Rational derivation of DA neuron subtypes from iPS cells for improved modelling of Parkinson's disease

从 iPS 细胞中合理推导 DA 神经元亚型以改进帕金森病模型

• 批准号: 9082946
• 负责人: Rajeshwar B Awatramani
• 金额: $ 63.42万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9082946
• 关键词: Agonist; Back; Cells; Coupled; Data; Derivation procedure; Development; Disease; Disease model; Dopamine; Dorsal; Embryo; Foundations; Future; Gene Expression; Gene Targeting; Generations; Genetic; Genetic Models; Goals; Heterogeneity; Human; Individual; Knowledge; Label; Laboratories; Light; Literature; Logic; Messenger RNA; Methods; Midbrain structure; Modeling; Molecular; Molecular Profiling; Mus; Mutation; Nerve Degeneration; Neurons; Parkinson Disease; Pathway interactions; Patients; Phenotype; Physiological; Physiology; Production; Property; Protocols documentation; Regimen; Role; SOX6 gene; Signal Transduction; Solid; Substantia nigra structure; Sum; Testing; Time; Tyrosine 3-Monooxygenase; Ventral Tegmental Area; base; beta catenin; design; dopaminergic neuron; dosage; feeding; human stem cells; improved; in vivo; induced pluripotent stem cell; loss of function; mRNA Differential Displays; motor symptom; mouse model; neuron development; pars compacta; postnatal; progenitor; public health relevance; research study; stem; stem cell technology; transcription factor; transcriptome; transcriptomics

 DESCRIPTION (provided by applicant): Dopamine (DA) deficiency, caused by DA neuron degeneration, underpins the devastating motor symptoms of Parkinson's disease (PD). DA neurons located in the ventral tier of the substantia nigra pars compacta (SNc), are particularly vulnerable, compared to those in the dorsal tier of the SNc or ventral tegmental area (VTA). Why these DA neurons display differential vulnerability remains enigmatic. Understanding the underlying mechanisms would shed light on degeneration as well as potential neuroprotective strategies to mitigate the disease. iPS-derived DA neurons are an important new method for modeling PD. Yet current protocols for generating DA neurons are not designed to generate specific DA subtypes, a critical requisite for modeling selective vulnerability. This gap exists because the molecular heterogeneity of midbrain DA neurons is not well understood. To elucidate the heterogeneity of DA neurons, we have recently used single cell molecular profiling, coupled with anatomical co-labeling studies, and revealed the existence of at least six distinct of DA neuron subtypes in mouse models. Here, we aim to use this knowledge to i. better understand DA neuron diversity in vivo ii. understand mechanisms that may influence the generation of DA neuron subtypes iii. derive and characterize two prominent DA neuronal subtypes, one located in the SNc and one in the VTA, from human iPS cells in a rational manner, and iv. use these DA neuron subtypes to examine selective vulnerability in the context of genetic PD mutations. In Aim1, we will examine how Wnt signaling may influence DA neuron subtype allocation. In Aim 2, having optimized the Wnt regimen, we will next use targeted gene manipulations to derive highly enriched cultures of two specific DA neuron subtypes, and then characterize those subtypes by physiological and transcriptomic approaches. Next, we will generate both DA neuron subtypes from iPS cells harboring a DJ-1 mutation and examine differential pathological effects on both, SNc as well as VTA DA neuron subtypes. In Aim 3, we will further characterize the phenotype of the two DA neuron subtypes in vivo. We will elucidate the projections, and complete transcriptomes of two murine DA neuron subtypes, taking advantage of genetically targeted mice. Information from this aim will further highlight the differences between these subtypes. Additionally, these results will feed back into Aims 1 and 2, to further optimize our DA neuron subtype derivation protocol. In sum, taking advantage of the combined expertise and extensive interactions of two labs, we propose a cohesive plan based on molecular logic, to derive distinct DA neuron subtypes from iPS cells and aim to improve modelling PD. These studies will open the future possibility of understanding the effects of a range of PD mutations on selective vulnerability.

 描述（由申请人提供）：由 DA 神经元变性引起的多巴胺 (DA) 缺乏是帕金森病 (PD) 破坏性运动症状的基础。与 SNc 背层或腹侧被盖区 (VTA) 的神经元相比，位于黑质致密部 (SNc) 腹侧层的 DA 神经元特别脆弱。为什么这些 DA 神经元表现出不同的脆弱性仍然是个谜。了解潜在的机制将有助于了解退化以及减轻疾病的潜在神经保护策略。 iPS 衍生的 DA 神经元是建模 PD 的重要新方法。然而，目前用于生成 DA 神经元的协议并不是为了生成特定的 DA 亚型而设计的，而特定的 DA 亚型是选择性脆弱性建模的关键前提。这种差距的存在是因为中脑 DA 神经元的分子异质性尚不清楚。为了阐明 DA 神经元的异质性，我们最近使用单细胞分子分析，结合解剖学联合标记研究，揭示了至少六种不同的 小鼠模型中的 DA 神经元亚型。在这里，我们的目标是利用这些知识来实现​​。更好地了解体内 DA 神经元多样性 ii．了解可能影响 DA 神经元亚型生成的机制 iii。以合理的方式从人类 iPS 细胞中衍生并表征两种主要的 DA 神经元亚型，一种位于 SNc，一种位于 VTA，以及 iv．使用这些 DA 神经元亚型来检查遗传 PD 突变背景下的选择性脆弱性。在 Aim1 中，我们将研究 Wnt 信号传导如何影响 DA 神经元亚型分配。在目标 2 中，优化了 Wnt 方案后，我们接下来将使用靶向基因操作来获得两种特定 DA 神经元亚型的高度富集培养物，然后通过生理学和转录组学方法表征这些亚型。接下来，我们将从携带 DJ-1 突变的 iPS 细胞中生成两种 DA 神经元亚型，并检查对 SNc 和 VTA DA 神经元亚型的不同病理影响。在目标 3 中，我们将进一步表征体内两种 DA 神经元亚型的表型。我们将利用基因靶向小鼠来阐明两种小鼠 DA 神经元亚型的预测和完整转录组。该目标的信息将进一步凸显这些亚型之间的差异。此外，这些结果将反馈到目标 1 和 2，以进一步优化我们的 DA 神经元亚型推导协议。总之，利用两个实验室的综合专业知识和广泛的互动，我们提出了一个基于分子逻辑的连贯计划，从 iPS 细胞中衍生出不同的 DA 神经元亚型，并旨在改进 PD 建模。这些研究将为了解一系列 PD 突变对选择性脆弱性的影响开辟未来的可能性。