Molecular Mechanisms of Defective Oligodendrocyte Differentiation in Down Syndrome

摘要 唐氏综合征(DS)是由21号染色体三倍体(HSA21)引起的，是最常见的遗传病 智力残疾的原因，发病率为每750名活产儿中就有一名。然而，这一增长究竟是如何 遗传物质导致智力残疾的原因尚不清楚。21三体已被证明可以改变基因 DS患者整个转录组的表达模式。其中一个变化是下调了对 调控少突胶质细胞(OL)谱系的基因网络。这种失调的基因表达识别出 受扰的OL产生作为观察到的白质缺陷的潜在潜在细胞机制 DS.少突胶质前体细胞(OPC)的产生依赖于Odon2的启动。 形态基因Sonic Hedgehog(SHH)。然而，在DS中，OPC起源的这两个关键组件都是 受伤了。三体细胞对SHH的促有丝分裂反应减弱。这降低了对SHH的反应性 可能会改变其下游基因靶标之一转录因子寡核苷酸的表达，该转录因子的表达 对于OPC规范来说是至关重要的。一旦被激活，寡核苷酸就会诱导一种复杂的转录产物的表达 控制OPC分化和最终成熟为OL的网络。除了最初的更改之外 Shh介导的激活、三体细胞中寡核苷酸的三倍体和错误表达可能合成了最初的 转录改变并进一步失调下游基因的表达。这个项目的目的是研究 这些已知干扰在三体细胞中的分子后果及其如何导致OPC的变化 分化整倍体和三体诱导多能干细胞(IPSCs)同基因对的研究进展 从DS患者体内分化为神经前体细胞(NPC)和OPC。在目标1中，我们将测试效果 SHH信号异常通过增加SHH信号的激活而影响DS中OPC的产生 通过应用平滑激动剂(SAG)和shRNA敲除初始SHH的途径 受体PTCH在DS中高表达。在目标2中，我们将重点研究寡核苷酸2的三倍体如何影响其 作为关键的转录因子，通过识别差异结合来调节转录网络 三体和整倍体细胞之间的转录因子，在OL发育过程中。作为对 转录因子三次复制对OPC相关转录网络的影响，我们将在 并利用加权基因共表达分析(WGCNA)鉴定OPC 包含通过芯片序列差异结合识别的基因的模块，并检查这些网络如何作为 整倍体细胞和三体细胞有很大的不同。这些目标加在一起将首次阐明 少突胶质细胞发育改变的分子基础可能导致 观察DS大鼠脑白质缺失情况。

ABSTRACT Down syndrome (DS) is caused by triplication of chromosome 21 (HSA21) and is the most common genetic cause of intellectual disability with a prevalence of 1 in 750 live births. However exactly how the increase in genetic material leads to the intellectual disability is unknown. Trisomy 21 has been shown to alter gene expression patterns across the entire transcriptome in DS. One of these alterations is the downregulation of network of genes regulating the oligodendrocyte (OL) lineage. This dysregulated gene expression identifies perturbed OL production as the potential underlying cellular mechanism of the observed white matter deficit in DS. Generation of oligodendrocyte precursor cells (OPCs) is dependent on the initiation of Olig2 by the morphogen Sonic hedgehog (SHH). However, in DS both of these crucial components of OPC genesis are impaired. Trisomic cells have a decreased mitogenic response to SHH. This decreased responsiveness to SHH may alter the expression of one of its downstream gene targets, the transcription factor Olig2, whose expression is crucial for OPC specification. Once activated, Olig2 induces the expression of a complex transcriptional network that controls the differentiation and eventual maturation of OPCs into OL. In addition to the initial altered SHH-mediated activation, the triplication and mis-expression of Olig2 in trisomic cells may compound the initial transcriptional changes and further dysregulate downstream gene expression. This project aims to examine the molecular consequences of these known perturbations in trisomic cells and how they lead to changes in OPC development by differentiating isogenic pairs of euploid and trisomic induced pluripotent stem cell (iPSCs) lines derived from people with DS into neural progenitor cells (NPCs) and OPCs. In Aim 1 we will test the effect dysfunctional SHH signaling has on OPC generation in DS by increasing the activation of the SHH signaling pathway both through application of a Smoothened agonist (SAG) and shRNA knockdown of the initial SHH receptor PTCH which is overexpressed in DS. In Aim 2 we will focus on how triplication of Olig2 affects its function as a key transcription factor regulating the transcriptional network by identifying differential binding of the transcription factor between trisomic and euploid cells throughout OL development. As a direct test of the effect transcription factor triplication has on OPC related transcriptional networks, we will perform RNA-seq at different OPC developmental stages and use weighted gene co-expression analysis (WGCNA) to identify the modules that contain the genes identified via ChIP-seq differential binding and examine how these networks as a whole differ between euploid and trisomic cells. Together these aims will elucidate, for the first time, the molecular underpinnings of alterations in oligodendrocyte development that may be leading to the observed white matter deficit in DS.