Pathological Mechanisms of Human Cerebellar Malformations

资助的父母资助摘要：许多小脑畸形已经在人类中被描述。除了运动和感觉整合缺陷外，大多数还会导致认知缺陷。令人惊讶的是，人们对这些畸形的发育基础知之甚少，特别是因为很少有关于正常或异常胎儿小脑发育的人类具体数据。本提案旨在利用人类胎儿样本和小鼠模型来推进人类小脑发育和畸形的知识。人类特异性数据将直接测试我们关于这些疾病原因的工作小鼠衍生假设的有效性，并加强正常发育数据的基础，这将为我们正在进行的人类小脑畸形遗传分析提供信息。我们将首次深入分析4-23孕周正常人类胎儿小脑发育，涵盖主要发育事件。然后，我们将检查人类胎儿Dandy-Walker畸形的病理，这是最常见的人类小脑畸形，影响约1/3000的活产婴儿。小鼠模型将与这些实验相结合，以评估发育病理学的机制。最后，我们将生成正常人类胎儿小脑神经元的第一个转录组数据。这些特定细胞类型的数据在目前可公开获得的大脑资源中严重缺失。我们的人类胎儿小脑神经元数据将与现有内源性小鼠小脑神经元发育数据集的转录组数据以及mES和hpsc衍生的小脑神经元发育数据集进行比较，以评估其作为模型系统的有效性。此外，这些数据还将与人类小脑畸形患者的外显子组数据相结合，以促进这些重要的和未充分研究的出生缺陷的基因发现。请求补充摘要：根据NOT-OD-19-071，本申请正在提交PA-19-056。本研究补充的目的是确定人类唐氏综合征样本的细胞，分子和形态小脑发育轨迹。发育概况将通过单细胞测序、组织学和免疫组织化学分析相结合，并辅以细胞培养分析来确定小脑颗粒祖细胞的有丝分裂特性。然后，唐氏综合症样本的数据将直接与实验室中正常和Dandy-Walker畸形发育性小脑样本的数据进行比较，并在亲本R01下生成。小脑发育不全是唐氏综合征患者最一致的表型之一，是这些患者神经系统表型的重要贡献者。然而，对先天性发育不全的小脑发育中断的了解甚少。我们将对唐氏综合症患者的小脑发育进行多模态描述，与我们已经生成的定义正常小脑发育的数据相媲美。了解唐氏综合征小脑发育轨迹如何以及何时与正常小脑和其他小脑畸形不同，将阐明儿童和成人唐氏综合征神经表型的细胞和电路基础。这些研究具有很高的影响力，具有相当大的转化潜力，可以确定唐氏综合征神经功能障碍的新治疗方法。他们还将生成关于模型系统（hipsc、类器官、动物模型）的发育阶段、细胞类型和分子特异性的基线数据。这些实验具体解决了包括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.