Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J].Neurochemical Research, 2003,28( 8): 1193-1199.
[1] Dempsey RJ, Baskaya MK, Dogan A. Attenuation of brain edema, blood-brain barrier breakdown, and injury volume by ifenprodil, a polyamine-site N-methyl-D-aspartate receptor antagonist, after experimental traumatic brain injury in rats [J]. Neurosurgery,2000,47: 399-404
[2] Lipton SA , Rosenberg PA. Excitatory amino acids as a final common pathway for neurologic disorders [J]. N Engl J Med, 1994,330: 613-622.
[3] Olney JW. Glutamate, a neurotoxic transmitter [J]. J Child Neurol, 1989,4:218-226
[4] Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies [J]. Curr Drug Targets, 2001,2: 241-271
[5] Montoliu C, Llansola M, Monfort P, et al. Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death [J]. Neurotox Res, 2001, 3(2): 179-88.
[6] Nath R, Scott M, Nadimpalli R, et al. Activation of apoptosis-linked caspase(s) in NMDA-injured brains in neonatal rats [J]. Neurochem Int, 2000, 36(2): 119-26.
[7] Okamoto S, Li Z , Ju C, et al. Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis [J]. PNAS, 2002, 99(6):3974-3979
[8] Sharp CD, Hines I, Houghton, J, et al. Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor [J]. Am J Physiol Heart Circ Physiol, 2003, 285: H2592-H2598.
[9] Low SJ and Roland CL..Review of NMDA antagonist-induced neurotoxicity and implications for clinical development. Int J Clin Pharmacol Ther. 2004,42(1): 1-14
[10] Lees KR, Asplund K, Carolei A, et al. Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomised controlled trial. GAIN International Investigators [J]. Lancet. 2000, 355(9219): 1949-54
[11] Sacco RL,DeRosa JT, Haley EC Jr, et al. Glycine antagonist in neuroprotection for patients with acute stroke: GAIN Americas: a randomized controlled trial [J]. JAMA.2001,285(13):1719-28
[12]Wu J, Anwyl R, Rowan MJ. β-amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus [J]. Neuroreport, 1995, 6(17): 2409-2413
[13]Hashimoto Y, Niikura T, Tajima H, et al. A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ [J]. Proc Natl Acad Sci USA, 2001, 98(11): 6336-6341.
[14] Hashimoto Y, Niikura T, Ito Y, et al. Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults [J]. Neurosci, 2001,21(23): 9235-9245.
[15]Tajima H, Kawasumi M, Chiba T, et al. A humanin derivative, S14G-HN, prevents amyloid-beta-induced memory impairment in mice [J]. J Neurosci Res., 2005, 79(5):714-723.
[16]Krejcova G, Patocka J, Slaninova J. Effect of humanin analogues on experimentally induced impairment of spatial memory in rats [J]. J Pept Sci., 2004,10(10): 636-639
[17]Mamiya T, Ukai M. [Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo [J]. Br J Pharmacol., 2001,134(8):1597-1599
[18] Hashimoto Y, Tsuji O, Niikura T, et al. Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death. J Neurochem, 2003, 84(4): 864-877.
[19] Zhai D, Luciano F, Zhu X, et al. Humanin binds and nullifies Bid activity by blocking its activation of Bax and Bak. J Biol Chem, 2005,280(16):15815-15824.
[20]Luciano F, Zhai D, Zhu X, et al. Cytoprotective peptide Humanin binds and inhibits pro-apoptotic Bcl-2/Bax-family protein BimEL. J Biol Chem., 2005, 280(16):15825-15835.
[21]Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation. Nature, 2003,423(6938): 456-461
[22]Kariya S, Hirano M, Furiya Y, et al. Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent [J]. Acta Neuropathol (Berl)., 2005, 109(4):367-372
[23]Kariya S, Takahashi N, Ooba N, et al. Humanin inhibits cell death of serum-deprived PC12h cells [J]. Neuroreport, 2002,13(6): 903-907.
