EXPRESSION PROFILING OF ENDOSOMAL PATHWAYS IN AD

AD 内体途径的表达谱

• 批准号: 6920489
• 负责人: STEPHEN D GINSBERG
• 金额: $ 28.6万
• 依托单位: NATHAN S. KLINE INSTITUTE FOR PSYCH RES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6920489
• 关键词: Alzheimer' s disease; Downs syndrome; autophagy; biomarker; cellular pathology; dentate gyrus; endocytosis; fibroblasts; gene expression; gene expression profiling; genetically modified animals; granule cell; human tissue; laboratory mouse; laser capture microdissection; lysosomes; microarray technology; molecular pathology; neuritic plaques; neurofibrillary tangles; neurogenetics; neurons; neuropathology; postmortem; pyramidal cells; tissue /cell culture; vesicle /vacuole

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.

该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。该设计旨在评估弱势群体内的基因表达水平，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，对具有早期内体异常的神经元进行基因表达检测，这是 AD 相关反应的第一个迹象。对人类死后大脑、称为 Ts65Dn 的唐氏综合症 (Ts21) 小鼠模型以及培养细胞进行人类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”。以这种方式，系统地应用 cDNA 阵列分析来表征 相对于这些大脑中幸存的神经元和正常对照大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。实验设计需要对已识别的神经元群体进行微抽吸，然后采用项目负责人实验室开发的新型单细胞 RNA 扩增方法，并结合定制设计的 cDNA 阵列分析。目标 1 包括评估人类死后大脑中选定的神经元群。目标 2 评估从 Ts65Dn 和二倍体小鼠获得的单个神经元群。目标 3 包括基因表达分析 培养的 Ts21 成纤维细胞的 App 水平通过小干扰 RNA (siRNA) 被降低。 Aim 4 利用 siRNA 技术敲低三体区域的其他基因，用于后续 cDNA 阵列分析。这种最先进的范式能够在选择性脆弱且相对不受神经变性影响的细胞类型中广泛、同时表示数百个基因，这些细胞类型代表了在病理进化的特定阶段在 AD 和 Ts21 大脑中观察到的一些最早的病理变化。