EXPRESSION PROFILING OF ENDOSOMAL PATHWAYS IN AD

这项建议的目的是描绘导致内吞、自噬和溶酶体系统(EALS)功能障碍的前驱细胞和分子事件，EALS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。这项设计是为了评估脆弱人群中的基因表达水平，同时避免来自其他细胞类型的潜在污染。在有早期内体异常的神经元中检测基因表达，这是AD相关反应的第一个迹象，与尚未受到影响的邻居和较不脆弱的神经元群体进行比较。人类海马神经元和新皮质神经元、小鼠海马神经元和新皮质神经元以及成纤维细胞的“分子指纹”在人死后的大脑、唐氏综合症(Ts21)小鼠模型Ts65Dn和培养细胞中进行。以此方式，系统地应用cDNA阵列分析来表征 在EALS病理开始时，相对于这些大脑中的备用神经元和正常对照大脑中未受影响的神经元，基因表达发生了变化。实验设计需要对已识别的神经元群体进行显微抽吸，然后在项目负责人的实验室中开发出一种新的单细胞RNA扩增方法，并结合定制设计的cDNA阵列分析。目标1包括从人死后大脑中选择神经元群体的评估。目的2评估从Ts65Dn和二倍体小鼠中获得的单个神经元群体。目标3由基因表达分析组成 培养的Ts21成纤维细胞通过小干扰RNA(SiRNA)下调App水平。AIM 4利用siRNA技术敲除三体区域中的其他基因，用于后续的cDNA阵列分析。这一最先进的范例能够在选择性脆弱和相对较少发生神经变性的细胞类型中广泛、同时表示数百个基因，这些细胞类型代表了AD和Ts21大脑在确定的病理进化阶段观察到的一些最早的病理变化。

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.