BETA AMYLOID AND MITOCHONDRIAL TOXICITY IN ALZHEIMER DISEASE

越来越多的证据表明，细胞内淀粉样β肽（Abeta）的积累是神经元扰动的关键因素。然而，Abeta通过其损害细胞特性导致神经元功能障碍的关键细胞内机制仍有待阐明。在过去的资助期间的工作支持ABAD和与AD相关的Abeta介导的线粒体功能障碍之间的联系：线粒体功能的早期缺陷。在靶向表达突变型APR和ABAD的转基因（Tg）小鼠的线粒体中发现了ABAD-AB复合物。我们发现了线粒体中Abeta的意外定位。我们的初步研究，使用生物化学和形态学方法（免疫印迹，双 用共聚焦显微镜和免疫电子显微镜进行免疫染色）证实了来自人AD脑和来自具有突变体人淀粉样前体蛋白（Tg mAPP）的靶向神经元过表达的转基因（Tg）小鼠的线粒体中的Abeta。此外，我们观察到易位的Abeta从内质网（ER）的线粒体在原代皮层神经元培养的Tg mAPP小鼠的大脑。与nonTg littlemates相比，在Tg mAPP小鼠的分离线粒体和脑中观察到线粒体功能障碍。我们假设这些观察结果的基本机制是由于两个事件引起的膜渗透性转换孔（MPTP）的Aft诱导的扰动：亲环素D（CypD）与线粒体内膜（即，与MPTP组分的相互作用），以及bax与线粒体外膜的增强的结合（从而改变外膜以及MPTP的渗透性），其触发半胱天冬酶的活化 途径和细胞色素c释放导致神经元凋亡。我们的具体目标是：1）描述A β定位于线粒体的参数，并将A β在线粒体中的水平与AD影响的脑区域（与备用区域相比）中的线粒体功能相关联; 2）确定A β输入到线粒体中的机制; 3）确定A β介导的线粒体功能障碍的机制; 4）使用转基因小鼠在体内分析Bax对A β诱导的细胞应激的贡献。这种竞争性更新是基于这样的假设，即线粒体提供了神经元内Abeta积累的位点，其增强了导致阿尔茨海默病中神经元扰动的细胞器功能障碍。这些研究将提供一种新的细胞内途径， 与AD相关的功能障碍。

Increasing evidence indicates that intracellular accumulation of amyloid-beta peptide (Abeta) is a key factor in neuronal perturbation. However, critical intracelluar mechanisms through which Abeta impairs cellular properties resulting in neuronal dysfunction remain to be elucidated. Working during the past grant period supports a link between ABAD and Abeta-mediated mitochondrial dysfunction relevant to AD: early defects of mitochondrial function. ABAD-AB complex was found in mitochondria of transgenic (Tg) mice with targeted neuronal expression of mutant APR and ABAD. We have found unexpected localization of Abeta in mitochondria. Our pilot study, using both of biochemical and morphologic methods (immunoblotting, double immunostaining with confocal microscope and immunoelectron microscopy) demonstrated Abeta in mitochondria from human AD brain and from transgenic (Tg) mice with targeted neuronal overexpression of mutant human amyloid precursor protein (Tg mAPP). Furthermore, we have observed translocation of Abeta from endpplasmic reticulum (ER) to the mitochondria in primary cortical neurons cultured from brains of Tg mAPP mice. Mitochondrial dysfunction was observed in isolated mitochondria and brains of Tg mAPP mice as compared with nonTg littlemates. We postulate that the basic mechanisms underlying these observations is Aft-induced perturbation of the membrane permeability transition pore (MPTP) due to two events: increased association of cyclophilin D (CypD) with the inner mitochondrial membrane (i.e., interaction with components of the MPTP), and enhanced association of bax with the outer mitochondrial membrane (thereby altering permeability of the outer membrane as well as MPTP), which triggers activation caspase pathway and cytochrome c release leading to neuronal apoptosis. Our specific aims are: 1) To delineate parameters of Abeta localization to mitochondria and to correlate levels of Abeta in mitochondria with mitochondrial function in AD-affected brain regions as compared with spared-regions; 2) To determine the mechanism of AB import into mitochondria; 3) To determine mechanisms of Abeta-mediated mitochondrial dysfunction; 4) To analyze the contribution of Bax to Abeta-induced cell stress in vivo using transgenic mice. This competitive renewal is based on the hypothesis that mitochondria provide a site for accumulation of intraneuronal Abeta which potentiates organelle dysfunction leading to neuronal perturbation in Alzheimer's disease. The proposed studies would provide a new intracellular pathway potentially leading to neuronal dysfunction relevant to AD.