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

 描述（由申请人提供）：多巴胺（DA）缺乏，由DA神经元变性引起，是帕金森病（PD）破坏性运动症状的基础。DA神经元位于腹侧层的黑质pars腹侧（SNc），是特别脆弱的，相比那些在背侧层的SNc或腹侧被盖区（VTA）。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神经元是用于PD建模的重要新方法。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，这是建模选择性脆弱性的关键条件。这种差距的存在，因为中脑DA神经元的分子异质性还没有得到很好的理解。为了阐明DA神经元的异质性，我们最近使用了单细胞分子分析，结合解剖学共标记研究，并揭示了至少六种不同的DA神经元的存在。 小鼠模型中的DA神经元亚型。在这里，我们的目的是利用这些知识，以我。更好地理解体内DA神经元的多样性理解可能影响DA神经元亚型生成的机制iii.以合理的方式从人iPS细胞衍生和表征两种突出的DA神经元亚型，一种位于SNc中，一种位于VTA中，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信号如何影响DA神经元亚型分配。在目标2中，已经优化了Wnt方案，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细胞产生两种DA神经元亚型，并检查对SNc和VTA DA神经元亚型的不同病理学影响。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。我们将阐明的预测，和完整的转录组的两个小鼠DA神经元亚型，利用基因靶向小鼠。从这一目标的信息将进一步突出这些亚型之间的差异。此外，这些结果将反馈到目标1和2中，以进一步优化我们的DA神经元亚型推导方案。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有凝聚力的计划，从iPS细胞中衍生出不同的DA神经元亚型，旨在改善PD建模。这些研究将开启未来了解一系列PD突变对选择性脆弱性的影响的可能性。

 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.