Generation and Characterization of Alzheimer Brain Cells

描述（由申请人提供）：遗传学方法为阿尔茨海默病（AD）的分子发病机制提供了重要见解。然而，只有约3%的AD是由于淀粉样前体蛋白（APP）或早老素1或2（PS1，PS2）的基因突变。我们建议使用诱导多能干细胞（iPS）生成人类体外模型，在该模型中，可以更准确地研究家族性和散发性AD的遗传和发育方面，并且可以确定后续药物发现的治疗靶点。提出了以下具体目的：具体目的1：从家族性和散发性AD受试者的皮肤成纤维细胞产生iPS细胞和神经元。我们已经成功地从PS1突变受试者的成纤维细胞中产生了分化的神经元。我们已经证明，这些神经元的分化导致它们获得明显的标准分子表型;即，A/42/40比率的变化）。这些神经元的初始基本标准化将包括，对于每个PS1突变，探索患者、受影响和未受影响的家庭成员以及携带相同突变或不同PS1突变的不同家庭中A42/40表型的个体内和个体间变异性。一个长期的目标将是神经胶质细胞和混合细胞培养物的产生。具体目的2：对AD iPS细胞系进行分子、生化和功能表征。我们已经定义了一个培养系统的AD iPS细胞衍生的神经元，包括必要的A <$42/40表型。我们现在将着手建立这些iPS细胞中AD相关分子的含量，同时寻求建立A <$42/40表型的细胞生物学基础。这将包括自噬途径的评估。具体目的3：鉴定家族性和散发性AD iPS细胞的转录谱。或者，该目的的主要目标是建立来源于上述iPS细胞系组的前脑神经细胞的基线分子表征。将对这些配置文件进行信息分析，以确定可能的AD相关网络，如Geschwind及其同事最近定义的那样。我们将研究来自患者iPS细胞的端脑神经细胞的体外细胞和分子表型如何变化，并且在与家族性或散发性AD相关的个体和突变中相似。 公共卫生相关性：遗传学方法为阿尔茨海默病（AD）的分子发病机制提供了重要见解。然而，只有约3%的AD是由于淀粉样前体蛋白（APP）或早老素1或2（PS1，PS2）的基因突变。我们建议使用诱导多能干细胞（iPS）生成人类体外模型，在该模型中，可以更准确地研究家族性和散发性AD的遗传和发育方面，并且可以确定后续药物发现的治疗靶点。

DESCRIPTION (provided by applicant): Genetic approaches have provided major insights into the molecular pathogenesis of Alzheimer's disease (AD). However, only about 3% of all of AD is due to genetic mutations in either amyloid precursor protein (APP), or presenilin 1 or 2 (PS1, PS2). We propose to generate a human in vitro model using induced pluripotent stem (iPS) cells, in which the genetic and developmental aspects of familial and sporadic AD can be studied more accurately and therapeutic targets can be identified for subsequent drug discovery. The following Specific Aims are proposed: Specific Aim 1: To generate iPS cells and neurons from skin fibroblasts from subjects with familial and sporadic AD. We have already succeeded in generating differentiated neurons from fibroblasts from subjects with PS1 mutations. We have demonstrated that differentiation of these neurons leads to their acquisition of an obvious standard molecular phenotype; i.e., a shift in the A¿ 42/40 ratio). The initial essential standardization of these neurons will include, for each PS1 mutation, the exploration of intra-individual and inter-individual variability in the A¿ 42/40 phenotype within patients, affectd and unaffected family members, and across different families that carry either the identical mutation or across different PS1 mutations. A longer-term goal will be the generation of glia and mixed cell cultures. Specific Aim 2: To perform molecular, biochemical and functional characterization of AD iPS cell lines. We have defined a culture system for AD iPS cell-derived neurons that includes the essential A¿ 42/40 phenotype. We will now proceed to establish the content of AD-related molecules in these iPS cells while seeking to establish the cell biological basis for the A¿ 42/40 phenotype. This will include an assessment of the autophagic pathway. Specific Aim 3: Identification of transcriptional profiles of familial and sporadic AD iPS cells. Or primary goal in this aim is to establish a baseline molecular characterization of forebrain neural cells derived from the panel of iPS cell lines specified above. Informatic analysis of these profiles will be performed in order to identify possible AD-related networks, as recently defined by Geschwind and colleagues. We will study how in vitro cellular and molecular phenotypes in telencephalic neural cells derived from patient iPS cells vary and are similar across individuals and mutations related to either familial or sporadic AD. PUBLIC HEALTH RELEVANCE: Genetic approaches have provided major insights into the molecular pathogenesis of Alzheimer's disease (AD). However, only about 3% of all of AD is due to genetic mutations in either amyloid precursor protein (APP), or presenilin 1 or 2 (PS1, PS2). We propose to generate a human in vitro model using induced pluripotent stem (iPS) cells, in which the genetic and developmental aspects of familial and sporadic AD can be studied more accurately and therapeutic targets can be identified for subsequent drug discovery.