[24]Kariya S, Takahashi N, Hirano M, et al. Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes [J]. Mol Cell Biochem. 2003,254(1-2): 83-89.
[25]Nishimoto I, Matsuoka M, Niikura T. Unravelling the role of Humanin [J]. Trends Mol Med, 2004,10(3): 102-105
[26]Zou P, Ding Y, Sha Y, et al. Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ_(1-40). Peptides, 2003,24(5): 679-685.
[27] Wang D, Li H, Yuan H, et al. Humanin delays apoptosis in K562 cells by downregulation of P38 MAP kinase [J]. Apoptosis. 2005,10(5):963-971
[28]Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link [J]. Nat Rev Mol Cell Biol., 2003, 4(7): 552-565
[29]Cebere A, Liljequist S. Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells [J]. Neurochemical Research, 2003,28( 8): 1193-1199
[30]Inomata Y, Nakamura H, Tanito M, et al. Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina [J]. J Neurochem, 2006,98(2): 372-385.
[31] Zhang Q, Zhang J. Effect of melatonin on the spatial and temporal changes of [Ca2+] i in single living cells of cortical neurons by laser scanning confocal microscopy [J]. Chin Med J (Engl)., 2000,113(6): 558-562
[32]SuzukiY, OnoY, HirabayashiY. Rapid and specific reactive oxygen species generation via NADPH oxidase activation durong Fas mediated apoptosis [J]. FEBS Lett, 1998,425(2):209-212.
[33]GotohY, Cooper JA. Reactive oxygen species and dimerization induced activation of apoptosis signal regulating kinase 1 in tumour necrosis factor alpha signal transduction [J]. J Biol Chem, 1998,273(28): 17477-17482.
[34]Koppenol WH, Moreno JJ, Pryor WA, et al. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide [J]. Chem Res Toxicol, 1992, 5(6): 834-842
[35]Amadoro G, Ciotti MT, Costanzi M, et al. NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK_MAPK activation [J]. PNAS, 2006,103(8): 2892-2897
[36]Hetman M, Gozdz A. Role of extracellular signal regulated kinases 1 and 2 in neuronal survival [J]. Eur J Biochem., 2004,271(11): 2050-2055
[37]Chu C, Alapat D, Wen X, et al. Ectopic expression of murine diphosphoinositol polyphosphate phosphohydrolase 1 attenuates signaling through the ERK1/2 pathway [J].Cell Signal. 2004,16(9):1045-1059
[38] Wang H, Yu SW, Koh DW, et al. Apoptosis-Inducing Factor Substitutes for Caspase Executioners in NMDA-Triggered Excitotoxic Neuronal Death [J]. J. Neurosci., 2004,24(48): 10963-10973
[39]Xu X, Chua CC, Gao J, et al. Humanin is a novel neuroprotective agent against stroke [J].Stroke., 2006,37(10): 2613-2619
[1]Selkoe DJ.The molecular pathology of Alzheimer's disease[J].Neuron,1991,6:487-498.
[2]Kawasumi M,Hashimoto Y,Chiba T,et al.Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults[J].Neurosignals,2002,11(5):236-250.
[3]Evans DA,Funkenstein HH,Albert MS,et al.Prevalance of Alzhemimer's disease in a community population of older persons.Higher than previously reported.JAMA,1989,262(18):2551-2556.
[4]Price DL,Sisodia SS.Mutant genes in familial Alzheimer's disease and transgenic models [J].Annu Rev Neurosci,1998,21:479-505.
[5]Katzman R,Kawas C.The epidemiology of dementia and Alzheimer disease.In Terry RD,Katzman R,Bick KL,editors.Alzheimer disease.New York:Raven Press,1994,P.105.
[6]罗焕敏,陈以慈.Alzheimer病病因学研究新进展及可能的发病机理[J].国外医学,老年医学分册,1996,17(1):22-28.
