Multimodal analysis of high-risk psychosis mutations in induced neuronal cells

描述（由申请人提供）：NCRCRG项目的目标是“创建多学科研究小组，与学术界和工业界合作，使用患者来源的重编程细胞来开发识别新靶点和开发新疗法的验证平台……以减轻精神疾病的负担。”在这里，我们建议使用来自诱导多能干细胞（iPS）的诱导神经元（iN）细胞来模拟由于三种已知会大大增加精神分裂症（SCZ）风险的工程或自然发生的突变导致的突触功能缺陷：NRXN1外显子缺失，22q11.2缺失和16p11.2重复。这项研究包括三个项目。Project 1将分析人类iN细胞的功能特征。对于每个突变，一个组件将研究具有工程突变的细胞与来自同一对照个体的未突变细胞，而另一个组件将研究来自5个具有感兴趣突变的SCZ患者与5个对照个体的iN细胞。项目2将分析小鼠模型中的相同突变，为人类发现提供跨物种验证，并尝试确定这些突变的突触表型在in细胞、培养的初级神经元和大脑切片（医学前额叶皮层）中是否相同。项目3将开发和优化本项目所需的干细胞方法（大规模实施iN细胞方案；针对大CNVs的新靶向突变策略；纯抑制性iN细胞的衍生；开发无小鼠iN细胞方案），也将需要基于本研究开发的病理生理模型开发未来的高通量筛选方法。这些实验将为重编程方法衍生的神经元的特征、每种突变产生的突触表型以及最终对包括SCZ在内的精神疾病风险的病理生理易感性提供新的见解。我们将确定这些突变是否产生不同的或至少部分重叠的突触表型。这两种观察结果都对未来的SCZ研究具有深远的意义。为了完成这项工作，我们汇集了来自斯坦福大学的优秀科学团队。Sudhof负责分子神经科学，Wernig负责干细胞生物学，Levinson负责精神分裂症遗传学)；罗格斯大学（罗格斯大学神经科学和干细胞生物学博士）；辛辛那提儿童医院医疗中心（Aronow博士，生物信息学和高含量成像）；礼来公司（Eli Lilly and Company）Isaac和Ursu负责电生理学，Merchant负责药物开发，Dage负责高含量成像和分析开发，Collier负责基因组学和系统生物学，Eastwood负责生物统计学)；Cellular Dynamics, Inc. （Swanson博士，代表干细胞生物学领域领先的生物技术公司）。这些项目代表了学术和工业机构的多学科努力，通过研究与SCZ相关的三种突变来深入了解精神疾病的病理生理学。

DESCRIPTION (provided by applicant): The goal of the NCRCRG program is "to create multidisciplinary research groups, in partnership with academia and industry, to use patient-derived reprogrammed cells to develop validated platforms for identifying novel targets and developing new therapeutics ... to reduce the burden of mental illness." Here we propose to use induced neuronal (iN) cells, derived from induced pluripotent stem (iPS) cells, to model defects in synaptic function due to three engineered or naturally-occurring mutations known to substantially increase the risk of schizophrenia (SCZ): NRXN1 exonic deletions, 22q11.2 deletions, and 16p11.2 duplications. The study includes three projects. Project 1 will analyze the functional characteristics of human iN cells. For each mutation, one component will study cells with engineered mutations vs. non-mutated cells from the same control individual, while a second component will study iN cells derived from 5 SCZ patients with the mutation of interest vs. 5 control individuals. Project 2 will analyze the same mutations in mouse models, providing both a cross-species validation of human findings, and an novel attempt to determine whether the synaptic phenotypes of these mutations are the same in iN cells, cultured primary neurons, and brain slices (medical pre-frontal cortex). Project 3 will develop and optimize stem cell methods that are required for this project (large-scale implementation of iN cell protocols; new targeted mutation strategies for large CNVs; derivation of pure inhibitory iN cells; development of a mouse-free iN cell protocol), and which will also be needed to develop future high-throughput screening assays based on the pathophysiological models developed in this study. These experiments will provide new insights into the characteristics of neurons derived by reprogramming method, into synaptic phenotypes produced by each of these mutations, and ultimately into the pathophysiology susceptibility to the risk of psychotic disorders including SCZ. We will determine whether these mutations produce distinct or at least partially overlapping synaptic phenotypes. Either observation has profound implications for future SCZ research. To accomplish this work, we have assembled an outstanding scientific team from Stanford University (Drs. Sudhof in molecular neuroscience, Wernig in stem cell biology and Levinson in genetics of schizophrenia); Rutgers University (Dr. Pang in neuroscience and stem cell biology); Cincinnati Children's Hospital Medical Center (Dr. Aronow in bioinformatics and high-content imaging); Eli Lilly and Company (Drs. Isaac and Ursu in electrophysiology, Merchant in drug development, Dage in high-content imaging and assay development, Collier in genomics and systems biology, and Eastwood in biostatistics); and Cellular Dynamics, Inc. (Dr. Swanson, representing a leading biotechnology company in stem cell biology). These projects represent a multidisciplinary effort of academic and industrial institutions to gain insight into te pathophysiology of psychotic disorders by studying three mutations that are associated with SCZ.