老年痴呆症中神经递质释放异常的机理

阿尔茨海默症(Alzheimer’s disease,AD)是以胞外淀粉样蛋白(amyloid-β,Aβ)沉积和胞内神经纤维缠结为病理特征的神经退行性疾病。AD典型症状的出现与中枢神经系统突触数量的减少密切相关,因此,明确AD早期突触数量还没有明显降低时突触功能失调的机制对AD的临床诊治具有十分重要的意义。寡聚Aβ、早老素功能缺失等因素造成的突触前神经递质释放异常很有可能是AD突触功能异常的上游机制。对AD中神经递质释放异常的现象和机制进行综述,并对这一领域存在的开放问题作一归纳。     

酵母双杂交筛选锥虫早老素蛋白相互作用蛋白

目的筛选寻找锥虫早老素蛋白相互作用蛋白,以了解锥虫早老素蛋白功能。方法体外表达锥虫早老素蛋白片段,装入pGBKT7诱饵质粒,与随机肽库系统共转化酵母,筛选阳性克隆并测序,通过与基因库锥虫功能序列比较,推导可能的相互作用蛋白。结果获得108个阳性克隆,对其中50个进行了序列测定和比较,获得最有可能的4个候选基因,分别为：丝/苏氨酸性磷酸酶2b催化亚基A2;钙激活蛋白,含锚蛋白重复序列蛋白以及一个具有与APP跨膜区结合位点特征序列的功能未知蛋白。结论成功利用随机肽库酵母双杂交系统筛选锥虫早老素蛋白相互作用基因,其相互作用仍有待进一步确认。     

早老素通过下调CDK4使HEK293T细胞周期阻滞

早老素（progerin）的累积导致儿童早老症（Hutchinson Gilford progeria syndrome, HGPS）的发生,并与正常衰老相关。早老素能使细胞内稳态失衡但分子机制仍有待深入研究。本研究旨在探讨早老素导入人胚胎肾293T细胞（human embryo kidney 293T cell, HEK293T）后细胞增殖、周期变化的分子机制。形态学观察发现过表达早老素的HEK293T细胞密度下降,(57±2.47)%细胞核形态皱缩。细胞增殖和周期实验证明早老素使细胞增殖减慢,发生G1/S期阻滞,G1细胞从 (42.3±1.31)%升至(47.2±1.26)%,而S期细胞从 (43.1±1.36)%降至 (38.5±1.42)%。Western印迹结果显示早老素的高表达引起p21蛋白表达上调（103.2±1.49）%,CDK4下调（63±1.52）%,而p53、ATM、CyclinE1以及p16等蛋白质水平均不变;HEK293T细胞中早老素的过表达导致γ H2AX水平下调（53±1.36）%,H2O2处理后变化趋势不变。我们的研究结果提示,早老素通过上调p21和下调CDK4使细胞发生周期阻滞,不能增加HEK293T细胞的损伤及衰老。     

阿尔茨海默症转基因动物模型脑组织病理学及免疫组化研究

日的通过组织病理学和免疫组织化学方法,验证阿尔茨海默症转基因动物模型。方法取转基因小鼠脑组织,冠状切面中１／３部位,脱水、包埋,进行组织病理学观察,并对相关抗体进行免疫组织化学研究。结果免疫组化显示在大脑皮层、小脑及海马的神经细胞有Ａβ沉淀形成。ＡＰＰ转基因鼠早老素的阳性细胞数及表达量多于对照鼠。刚果红染色可见大脑皮层间有淀粉样物质形成。结论从病理学角度验证此模型的表型与人类病变的相似性,并证明早老素－１可加速淀粉样沉淀的形成,二者的作用是相互的。     

果蝇早老素基因的研究进展

果蝇早老素(Drosophila presenilin,DPS)是一种具有天冬氨酸水解酶活性的蛋白,在果蝇生长发育的各阶段都有表达,对果蝇的生长发育和调节胞内Ca2+平衡具有重要的作用。果蝇早老素功能的缺失能够导致Notch信号下降、神经元凋亡和胞质钙浓度上升,最终造成果蝇长时程记忆损伤和认识丧失。果蝇早老素的这些功能使之成为研究果蝇阿尔茨海默病(Alzheimer’s disease,AD)模型最重要的着手点之一,为阐明AD的病理提供了大量有价值的信息。文章概述了DPS基因与AD相关的功能。     

