Identifying Factors Regulating Medium Spiny Neuron Differentiation or Maintenance as Therapeutic Targets for Huntington's Disease using Induced Pluripotent Stem Cells

摘要 亨廷顿病(HD)是一种致命性、显性遗传性神经退行性疾病，主要影响 纹状体和皮质中的神经元，目前还没有有效的治疗方法。先天性心脏病是由一种 亨廷顿蛋白基因中CAG的扩增导致编码蛋白中的多谷氨酰胺(PolyQ)扩增 (HTT)，CAG扩张超过38次的患者表现出舞蹈、心理问题、 和认知能力下降。突变型HTT的表达导致选择性神经元功能障碍和变性 尽管它的表达模式无处不在。干细胞研究的最新进展表明，患者诱导 多能干细胞(IPSCs)可能提供新的疾病模型和新的疾病治疗方法。这些 研究将利用来自HD患者的IPSCs(HD-IPSCs)作为HD的人体模型。使用遗传 工程上，我们获得了用于HD建模的等位基因HD-IPSC系列(CAG重复21，45，72， 100)。了解慢性萎缩性胃炎患者CAG重复序列扩增依赖性疾病表型的分子基础 ，我们对HD iPSCs和HD神经干细胞(NSCs)进行了转录转录分析，并与 同基因对照。差异基因表达和通路分析表明转化生长因子-β和Netrin-1是最高的 参与神经元发育的失调通路和失调基因被丰富 背侧纹状体的形成。被破坏的纹状体和神经元网络可以被调节到 纠正HD表型并提供治疗靶点。因此，经过校正的同基因HD-IPSCs 等位基因提供了对疾病过程的机械性见解，并允许识别新的治疗方法 HD的目标。事实上，我们的研究表明，导致介质成熟或保持的因素 多刺神经元(MSN)可能改善亨廷顿病的表型。我们已经发现了Netrin 导致MSN成熟率提高，自发电活动增加， DARPP-32水平。我们将在本申请中调查以下目标：具体目标1.我们将 描述正常IPSCs、HD-IPSCs和经基因矫正的HD-IPSCs的细胞和功能缺陷 使用组学方法将IPSCs分化为中等刺状神经元；特定目标2.使用DARPP-32 在成熟的MSN中指导基因表达的基因组元件，我们将开发一个成熟的标记 并确定在该细胞HD模型中调节MSN分化和维持的因素； 具体目标3.我们将确定促进MSN差异化或维护的因素是否会改善HD 这种疾病的小鼠模型的表型。HD的治疗目标将被确定和新的治疗方法 将会被探索。

ABSTRACT Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder that primarily affects neurons in the striatum and cortex, and for which there is currently no effective treatment. HD is caused by a CAG expansion in the huntingtin gene leading to a polyglutamine (polyQ) expansion in the encoded protein (HTT), and patients with a CAG expansion greater than 38 repeats exhibit chorea, psychological problems, and cognitive decline. Expression of mutant HTT leads to selective neuronal dysfunction and degeneration despite its ubiquitous expression pattern. Recent advances in stem cell research suggest that patient induced pluripotent stem cells (iPSCs) may provide novel models of disease and new treatments for diseases. These studies will utilize iPSCs derived from HD patients (HD-iPSCs) as a human model of HD. Using genetic engineering, we generated an isogenic allelic HD-iPSC series for HD modeling (CAG repeat of 21, 45, 72, 100). To understand the molecular basis for the CAG repeat expansion dependent disease phenotypes in NSCs, we performed transcriptomic analysis of HD iPSCs and HD neural stem cells (NSCs) compared to isogenic controls. Differential gene expression and pathway analysis pointed to TGF-β and netrin-1 as the top dysregulated pathways, and dysregulated genes were enriched for those involved in neuronal development and the formation of the dorsal striatum. The disrupted striatal and neuronal networks could be modulated to correct HD phenotypes and provide therapeutic targets. Therefore the isogenic HD-iPSCs with corrected alleles provides mechanistic insights into the disease process and allows the identification of novel therapeutic targets for HD. Indeed our studies suggest that factors that lead to the maturation or maintenance of medium spiny neurons (MSNs) are likely to ameliorate Huntington's disease phenotypes. We have found that netrin leads to enhanced rate of maturation of MSNs with increased spontaneous electrical activity and increased levels of DARPP-32. We will investigate the following aims in this application: Specific Aim 1. We will characterize the cellular and functional deficits in normal iPSCs, HD-iPSCs, and genetically corrected HD- iPSCs differentiated into medium spiny neurons using “omics” approaches; Specific Aim 2. Using DARPP-32 genomic elements that direct gene expression specifically in mature MSNs, we will develop a marker of mature MSNs and identify factors that mediate differentiation and maintenance of MSNs for this cellular HD model; Specific Aim 3. We will determine if factors that promote MSN differentiation or maintenance ameliorate HD phenotypes in mouse models of the disease. Therapeutic targets will be identified and new treatments for HD will be explored.