EXPRESSION PROFILING OF ENDOSOMAL PATHWAYS IN AD

该建议的目的是描述导致内吞、自噬和溶酶体系统（EALS）功能障碍的先行细胞和分子事件，这是已知在散发性阿尔茨海默病（AD）中发生的最早的细胞紊乱。该设计旨在评估脆弱人群中的基因表达水平，同时避免其他细胞类型的潜在污染。与尚未受影响的邻居和较不脆弱的神经元群体相比，在具有早期内体异常的神经元中测定基因表达，早期内体异常是AD相关反应的第一个迹象。人海马神经元和新皮质神经元、小鼠海马和新皮质神经元以及成纤维细胞的“分子指纹”在人死后脑、唐氏综合征（Ts 21）的小鼠模型（称为Ts 65Dn）和培养的细胞中进行。以这种方式，系统地应用cDNA阵列分析来表征 相对于这些脑中的备用神经元和正常对照脑中未受影响的神经元，在EALS病理学开始时基因表达发生变化。实验设计需要识别神经元群体的微抽吸，然后在项目负责人的实验室中开发的新的单细胞RNA扩增方法与定制设计的cDNA阵列分析相结合。目的1是从人死后脑中选择神经元群体进行评估。目的2评价从Ts65Dn和二倍体小鼠获得的单个神经元群体。目的3包括基因表达分析， 通过小干扰RNA（siRNA）敲低App水平的培养的Ts21成纤维细胞。目的4利用siRNA技术敲除三体区域的其他基因，为后续的cDNA阵列分析奠定基础。这种最先进的范例使得能够广泛地、同时地代表数百种基因，这些基因在选择性地易受神经变性影响且相对不受神经变性影响的细胞类型中，这些细胞类型代表了在病理学演变的限定阶段在AD和Ts21脑中观察到的一些最早的病理学变化。

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.