锥虫早老素蛋白亲水区的克隆和表达纯化

目的体外表达锥虫早老素蛋白亲水区肽段,以制备抗血清用于功能研究。方法根据锥虫早老素蛋白二级结构特性,设计引物分别扩增N端及C端片段的亲水区肽段基因,装入原核表达载体进行表达,并通过变性纯化方法获得足够量表达蛋白,制备兔抗血清。结果成功扩增并克隆锥虫早老素蛋白亲水区片段L2及L7．并分别采用变性磁珠法和变性树脂法进行大量蛋白产物纯化,浓缩纯化产物制备兔抗血清经Westem blot杂交验证,出现目的蛋白大小阳性条带。结论成功表达锥虫早老素蛋推测N端及C端亲水肽段,并成功制备抗血清．可用于锥虫早老素蛋白功能分析。     

我国科学家发现阿尔茨海默症致病的新机制

2006年11月19日,国际著名学术期刊NatMed网络版在线发表了我国科学家关于β淀粉样蛋白产生过程新机制的最新研究成果。中国科学院上海生命科学研究院生物化学与细胞生物学研究所裴钢院士研究组经多年研究后发现,β2-肾上腺素受体被激活后,增强γ-分泌酶的活性,进而能够增加导致阿尔茨海默症的β淀粉样蛋白的产生。这项发现揭示了阿尔茨海默症致病的新机制,并且提示β2-肾上腺素受体有可能成为研发阿尔茨海默症的治疗药物的新靶点。阿尔茨海默症(老年痴呆症)是一种以进行性认知障碍和记忆力损害为主的中枢神经系统退行性疾病,患者初期出现记…     

《科学》：研究提出早老性痴呆发病新机制

美国麻省总医院和哈佛医学院的科学家称,阿尔茨海默症（早老性痴呆症）患者大脑内的淀粉斑让朊蛋白变形。导致大脑的星形胶质细胞变得过度活化,使大脑不能正常工作,因此,造成了阿尔茨海默症。这个发现对阿尔茨海默症背后的大脑机制提出了新的解释,为研发新药物提供了线索,相关研究论文发表在近期出版的《科学》杂志上。     

早老蛋白功能研究进展

分子遗传研究表明早老蛋白基因（PS）突变是家族性阿尔茨海默疾病家系中最常见的遗传性基因突变。因此PS功能的研究成为近年来阿尔茨海默疾病研究领域的热点。本文综述了PS参与调节Notch信号通路和Wnt信号通路信号传导的研究进展,以及PS与神经细胞程序性死亡的关系,详细讨论了PS即γ分泌酶这一假说,并概述了PS的其它相互作用蛋白及相互作用的生物学意义。     

阿尔茨海默症基因研究态势分析

阿尔茨海默症是一种起病隐匿的进行性发展的神经系统退行性疾病。随着全球老龄化日趋严重,阿尔茨海默症严重威胁人民的生命健康与生活质量。阿尔茨海默症成因复杂,治疗阿尔茨海默症仍然是重要的医学难题。通过对Web of Science核心合集中收录的阿尔茨海默症基因研究相关的文献,利用文献计量的方法,进行研究趋势、研究人员、研究机构、国家和研究热点等方面进行态势分析,为相关领域的研究人员提供重要的参考。     

治疗阿尔茨海默病的早老素/γ-分泌酶抑制剂和调节剂

阿尔茨海默病（Alzheimer’s disease,AD）又称老年痴呆症,是一种中枢神经系统（central nervous system,CNS）退行性疾病。β-淀粉样蛋白（β-amyloid,Aβ42）被认为在阿尔茨海默病（AD）的发生、发展过程中起核心作用。Aβ42由APP经β-和γ-分泌酶相继切割产生。γ-分泌酶是一个蛋白酶复合体,早老素（presenilin,PS）是γ-分泌酶的催化组分。因此,抑制PS/γ分泌酶的活性是治疗AD的关键,因而PS/γ分泌酶也是治疗AD的主要靶点。根据这些理论,人们提出了一些治疗AD的新方法,其中PS/γ-分泌酶抑制剂和调节剂成为近年来人们关注的焦点。     

APPswe／PS1dE9／TAU三转基因阿尔兹海默病大鼠模型的建立

目的大鼠的大脑比小鼠更大,是研究神经系统的重要模型。建立APPswe／PS1dE9／TAU三转基因大鼠,发展能更全面表现人类阿尔兹海默病表型的动物模型。方法构建人PrP—hAPP695K595N／M596L、PrP-hPS1dE9和PDGF-TAU转基因表达载体,显微注射法制备转基因大鼠。PCR法鉴定转基因首建鼠及其子代基因型。Western blot检测转基因大鼠脑组织中人APP、PS1和TAU蛋白的表达。Morris水迷宫检测6月龄三转基因大鼠学习记忆能力改变。APP、PHF—TAU免疫组织化学染色观察三转基因大鼠脑组织APP及TAU的表达。结果得到1个同时高表达人APP、PS1和TAU三个基因的转基因大鼠品系。转基因大鼠6月龄已经出现显著的行为学改变：学习记忆能力下降,病理学改变表现为过度磷酸化TAU增多和神经元胞浆内AB表达异常增加。结论成功建立了APPswe／PS1dE9／TAU三转AD大鼠,可做为新一代工具动物模型用于基础医学和AD转化医学研究。     

