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

摘要 亨廷顿氏病（HD）是一种致命的、显性遗传的神经退行性疾病，主要影响 纹状体和皮质中的神经元，并且目前没有有效的治疗方法。HD是由一种 亨廷顿蛋白基因中的CAG扩增导致编码蛋白中的多聚谷氨酰胺（polyQ）扩增 (HTT)CAG扩增大于38次重复的患者表现出舞蹈病，心理问题， 和认知能力下降突变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， 多能干细胞（iPSC）可以提供新的疾病模型和新的疾病治疗方法。这些 研究将利用源自HD患者的iPSC（HD-iPSC）作为HD的人类模型。利用遗传 工程化，我们产生了用于HD建模的等基因等位基因HD-iPSC系列（CAG重复21，45，72， 100）。了解CAG重复扩增依赖性疾病表型的分子基础， 为了研究HD iPSC和HD神经干细胞的转录组学，我们进行了HD iPSC和HD神经干细胞（NSC）的转录组学分析，并与对照组进行了比较。 同基因对照差异基因表达和途径分析指出，TGF-β和netrin-1是最高的 异常调节的途径，以及那些参与神经元发育的基因被富集 以及背侧纹状体的形成。被破坏的纹状体和神经元网络可以被调节， 纠正HD表型并提供治疗靶点。因此，具有校正的HD-iPSC的同基因HD-iPSC 等位基因提供了对疾病过程的机制见解，并允许鉴定新的治疗药物。 HD的目标。事实上，我们的研究表明，导致培养基成熟或维持的因素 多刺神经元（MSNs）可能改善亨廷顿病的表型。我们发现奈特林 导致MSN的成熟率提高，自发电活动增加， DARPP-32的水平。我们将在本申请中研究以下目标：具体目标1。我们将 表征正常iPSC、HD-iPSC和遗传校正的HD-iPSC中的细胞和功能缺陷。 使用“组学”方法使iPSC分化成中等多刺神经元;具体目标2.使用DARPP-32 基因组元件，指导基因表达，特别是在成熟的MSN，我们将开发一个标记， MSNs，并鉴定介导该细胞HD模型的MSNs分化和维持的因子; 具体目标3。我们将确定促进MSN分化或维持的因素是否能改善HD 在疾病的小鼠模型中的表型。将确定治疗目标，并为HD提供新的治疗方法 将被探索。

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.