Investigating Trisomy 21 Impact on Human Neural Cell Development and Function Using "Trisomy Silencing" in vitro

项目摘要 唐氏综合征（DS），21三体，是最常见的智力障碍，影响数百万人，也是一种形式 早发性阿尔茨海默病（AD）。对DS病理学的理解一直受到复杂性的阻碍， 由21号染色体（chr 21）上约250个基因的过表达引起。许多报告描述了各种各样的 发育和功能性病变的证据然而，有相互矛盾的证据，即使在 哪些大脑区域或细胞类型受到影响。此外，很少有研究涉及病理学何时出现， 当它是可逆的。因此，更好地了解DS对大脑发育的确切影响， 当所述影响发生时，识别特定脑细胞类型中的机械原因是至关重要的。 来自小鼠模型、人诱导多能干细胞（iPSC）和死后DS脑样本的报告 关于DS对脑发育和功能的影响存在争议。此外，当在 在发育过程中，DS的影响发生，并且chr 21基因负责是未知的。初步结果 表明同基因二体和三体细胞系之间的克隆变异性太大，无法建立任何 即使在类器官变异性得到很好控制的情况下，三体性也会对神经发育产生影响。令人惊讶的是，AD- 仍观察到相关Aβ病理学。为了避免细胞系之间的变异性，我们的策略将是 在几种三体细胞系中操纵chr 21的过表达。通过将诱导型XIST转基因插入到 我们的实验室已经证明了额外的chr 21使用内源性机制进行全面的染色体沉默 用于剂量补偿。我们的XIST诱导的“三体沉默”系统通过允许染色体的突变来减少变异性。 研究几乎相同的细胞，有或没有额外的chr 21表达。利用这个系统，劳伦斯实验室 表明DS可能通过延长神经祖细胞（NPC）的命运来延迟神经发生， 神经元单层分化延长NPC周期已知会改变细胞命运，因此我们将 检查可能改变脑细胞类型组成的神经发育的后期阶段。与此同时，我们将 检查三体沉默是否可以纠正DS类器官中已建立的AD相关细胞病理学。 我们还将使用这些方法来研究21三体对特定细胞中整体转录的影响。 与细胞病理学有关的类型。DS研究的当前主题是21三体引起非常广泛的变化 非Chr 21基因的表达。通过转录组学，许多研究报告， 影响可能会改变特定的基因/途径，影响不同神经细胞的发育和活动。 类型然而，我们实验室的工作表明，样本之间的其他差异，而不是由于三体，可能解释 这种“全球扰动”的大部分。这个建议的假设是21三体改变了相对的 在发育过程中大脑中神经细胞类型的比例和/或破坏特定神经细胞中的通路 类型使用XIST系统，我将更好地确定21三体如何以及何时改变发育， 在DS中的特定神经细胞类型的转录组，同时提供有关基因的机制见解。

PROJECT SUMMARY Down syndrome (DS), trisomy 21, is the most common intellectual disorder affecting millions and also is a form of early-onset Alzheimer Disease (AD). Understanding of DS pathology has been hindered by the complexity caused by overexpression of the ~250 genes on chromosome 21 (chr21). Many reports describe a diverse range of developmental and functional pathologies in the DS brain. However, there is conflicting evidence even as to which brain regions or cell types are impacted. Furthermore, few studies address when pathology arises and when it remains reversible. Therefore, better understanding of the exact impact of DS on brain development and function, when said effects occur, and identification of mechanistic causes in specific brain cell types is critical. Reports from mouse models, human induced pluripotent stem cells (iPSCs), and post-mortem DS brain samples regarding the impact of DS on brain development and function are conflicted. Furthermore, when during development the impacts of DS occur and which chr21 genes are responsible are unknown. Preliminary results show that the clonal variability between isogenic disomic and trisomic cell lines was too great to establish any neurodevelopmental effect of trisomy even when organoids variability was well controlled. Surprisingly, AD- related Aβ pathology was still observed. To circumvent variability between cell lines our strategy will be to manipulate over-expression of chr21 in several trisomic cell lines. By inserting an inducible XIST transgene into the extra chr21, our lab has demonstrated comprehensive chromosomal silencing using endogenous machinery for dosage compensation. Our XIST-inducible system for “trisomy silencing” reduces variability by allowing the study of nearly identical cells, with or without extra chr21 expression. Using this system, the Lawrence lab has demonstrated that DS potentially delays neurogenesis by prolonging neural progenitor cell (NPC) fate during neuronal monolayer differentiation. Prolonged NPC cycling is known to alter cell fate and therefore we will examine later stages of neurodevelopment that may alter brain cell-type composition. At the same time, we will examine if trisomy silencing can correct an established AD-related cell pathology in DS organoids. We will also use these approaches to examine the impact of trisomy 21 on global transcription in specific cell types relating to cell pathologies. A current theme in DS research is that trisomy 21 causes very broad changes in expression of non-chr21 genes. Through transcriptomics many studies have reported that genome-wide impacts may alter specific genes/pathways, impacting both the development and activity of different neural cell types. However, work in our lab suggests other differences between samples, not due to trisomy, may account for much of this “global perturbation”. The hypothesis for this proposal is that trisomy 21 alters the relative proportions of neural cell types in the brain during development and/or disrupts pathways in specific neural cell types. Using the XIST-system I will better determine how and when trisomy 21 alters the development and transcriptome of specific neural cell types in DS, while providing mechanistic insights into genes involved.