自噬在淀粉样蛋白-β异常聚集于视网膜色素上皮细胞中的作用研究

Age-related macular degeneration (AMD) is a degenerative disease in the eye,which causes irreversible blindness in elderly and is one of the major causes of blindness in developed countries. Drusen is pathological hallmark of AMD, of which drusen accumulates in the subretinal pigment epithelium (RPE) space. β-amyloid (Aβ) peptide, a major molecular signature in the brain of Alzheimer’s disease（AD）, have been identified as one of the major components in drusen as well as in RPE cells in the retina of AMD. Recent studies suggest that, Aβ might play an important role in the drusen formation and pathogenesis of AMD. Our previous data together with the current study support the notion that Aβ accumulation and Aβ-mediated toxity may involved in the formation of dursen...Autophagy is the major pathway involved in the degradation of proteins and organelles, cellular remodeling, and survival during nutrient starvation. Autophagosomal dysfunction has been implicated in an increasing number of diseases from cancer to bacterial and viral infections and more recently in neurodegeneration.While a decrease in autophagic activity appears to interfere with protein degradation and possibly organelle turnover, increased autophagy has been shown to facilitate the clearance of aggregation-prone proteins and promote neuronal survival in a number of disease models. Autophagy has gained increasing attention in neuronal cell biology.Neuronal autophagy is induced early in AD and before Aβ deposits extracellularly. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes.Autophagy appears impaired in the final stages of neurodegenerative disease. All these give us a clue that autophagy is involved in Aβ metabolism and thus could represent a therapeutic target in AD. However, it is still unknown the role of autophagy in the aggregation of Aβ in RPE...We design the experiments by using transgenic mice overexpressed human APP (Tg2576)and stable GFP-LC3-expressed APRE-19 transfected with human APP or Aβ. First, we detected age-related autophagy changes in RPE layer and whether these changes play a role in the aggregation of Aβ in transgenic mice and wild type control mice. Transmission electron microscopy(TEM) was used to observe the age-related ultrastructure and AVs of RPE in Tg2576 and Western-blot analysis to explore the expression of LC3 and Atg6 protein in RPE of Tg2576 in different age group.Then we estabalish a dynamic in- vitro system to observe autophagy.Meanwhile expression of Aβ and LC3 in different circumstance was examined by Western-blot.At last,we use autophagy inhibitor and inducer to show the role of autophagy in the aggregation of Aβ in vivo and in vitro respectively.

淀粉样蛋白-β（Aβ）异常聚集于视网膜色素上皮（RPE）细胞并致细胞损伤。自噬是胞内蛋白质降解的主要途径，影响Aβ在中枢神经元的聚集。本项目从蛋白质异常聚集和降解入手，提出“自噬影响Aβ异常聚集于RPE”的工作设想。首先应用不同月龄过表达APP的转基因小鼠，采用免疫荧光、透射电镜、免疫印迹等技术，观察RPE自噬活性在Aβ聚集过程中的时空变化；继而转染APP/Aβ至稳定表达GFP-LC3的APRE19细胞，活细胞示踪自噬、结合亚细胞定位，明确与自噬相关的Aβ在细胞内的代谢途径；通过自噬干预（特异性激动剂和抑制剂），探讨调控RPE细胞自噬对Aβ异常聚集及细胞功能的作用及机制。通过本研究明确①RPE细胞自噬活性在Aβ异常聚集过程中的变化②调控RPE细胞自噬对Aβ异常聚集及RPE细胞功能作用，有望为探索RPE保护的新靶点提供实验依据与理论基础。

年龄相关性黄斑变性(Age-related macular degeneration，AMD)可引起严重的视力丧失，位列发达国家老年人群中第一位不可逆性致盲眼病 。 AMD 根据病理特点不同可分为干性和湿性。异常蛋白质聚集形成玻璃膜疣(drusen)是干性 AMD 的典型病理特征。视网膜色素上皮 (retina pigment epithelium cell， RPE) 细胞内和/或 RPE细胞外发生异常蛋白聚集，导致 RPE 功能障碍，进而诱发氧化应激、炎症反应、引起血管生成因子和血管生成拮抗因子失衡，最终导致 AMD。 对于 AMD—特征性伴随蛋白质错误折叠并发生聚集，研究关注的焦点一直是蛋白质异常聚集发生机制以及有效清除方式。淀粉样蛋白-β（Aβ）异常聚集于视网膜色素上皮（RPE）细胞并致细胞损伤。蛋白质异常聚集发生机制以及有效清除方式是当前研究的热点和难点。针对以上问题，本项目完成了以下工作： 利用过表达人源 APP 转基因动物和自噬细胞示踪、工具药物，从体内和体外两个层次、正反两个方面进行以下研究：①体内应用两种 APP 过表达转基因小鼠，采用免疫荧光、透射电镜、免疫印迹等技术，观察Aβ 聚集过程中自噬活性 （自噬标志物/关键自噬相关基因蛋白）变化的时空特点；②体外建立自噬示踪联合 APP/Aβ 过表达细胞模型，转染APP/Aβ至稳定表达GFP-LC3的APRE19细胞，活细胞示踪自噬、结合亚细胞定位，动态观察与自噬相关的 Aβ在细胞内产生和代谢的过程及自噬与细胞外 Aβ 生成的关系；明确与自噬相关的Aβ在细胞内的代谢途径；③根据体内及体外对上述病理过程中 RPE 自噬活性的动态变化情况， 采用自噬特异性激动剂以增加内源性自噬、自噬特异性抑制剂以抑制内源性自噬，研究自噬干预对 Aβ 聚集及 RPE 功能的作用及其机制。上述研究对明确Aβ在RPE异常聚集过程有重要作用，有助于深入和丰富对Aβ聚集致细胞毒性作用的认识，并有可能为 drusen 形成---异常蛋白质聚集，提供新的研究方向，拓展了以自噬为调控靶点的AMD防治思路。