[7]Hardy JA,Higgins GA.Alzheimer's disease:the amyloid cascade hypothesis.Science,1992,256:184-185.
[8]Lashuel HA,Hartley D,Petre BM,et al.Neurodegenerative disease:amyloid pores from pathogenic mutations[J].Nature,2002,418(6895):291.
[9]Zou P,Ding Y,Sha Y,et al.Humanin peptides block calcium influx of rat hippocampal neurons by altering fibrogenesis of Aβ_1-40[J].Peptides,2003,24(5):679-685.
[I0]Ying G,Iribarren P,Zhou Y,et al.Humanin,a newly identified neuroprotective factor,uses the G protein-coupled Formylpeptide receptor-like-1 as a functional receptor[J].The Journal of Immunology,2004,172:7078-7085.
[11]Gearing M,Mori H,Mirra SS.Abeta-peptide length and apolipoprotein E genotype in Alzheimer's disease[J].Ann Neurol,1996,39(3):395-399.
[12]Steiner H,Capell A,Leimer U,et al.Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease[J].Eur Arch Psychiatry Clin Neurosci,1999,249(6):266-270.
[13]Xia W.Role of presenilin in gamma-secretase cleavage of amyloid precursor protein[J].Exp Gerontol,2000,35(4):453-460.
[14]Hashimoto Y,Niikura T,Ito Y,et al.Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer;s disease-relevant insults[J].Neurosci,2001,21(23):9235-9245.
[15]Fraser PE,Yu G,Levesque L,et al.Presenilin function:connections to Alzheimer's disease and signal transduction[J].Biochem Soc Syrup,2001,(67):89-100.
[16]Kulic L,Walter J,Multhaup G,et al.Separation of presenilin function in amyloid beta-peptide generation and endoproteolysis of Notch[J].Proc Natl Acad Sci U S A,2000,97(11):5913-5918.
[17]Yoshida H,Hastie C J,McLauchlan H,et al.Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase(JNK)[J].J Neurochem,2004,90(2):352-358.
[18]Loo DT,Copani A,Pike C J,et al.Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.Proc Natl Acad Sci U S A,1993,90(17):7951-7955.
[19]廖海妮,张宗玉,童坦君.Alzheimer病与细胞凋亡 生命的化学 1999,19(3):141-143.
[20]Cummings JL,Vinters HV,Cole GM,et al.Alzheimer's disease:Etiologies,pathophysiology,cognitive reserve,and treat opportunities[J].Neurology,1998,51(1Suppl 1):S2-17;discussion S65-7.
[21]Niikura T,Hashimoto Y,Tajima H,et al.Death and survival of neuronal cells exposed to Alzheimer's insults[J].J Neurosci Res,2002,70(3):380-391.
[22]Hashimoto Y,Ito Y,Niikura T,et al.Mechanism of neuropretection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein[J].Biochem Biophys Res Commun,2001,283(2):460-468.
[23]Hashimoto Y,Niikura T,Tajima H,et al.A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ[J].Proc Natl Acad Sci USA,2001,98(11):6336-6341.
[24]Mamiya T,Ukai M.[Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo[J].Br J Pharmacol,2001,134(8):1597-1599.
[25]Jung SS,Van Nostrand WE.Humanin rescues human cerebrovascular smooth muscle cells from Abeta-induced toxicity[J].J Neurochem,2003,84(2):266-272.
[26]Kariya S,Takahashi N,Hirano M,et al.Humanin improves impaired metabolic activity and prolongs survival of serum-deprived human lymphocytes[J].Mol Cell Biochem.2003,254(1-2):83-89.
[27]Kariya S,Takahashi N,Ooba N,et al.Humanin inhibits cell death of serum-deprived PC12h cells[J].Neuroreport,2002,13(6):903-907.
