Pathological Mechanisms of Human Cerebellar Malformations

资助父母基金摘要：在人类中已经描述了大量的小脑畸形。除运动和感觉整合缺陷外，大多数还会导致认知障碍。令人惊讶的是，人们对这些畸形的发育基础知之甚少，特别是因为几乎没有关于正常或异常胎儿小脑发育的人类特定数据。这项建议寻求利用人类胎儿样本和小鼠模型来促进对人类小脑发育和畸形的了解。人类特有的数据将直接测试我们工作中的老鼠衍生的关于这些疾病原因的假说的有效性，并加强正常发育数据的基础，这些数据将为我们正在进行的人类小脑畸形的遗传分析提供信息。我们将首次对妊娠4-23周的正常人类胎儿小脑发育进行深入分析，涵盖主要发育事件。然后，我们将研究人类胎儿Dandy-Walker畸形的病理。Dandy-Walker畸形是最常见的人类小脑畸形，影响到约1/3000的活产。将结合这些实验产生小鼠模型，以评估发育病理学的机制。最后，我们将为正常的人胎小脑神经元生成第一个转录组数据。这些细胞类型的特定数据在目前公开可用的大脑资源中严重缺失。我们的人胎小脑神经元数据将与现有的内源性小鼠发育小脑神经元以及MES和hPSC衍生的小脑神经元到发育的转录组数据进行比较，以评估它们作为模型系统的有效性。此外，这些数据还将与来自人类小脑畸形患者的外显子组数据整合，以促进这些重要的和未被研究的出生缺陷的基因发现。请求补充材料摘要：本申请是根据NOT-OD-19-071向PA-19-056提交的。本研究补充资料的目的是确定人类唐氏综合症样本的细胞、分子和形态的小脑发育轨迹。发育概况将通过结合单细胞测序、组织学和免疫组织化学分析来生成，并辅之以细胞培养分析，以确定小脑颗粒前体细胞的有丝分裂特性。然后，来自唐氏综合症样本的数据将直接与实验室提供的正常和Dandy-Walker畸形发育小脑样本的轮廓进行比较，并在父母R01下生成。小脑发育不良是唐氏综合症患者最一致的表型之一，是这些患者神经表型的重要组成部分。然而，对于导致先天性发育不全的小脑发育障碍，人们知之甚少。我们将对唐氏综合症患者的人类小脑发育进行多模式描述，与我们已经产生的定义正常小脑发育的数据进行比较。了解唐氏综合症小脑发育轨迹如何以及何时与正常和其他小脑畸形不同，将阐明儿童和成人唐氏综合症神经学表型的细胞和回路基础。这些研究具有很高的影响力，具有相当大的翻译潜力，可以确定治疗唐氏综合症神经缺陷的新方法。他们还将生成模型系统(HiPSCs、有机物、动物模型)的发育阶段、细胞类型和分子特异性的基线数据。这些实验具体涉及包含项目研究目标的组成部分1和组成部分2。

Abstract of the funded parent grant: Numerous cerebellar malformations have been described in humans. Most cause cognitive, in addition to motor and sensory integration deficits. Surprisingly little is understood regarding the developmental basis of these malformations, particularly since little human specific data is available for normal or abnormal fetal cerebellar development. This proposal seeks to advance knowledge of human cerebellar development and malformations using human fetal samples and mouse models. The human-specific data will directly test the validity of our working mouse-derived hypotheses regarding the causes these disorders and strengthen the foundation of normal developmental data which will inform our ongoing genetic analyses of human cerebellar malformations. We will conduct the first in-depth analysis of normal human fetal cerebellar development from 4-23 Gestational Weeks, covering major developmental events. We will then examine the pathology of human fetal Dandy-Walker malformation the most common human cerebellar malformation, affecting ~1/3000 live births. Mouse models will be generated in conjunction with these experiments to assess the mechanisms of the developmental pathology. Finally, we will generate the first transcriptome data for normal human fetal cerebellar neurons. These cell-type specific data are critically missing from current publicly available brain resources. Our human fetal cerebellar neuron data will be compared to transcriptome data from existing datasets of endogenous mouse developing cerebellar neurons as well as mES and hPSC-derived cerebellar neurons to development to assess their validity as model systems. Further, the data will also be integrated with exome data from human cerebellar malformation patients to facilitate gene discovery for these important and understudied birth defects. Abstract of Requested Supplement: This application is being submitted for PA-19-056 in accordance with NOT-OD-19-071. The purpose of this research supplement is to define the cellular, molecular and morphological cerebellar developmental trajectories in human Down Syndrome samples. Developmental profiles will be generated through a combination of single cell sequencing, histological and immunohistochemical analyses and complemented with cell culture assays defining the mitogenic properties of cerebellar granule progenitors. Data from Down Syndrome samples will then be directly compared to profiles from normal and Dandy-Walker malformation developmental cerebellar samples available in the lab and generated under the parent R01. Cerebellar hypoplasia is one of the most consistent phenotypes in Down Syndrome patients that is a significant contributor to neurological phenotypes in these patients. Yet, very little is understood about the developmental disruption of cerebellar development that underlies the congenital hypoplasia. We will produce a multi-modal description of human cerebellar development in Down Syndrome, comparable to data we are already generating to define normal cerebellar development. An understanding how and when Down Syndrome cerebellar developmental trajectories differ from normal and other cerebellar malformations will elucidate the cellular and circuit underpinnings of pediatric and adult Down Syndrome neurological phenotypes. The studies are of high impact with considerable translational potential to identify new therapeutic approaches for neurological deficits in Down Syndrome. They will also generate baseline data human data to the developmental stage-, cell type-, and molecular-specificity of model systems (hiPSCs, organoids, animal models). These experiments specifically address Component 1 and Component 2 of the INCLUDE Project research objectives.