Modeling Pathogenesis of Huntington's disease using patient-derived induced pluri

描述（由申请人提供）：亨廷顿病（HD）是一种无法治愈的遗传性神经退行性疾病，由亨廷顿蛋白（Htt）基因外显子1 CAG重复序列异常扩增引起。目前尚不清楚为什么突变Htt对神经元，特别是纹状体中3-氨基丁酸（GABA）投射神经元的中棘神经元（msn）有毒。人们普遍认为，对HD发病机制缺乏了解的主要原因之一是缺乏可靠的实验模型。HD的细胞培养模型对于了解HD的分子机制至关重要，并已在许多研究中得到应用。虽然先前开发的HD稳定细胞系表现出HD的一些病理特征，但几乎没有一个具有足够的神经元标记物或其他神经元特性，因此它们不是HD建模的理想选择。为了更好地了解HD的发病机制，还需要新的HD细胞模型。近年来，在将人类体细胞重编程为诱导多能干细胞（iPS）方面的快速进展，使得从HD患者身上产生iPS细胞模型成为可能。然而，由于目前的HD iPS细胞系含有多个用于传递多种重编程因子的病毒插入物，因此这些细胞是否能够很好地模拟HD是值得怀疑的，因为多个病毒插入物可能会破坏内源性基因表达并导致异常的细胞分化。为了克服这个问题，我们最近从HD患者成纤维细胞中产生了无病毒和无转基因的iPS细胞，在这个项目中，我们建议使用这些iPS细胞来模拟HD。我们假设这些不含任何外源DNA序列的HD iPS细胞，在适当的条件下，可以成功诱导分化为成熟神经元，特别是gaba能神经元。此外，我们假设它们会在细胞培养中复制HD在神经元成熟和衰老过程中的主要病理特征。为了检验这些假设，提出了以下具体目标，其中两名本科生将参与研究。1. 描述无病毒或转基因插入物的HD患者来源的iPS细胞系。2. 诱导iPS细胞向gaba能神经元分化。3. 目的：观察来自HD iPS细胞的gaba能神经元的特征。小灵通398/2590 （Rev. 11/07）页延续格式页

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an incurable inherited neurodegenerative disorder resulting from an abnormal expansion of CAG repeats in exon 1 of the huntingtin (Htt) gene. It remains unclear why mutant Htt is toxic to neurons, especially medium spiny neurons (MSNs) that are 3-aminobutyric acid (GABA) projection neurons in the striatum. It has been widely accepted that one of the major reasons for the lack of understanding of HD pathogenesis is the paucity of reliable experimental models. Cell culture models of HD are essential for understanding the molecular mechanisms of HD and have been employed in a number of studies. Although the HD stable cell lines developed previously exhibit some of the pathological features of HD, almost none of them possess adequate neuronal markers or other neuronal properties and thus they are not ideal for modeling HD. To better understand the pathogenesis of HD, additional novel cell models of HD are required. Recent rapid advances in reprogramming human somatic cells into induced pluripotent stem (iPS) cells have made it possible to generate iPS cell models from HD patients. However, since the current HD iPS cell lines contain multiple viral inserts that are used for delivering several reprogramming factors, it is questionable whether these cells can model HD well because the multiple viral inserts may disrupt endogenous gene expression and cause abnormal cellular differentiation. To overcome this problem, we recently generated virus- and transgene-free iPS cells derived from HD patient fibroblasts and, in this project, we propose to model HD using these iPS cells. We hypothesize that these HD iPS cells, free of any exogenous DNA sequence, can be successfully induced to differentiate into mature neurons, especially GABAergic neurons, once adequate conditions are given. Additionally, we hypothesize that they will reproduce the major pathological features of HD across neuronal maturation and aging in cell culture. To test these hypotheses the following specific aims are proposed, in which two undergraduate students will participate in the studies. 1. To characterize HD patient-derived iPS cell lines free of viral or transgenic inserts. 2. To induce the iPS cells to differentiate into GABAergic neurons. 3. To characterize the GABAergic neurons derived from the HD iPS cells. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Huntington's disease is an inherited neurological disorder that selectively causes nerve cells to die in the specific brain regions and effective therapy for this disease is still unavailable at present. Although various cell and animal models of Huntington's disease have been created over last decade, so far there is no ideal model that can replicate all of essential pathological features of this disease. This project is designed to determine whether the nerve cells that are converted from patient induced pluripotent stem cells can mimic major aspects of pathology of this disorder in cell culture.