Presenilin 1 interacts with acetylcholinesterase and alters its enzymatic activity and glycosylation

Presenilin 1 (PS1) plays a critical role in the gamma-secretase processing of the amyloid precursor protein to generate the beta-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships.     

动态观察姜黄素对APP/PS1双转基因小鼠尿液AD7C-NTP浓度的影响

目的通过动态检测小鼠尿液中AD7C-NTP的浓度变化,了解姜黄素在阿尔茨海默病治疗方面的作用。方法 3月龄APP/PS1双转基因小鼠35只,随机分为5组,每组7只,分别为模型组,阳性对照组(罗格列酮组),姜黄素大、中、小剂量组;另选用同月龄同背景的C57BL/6J小鼠7只作为正常对照组。以上6组连续灌胃3个月,分别于灌胃前、灌胃30d、灌胃60d和灌胃90d收集小鼠尿液,应用酶联免疫吸附试验(ELISA)检测尿AD7C-NTP浓度的变化。结果不同时间点各组小鼠尿AD7C-NTP浓度的动态存在波动,各治疗组治疗2个月与治疗1个月相比,AD7C-NTP浓度均有所下降(P0.05,P0.01);同一时间点小鼠尿AD7C-NTP浓度组间比较:治疗2个月后,西药组,姜黄素大、小剂量组与模型组相比AD7C-NTP浓度有所下降(P0.05,P0.01)。结论姜黄素可降低AD模型小鼠尿液内AD7C-NTP浓度,延缓AD的进展。     

Age and Sex Dependent Alteration in Presenilin Expression in Mouse Cerebral Cortex

(1) Presenilin (PS) expression is regulated by several cellular and extracellular factors which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease (AD), which is linked to mutations in PS genes. (2) We have analyzed the effect of age and sex on PS expression by northern hybridization and western blot analysis using the cerebral cortex of adult (24 ± 2 weeks) and old (65 ± 5 weeks) mice. (3) Our results demonstrate that PS1 was downregulated and PS 2 was upregulated in old mice of both sexes. The level of PS 1 was relatively higher and that of PS 2 was lower in female than male mice of same age group. Taken together, these findings show age and sex dependent alteration in PS expression, which in turn may influence the signal transduction pathways and consequently brain functions.     

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.     

Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimer's disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus-mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild-type or mutant (N141I) PS2-expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control beta-galactosidase-expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine-induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild-type counterpart. Staurosporine-induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl-2 protein expression was specifically down-regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C- and N-terminal fragments of PS2. Additionally, expression of mutant, but not wild-type PS2, increased the production of beta-amyloid protein (Abeta) 42. These data collectively suggest that the pro-apoptotic effect of PS2 is mediated by down-regulation of Bcl-2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.     

阿尔兹海默病小鼠模型的磁共振影像学分析

目的分析和研究阿尔兹海默病（AD）转基因模型小鼠（APP/PS1）活体脑部影像学特征。方法本研究利用7.0 T高场强磁共振成像（MRI）技术,对1、3、5、7、9和11月龄AD转基因小鼠模型及对照组活体脑组织的微观结构变化进行对比研究。定量分析了脑组织顶叶皮层及海马区的横向弛豫时间（T2）、表观扩散系数（ADC）和各向异性分数（FA）随AD小鼠年龄变化情况。结果从9月龄开始AD转基因小鼠顶叶皮层及海马区可见散点状低信号区,并且随年龄增长逐渐增多;AD转基因小鼠顶叶皮层和海马区的T2磁豫时间在1～9月龄过程中有减小趋势,但与对照组无显著性差异;顶叶皮层及海马区ADC值的计算结果表明,7～11月龄AD转基因小鼠的ADC值明显下降（P≤0.05）;同样APP/PS1小鼠的FA值从5月龄就开始降低（P≤0.05）,并且这种差异一直持续到11月龄。结论高场强MRI能够显示AD病变出现早期小鼠顶叶皮层及海马区FA值的明显改变,揭示FA值对早期痴呆症临床诊断具有一定的参考价值。