EXPRESSION PROFILING OF ENDOSOMAL PATHWAYS IN AD

本提案的目的是描述导致内吞、自噬和溶酶体系统（EALS）功能障碍的先前细胞和分子事件，这是已知最早发生在散发性阿尔茨海默病（AD）中的细胞紊乱。设计目的是评估易感人群的基因表达水平，同时避免其他细胞类型的潜在污染。与尚未受影响的相邻神经元和较不脆弱的神经元群体相比，检测早期内体异常神经元的基因表达，这是ad相关反应的第一个迹象。人类海马神经元和新皮层神经元、小鼠海马神经元和新皮层神经元以及成纤维细胞的“分子指纹”在人类死后的大脑、称为Ts65Dn的唐氏综合症小鼠模型（Ts21）和培养细胞中进行。以这种方式，cDNA阵列分析被系统地应用于表征

The aim of this proposal is to delineate antecedent cellular and molecular events that lead to the dysfunction of the endocytic, autophagic, and lysosomal systems (EALS), the earliest cellular disturbances known to occur in sporadic Alzheimer's disease (AD). The design is to assess gene expression levels within vulnerable populations while avoiding potential contamination from other cell types. Gene expression is assayed in neurons with early endosomal abnormalities, the first sign of AD-related responses, as compared to not-as-yet affected neighbors and to less vulnerable neuronal populations. A "molecular fingerprint" of human hippocampal neurons and neocortical neurons, mouse hippocampal and neocortical neurons, and fibroblasts is performed on human postmortem brains, a mouse model of Down's syndrome (Ts21) termed Ts65Dn, and in cultured cells. In this manner, cDNA array analysis is applied systematically to characterize gene expression changes at the inception of EALS pathology relative to spared neurons in these brains and to unaffected neurons in normal control brains. The experimental design entails microaspiration of identified neuronal populations followed by a novel single cell RNA amplification methodology developed in the laboratory of the Project Leader combined with custom-designed cDNA array analysis. Aim 1 consists of assessment of select neuronal populations from human postmortem brains. Aim 2 evaluates individual neuronal populations obtained from Ts65Dn and diploid mice. Aim 3 consists of gene expression analysis of cultured Ts21 fibroblasts with App levels knocked down via small interference RNA (siRNA). Aim 4 utilizes siRNA technology to knockdown other genes in the trisomic region for subsequent cDNA array analysis. This state-of-the-art paradigm enables an extensive, concurrent representation of hundreds of genes in selectively vulnerable and relatively spared cell types to neurodegeneration that represent some of the earliest pathological changes observed in AD and Ts21 brains at defined stages of pathology evolution.