[28]Terashita K,Hashimoto Y,Niikura T,et al.Two serine residues distinctly regulate the rescue function of Humanin,functiona;potentiation by isomerization and dimerization[J].J Neurochem,2003,85(6):1521-1538.
[29]Yamagishi Y,Hashimoto Y,Niikura T,et al.Identification of essential amino acids in Humanin,a neuroprotective factor against Alzheimer's disease-relevant insults[J].Peptide, 2003,24(4): 585-595.
[30]Caricasole A, Bruno V, Cappuccio I, et al. A novel rat gene encoding a Humanin-like peptide endowed with broad neuroprotective activity [J]. FASEB J, 2002, 16(10):1331-1333.
[31]Guo B, Zhai D, Cabezas E, et al. Humanin peptide suppresses apoptosis by interfering with Bax activation [J]. Nature, 2003,423(6938): 456-461.
[32]Yadav VK, Muraly P, Medhamurthy R. Identification of novel genes regulated by LH in the primate corpus luteum: insight into their regulation during the late luteal phase. Mol Hum Reprod, 2004, Epub 2004 Jul 02.
[33]Tajima H, Niikuraa T, Hashimoto Y, et al. Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-relatted insults [J]. Neurosci Lett, 2002, 324(3): 227-231.
[34] Hashimoto Y, Niikura T, Ito Y, etal. Multiple mechanisms underlie neurotoxicity by different type Alzheimer's disease mutations of amyloid precursor protein [J]. J Biol Chem,2000,275(44): 34541-34551.
[35]Ikonen M, Liu B, Hashimoto Y, et al. Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis [J].Proc Natl Acad Sci U S A,2003,100(22): 13042-13047
[36]Maximov V, Martynenko A, Hunsmann G,et al. Mitochondrial 16S rRNA gene encodes a functional peptide, a potential drug for Alzheimer's disease and target for cancer therapy [J].Med Hypotheses, 2002, 59(6): 670-673.
[37]von Heijne G. Signal sequences. The limits of variation [J]. J Mol Biol,1985, 184(1):99-105.
[38]Hashimoto Y, Terashita K, Niikura T, et al. Humanin antagonists: mutants that interfere with dimerization inhibit neuroprotection by Humanin [J]. Eur J Neurosci, 2004, 19(9):2356-2364.35
[39]Nishimoto I, Matsuoka M, Niikura T. Unravelling the role of Humanin [J]. Trends Mol Med, 2004,10(3): 102-105.
[40]Nishimoto I. ["Death and survival of neuronal cells exposed to Alzheimer's disease-relevant insults"] [J]. Nippon Yakurigaku Zasshi, 2002,120(1): 11P-15P.
[41]Jobling MF, Huang X, Stewart LR, et al. Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide prP(106-126) [J]. Biochemistry, 2001,40(27): 8073-8084.
[42]Rottkamp CA, Raina AK, Zhu X, et al. Redox-active iron mediates amyloid-β toxicity [J]. Free Radic Biol Med, 2001, 30(4): 447-450.
[43]O'Donovan CN, Tobin D, Cotter TG. Priori protein fragment PrP-(106-126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells [J].J Biol Chem,2001,276(47):43516-43523.
[44]Sponne I, Fifre A, Koziel V, et al. Humanin rescues cortical neurons from prion-peptide-induced apoptosis [J]. Mol Cell Neurosci, 2004,25(1): 95-102.
[45]Pillot T, Drouet B, Pincon-Raymond M,et al. A nonfibrillar form of the fusogenic prion protein fragment [118-135] induces apoptotic cell death in rat cortical neurons [J]. J Neurochem, 2000,75(6):2298-308.
