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Title: 部分一:低氧加重阿尔茨海默病病理进程的机制研究 部分二:自噬在肌萎缩侧索硬化中病理机制的研究
Author: 李靓
Degree Level: 博士
Issued Date: 2008-05-05
Degree Grantor: 中国科学院上海生命科学研究院
Place of Degree Grantor: 上海生命科学研究院
Supervisor: 乐卫东
Keyword: 主题词 ; 阿尔茨海默病 ; 低氧 ; APPswe+PS1A246E转基因小鼠 ; β淀粉样前体蛋白 ; β淀粉样多肽 ; 自噬 ; 肌萎缩侧索硬化(ALS) ; 运动神经元 ; 有髓神经纤维
Alternative Title: The role of hypoxia in the pathogenesis of Alzheimer’s Disease The role of autophagy in Amyotrophic lateral sclerosis
Major: 神经生物学
Abstract: 目的:阿尔茨海默病(Alzheimer’s Disease, AD)是一种最为常见的老年神经退行性疾病,其特点是起病潜隐,病程迁延,受累的神经元呈现不可逆转的进行性变性,导致认知记忆功能出现障碍,最终患者丧失生活自理能力。AD的发病机制十分复杂,目前的研究还无法揭示AD发病的根本原因。已知某些特定基因的变异可导致AD或增加AD发病的风险。然而越来越多的研究认为环境因素在AD的发病过程中也起着重要的作用。例如经历过中风或者脑梗阻的病人,其AD的发病率明显升高;病理学研究显示大多数AD患者缺血性脑损伤和AD病理共存,此外临床研究发现缺血性脑血管病患者脑中Aβ的水平有明显增加。这些研究提示了低氧(hypoxia)可能会影响β淀粉样前体蛋白(Amyloid precursor protein, APP)的代谢,加速老年斑的沉积,是AD发病的危险因子。尽管如此,低氧加快AD发病进程的分子机制还没有完全明确,需要我们进一步的深入研究。 方法:我们利用表达APPswe突变蛋白和Presenilin1(PS1)A246E 突变蛋白的APPswe+PS1A246E双转基因AD小鼠模型和细胞模型,结合低氧干预的手段,建立AD小鼠的低氧模型,研究低氧影响AD发病进程的作用机制。实验主要分为以下3个部分: (1)低氧对β淀粉样多肽(Aβ)以及老年斑形成的影响;(2)低氧在AD小鼠脑部APP剪切的中的作用;(3)低氧改变APP异常代谢的分子机制。 结果:我们发现长期的间歇性低氧干预,能够增加APP蛋白的β剪切,加快APPswe+PS1A246E 转基因小鼠脑部Aβ的产生以及老年斑沉积。进一步研究发现低氧干预能增加小鼠脑部γ分泌酶组分APH-1a的水平,继而引起γ分泌酶活性的提高。此外,我们还证明低氧干预能够激活小鼠脑部细胞的自噬,而细胞内自噬的激活会增加APP蛋白代谢中间产物C99的γ剪切,从而引起Aβ水平的上升。利用细胞低氧模型研究,我们发现在SH-SY5Y细胞中,绿色荧光标记的C99(Green Fluorescent Protein, GFP-C99)和Monodansylcadaverine (MDC) 标记的自噬小体有部分的共定位;用自噬的抑制剂3-methyladenine (3-MA)预处理SH-SY5Y细胞后,可以显著减弱低氧引起的Aβ水平的增加,提示自噬的确参与了低氧介导的APP代谢异常。 结论:综上所述,我们的研究发现低氧能够影响APP的代谢,促进APP蛋白的β和γ剪切,从而增加Aβ的产生,最终加快了AD发病的进程。 目的: 肌萎缩侧索硬化 (Amyotrophic lateral sclerosis, ALS)是一种导致运动神经元变性的神经退行性疾病,随着运动神经元的丢失,患者逐渐丧失活动能力,最后危及生命。约有20%的家族性ALS是由铜/锌超氧化物歧化酶1(Cu, Zn-superoxide dismutase, SOD1)基因突变造成,但是相关病理机制还没有完全明确。最近研究发现,突变的SOD1蛋白可以通过细胞自噬(autophagy)通路降解,为了进一步探讨自噬在ALS病理过程中的意义,我们首先对转SODG93A基因的ALS小鼠模型脊髓部位的自噬情况进行了研究。 方法:我们通过检测ALS小鼠脊髓部位细胞自噬标记物LC3-II的表达和分布,以及电镜观察,研究不同病理阶段的ALS小鼠脊髓部位细胞自噬的激活情况。 结果:免疫印迹结果显示,ALS小鼠脊髓部位自噬的标记物LC3-II的表达从90天起就有明显的增加,免疫组化显示LC3-II在运动神经元胞体内呈点状分布;电镜结果显示ALS小鼠脊髓运动神经元胞体内自噬小体(autophagic vacuoles, AVs)的数量与正常对照相比没有明显变化,疾病末期组(140天)小鼠脊髓运动神经元胞体内自噬小体的数量比其同年龄野生型小鼠略有增加。进一步研究表明,ALS小鼠脊髓前角部位的有髓神经纤维内的自噬小体要显著多于正常对照,这种变化在ALS小鼠90天时就已经出现,120天时更为显著。 结论:自噬在ALS小鼠的脊髓部位有明显的激活,这种激活作用主要发生在运动神经元的有髓神经突触,表明突触是ALS运动神经元病变最先累及的部位
English Abstract: Objectives: Alzheimer disease (AD) is the most common neurodegenerative disorder in aged people characterized by neuronal loss and cognitive impairment. It is known that familial AD can be caused by certain gene mutations while sporadical forms are associated with several genetic and environmental risk factors. However, the detailed mechanisms involved in the pathogenesis of AD are still largely unknown. Recently, circumstantial evidences suggest environmental factors to be significant contributor for the development of AD. The greatly increased incidence of AD following ischemic cerebrovascular disease indicates that hypoxia is a risk factor that may accelerate AD pathogenesis by altering amyloid precursor protein (APP) processing. However, the exact molecular mechanisms underlying the hypoxia mediated AD pathogenesis are yet to be elucidated. Method:In the present study, we applied APPswe+PS1A246E double transgenic mice model and cell model to investigate the molecular mechanism underlying the