Molecular and Cellular Mechanisms of Miller-Dieker Syndrome

描述（由申请人提供）： 大脑皮层的正常发育需要一系列复杂的细胞事件，包括特化、增殖、迁移和分化，以建立适当的结构和功能。破坏这些关键发育过程的突变会导致皮质畸形。尽管它们的流行和社会负担，我们对导致皮质畸形的突变如何破坏大脑发育的理解仍然有限。米勒-迪克尔综合征（MDS）是一种严重的发育障碍，其特征在于颅面畸形、脑体积减小（小头畸形）、几乎不存在皮质折叠（无脑畸形）和破坏性神经学后果，如智力迟钝和难治性癫痫。MDS是由人类条带17p13.3的大杂合缺失引起的，携带几十个基因，包括PAFAH 1B 1/LIS 1。PAFAH 1B 1中较小的缺失或突变是孤立性无脑畸形序列（ILS）的主要原因，其表现出不太严重的无脑畸形，没有其他异常。对Pafah 1b 1突变小鼠的分析揭示了神经元迁移的缺陷，这被认为是无脑畸形的主要细胞缺陷。然而，17p13.3基因座中的大多数其他基因在皮质发育或MDS发病机制中的作用尚未得到研究。此外，由于缺乏重现MDS的完整遗传缺陷或临床表现的小鼠模型，尚不清楚神经干细胞的诱导、增殖或分化是否也被破坏，如MDS患者中小头畸形表型所预期的那样。此外，最近的研究表明，人类和小鼠的大脑皮层发育存在重大差异，小鼠的大脑天生是无脑的。这些限制使得有必要使用人脑组织和患者来源的细胞来研究人类进化的复杂过程以及它们如何在皮质畸形中被破坏。为了实现这一目标，我从MDS和ILS患者中产生了诱导多能干细胞（iPSC）。本项目的目的是利用体外人类干细胞模型和体外人类发育中的皮质组织研究MDS的细胞和分子机制。在研究期的初始阶段（K99），将通过比较患者iPSC体外皮质发育期间的MDS和ILS表型来鉴定LIS 1依赖性和独立性细胞缺陷。此外，将进行细胞类型特异性基因表达分析，以鉴定除PAFAH 1B 1外可能影响皮质发育和MDS进展的基因。随后的研究（K99+ R 00）将集中于在iPSC和人体组织中使用细胞生物学和遗传学方法相结合的新候选物的系统功能验证。本文提出的实验将建立和表征ILS和MDS的新型干细胞模型，定义这些疾病的细胞基础，并阐明MDS中缺失的基因如何影响脑发育。这项工作将提高我们对人类皮质发育和MDS发病机制的基本理解，并可能导致对主要发育障碍类别（包括小头畸形和无脑畸形）的新疗法的确定。

DESCRIPTION (provided by applicant): Normal development of the cerebral cortex requires a complex series of cellular events, including specification, proliferation, migration and differentiation, to establish the proper structure and function. Mutations that disrupt these key developmental processes give rise to cortical malformations. Despite their prevalence and societal burden, our understanding of how mutations that cause cortical malformations disrupt brain development is still limited. Miller Dieker Syndrome (MDS) is a severe developmental disorder, characterized by craniofacial dysmorphisms, reduced brain size (microcephaly), nearly absent cortical folding (lissencephaly) and devastating neurological consequences such as mental retardation and intractable epilepsy. MDS is caused by large heterozygous deletions of human band 17p13.3, harboring several dozen genes, including PAFAH1B1/LIS1. Smaller deletions or mutations in PAFAH1B1 are the major cause of Isolated Lissencephaly Sequence (ILS), which exhibits less severe lissencephaly and no additional abnormalities. Analyses of Pafah1b1 mutant mice revealed defects in neuronal migration, which is considered to be the main cellular deficiency in lissencephaly. However, the roles of most of the other genes in 17p13.3 locus in cortical development or MDS pathogenesis have not been examined. In addition, due to a lack of mouse models that recapitulate the complete genetic defects or clinical manifestations of MDS, it is unknown whether induction, proliferation or differentiation of neural stem cells is also disrupted, as might be expected for the microcephaly phenotype in MDS patients. Moreover, recent work has shown critical differences between cortical development in humans and mice, whose brains are naturally lissencephalic. These limitations necessitate the use of human brain tissue and patient-derived cells to study the complex processes that have evolved in human and how they are disrupted in cortical malformations. Towards that aim, I generated induced pluripotent stem cells (iPSCs) from MDS and ILS patients. The goal of this project is to investigate the cellular and molecular mechanisms of MDS using human stem cell models in vitro and human developing cortical tissues ex vivo. During the initial phase of the research period (K99), LIS1-dependent and independent cellular deficiencies will be identified by comparing MDS and ILS phenotypes during in vitro cortical development from patient iPSCs. In addition, cell type-specific gene expression analysis will be done to identify genes besides PAFAH1B1 that are likely to impact cortical development and MDS progression. Subsequent studies (K99+R00) will focus on systematic functional validation of novel candidates using a combination of cell biological and genetic approaches in iPSCs and human tissues. The experiments proposed here will establish and characterize novel stem cell models of ILS and MDS, define the cellular basis for these disorders and elucidate how genes deleted in MDS affect brain development. This work will improve our fundamental understanding of human cortical development and MDS pathogenesis and may lead to the identification of new therapies for major classes of developmental disorders, including microcephaly and lissencephaly.