[46]Granata R, Trovato L, Garbarino G, et al. Dual effects of IGFBP-3 on endothelial cell apoptosis and survival: Involvement of the sphingolipid signaling pathways [J]. FASEB J.2004 Jul 9 [Epub ahead of print]
[47] Liu B, Lee HY, Weinzimer SA, et al. Direct functional interactions between insulin-like growth factor-binding protein-3 and retinoid X receptor-alpha regulate transcriptional signaling and apoptosis [J]. J Biol Chem, 2000,275(43):33607-33613.
[48] Kim HS, Ingermann AR, Tsubaki J, et al. Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells [J]. Cancer Res, 2004, 64(6):2229-2237.
[49] Rajah R, Valentinis B, Cohen P. Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGF-independent mechanism [J]. J Biol Chem, 1997, 272(18):12181-12188.
[50]Rensink AA, Gellekink H, Otte-Holler I, et al. Expression of the cytokine leukemia inhibitory factor and pro-apoptotic insulin-like growth factor binding protein-3 in Alzheimer's disease [J]. Acta Neuropathol (Berl), 2002,104(5): 525-533.
[51] Zheng H, Jiang M, Trumbauer ME, et al. beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity [J]. Cell, 1995, 81(4):525-531.
[52] Heber S, Herms J, Gajic V, et al. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members [J]. J Neurosci, 2000, 20(21):7951-7963.
[53]Rohn TT, Ivins KJ, Bahr BA,et al. A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis [J]. J Neurochem, 2000, 74(6): 2331-2342.
[54]Sudo H, Jiang H, Yasukawa T, et al. Antibody-regulated neurotoxic function of cell-surface beta-amyloid precursor protein [J]. Mol Cell Neurosci, 2000, 16(6): 708-723.
[55] Sudo H, Hashimoto Y, Niikura T, et al. Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein [J]. Biochem Biophys Res Commun, 2001, 282(2):548-556.
[56]Hashimoto Y,Tsuji O,Niikura T,et al.Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death[J].J Neurochem,2003,84(4):864-877.
[57]Kawasumi M,Hashimoto Y,Chiba T,et al.Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults[J].Neurosignals,2002,11(5):236-250.
[58]Hashimoto Y,Niikura T,Chiba T,et al.The cytoplasmic domain of Alzheimer's amyloid-beta protein precursor causes sustained apoptosis signal-regulating kinase 1/c-Jun NH2-terminal kinase-mediated neurotoxic signal via dimerization[J].J Pharmacol Exp Ther,2003,306(3):889-902.
[59]Scheinfeld MH,Roncarati R,Vito P,et al.Jun NH2-terminal kinase(JNK)interacting protein 1(JIP1)binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein(APP)[J].J Biol Chem,2002,277(5):3767-3775.
[60]Matsuda S,Yasukawa T,Homma Y,et al.c-Jun N-terminal kinase(JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK[J].J Neurosci,2001,21(17):6597-6607.
[61]Niikura T,Hashimoto Y,Tajima H,et al.A tripartite motif protein TRIM11 binds and destabilizes Humanin,a neuroprotective peptide against Alzheimer's disease-relevant insults[J].Eur J Neurosci,2003,17(6):1150-1158.
[62]Brenneman DE,Hauser J,Neale E,et al.Activity-dependent neurotrophic factor:structure-activity relationships of femtomolar-acting peptides[J].J Pharmacol Exp Ther,1998,285(2):619-627.
[63]Mark R J,Keller JN,Kruman I,et al.Basic FGF attenuates amyloid beta-peptide-induced oxidative stress,mitochondrial dysfunction,and impairment of Na+/K+-ATPase activity in hippocampal neurons[J].Brain Res,1997,756(1-2):205-14.
[64]Dore S,Kar S,Quirion R.Insulin-like growth factor I protects and rescues hippocampal neurons against beta-amyloid- and human amylin-induced toxicity[J].Proc Natl Acad Sci U S A,1997,94(9):4772-47777.
[65]Adams JM,Cory S.Life-or-death decisions by the Bcl-2 protein family[J].Trends Riochem Sci,2001.26(1):61-66