hypoxia mediated acceleration of AD pathogenesis. Our study mainly focued on three aspects: (1) the effect of hypoxia on amyloid β (Aβ) generation and plaque formation in APPswe+PS1A246E double transgenic mice; (2) the change of APP processing in AD mice under hypoxic condition; (3) the molecular mechanisms of abnormal APP processing induced by hypoxia. Results: We demonstrated that repeated hypoxia increased Aβ generation and neuritic plaques formation through elevating β-cleavage of APP in APPswe+PS1A246E double transgenic mice. We also found that hypoxia enhanced the expression of APH-1a, a component of γ-secretase complex, which in turn may lead to increase in γ-cleavage activity. Furthermore, we demonstrated that repeated hypoxia treatment can activate macroautophagy (hereinafter autophagy), which may contribute to the increase in Aβ production since pretreatment with macroautophagy inhibitor 3-methyladenine significantly blocked chemical hypoxic condition-induced increase in Aβ production in SH-SY5Y cells. Conclusion: Taken together, our results suggest that hypoxia plays an important role in modulating the APP processing by facilitating both β and γ-cleavage which may result in a significant increase of Aβ generation. Objective: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons (MNs) leading to paralysis and death. About 20% familial cases of ALS (fALS) are caused by Cu, Zn-superoxide dismutase (SOD1) gene mutation. There is evidence suggesting that macroautophagy can degrade mutated SOD1 in vitro. To investigate the role of autophagy in the pathogenesis of ALS, we examined the status of macroautophagy in the spinal cord of SOD1G93A transgenic mice at different stages. Method: We applied western-blot and inmmunochemistry to investigate LC3 processing in spinal cord as well as its distribution in MNs. In addition, we determined the number of autophagic vacuoles (AVs) in different part of MNs by electrical microscopy in the spinal cord of ALS mice at different stages. Results: Western-blot showed the protein level of LC3-II in spinal cord of ALS mice was increased; and immunohistochemical analysis showed that LC3 immunofluorescence turned into a population of puncta in MNs of ALS mice; EM image showed that AVs in the cell bodies of MNs of ALS mice were not significantly altered until the mice reached at the terminal stage (140 d) when we found a few more AVs appeared. However, the number of AVs was significantly increased in myelinated nerve fiber in the spinal cord of ALS mice at presymptomatic stage (90 d). This increase became even more significant when the mice reached at the onset stage (120 d). Conclusion: Our data demonstrate that autophagy is activated in spinal cord of SOD1G93A mice indicating a possible role of macroautophagy in the pathogenesis of ALS. In addition, the early change in myelinated nerve fiber of the MNs indicates that the neurites are most vulnerable which is the earliest part involve in the pathogenesis of ALS.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2370
Appears in Collections:神经所(总)_学位论文

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Recommended Citation:
部分一:低氧加重阿尔茨海默病病理进程的机制研究 部分二:自噬在肌萎缩侧索硬化中病理机制的研究.李靓[d].中国科学院上海生命科学研究院,2008.20-25
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