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

NCRCRG计划的目标是“与学术界和工业界合作，创建多学科研究小组，使用患者来源的重编程细胞开发经验证的平台，用于识别新靶点和开发新疗法。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）细胞来模拟突触功能的缺陷，这是由于三种工程或自然发生的突变，已知会大大增加精神分裂症（SCZ）的风险：NRXN 1外显子缺失，22q11.2缺失和16p11.2重复。该研究包括三个项目。项目1将分析人类iN细胞的功能特征。对于每种突变，一个组件将研究具有工程化突变的细胞与来自相同对照个体的非突变细胞，而第二个组件将研究来自5名具有目标突变的SCZ患者与5名对照个体的iN细胞。项目2将在小鼠模型中分析相同的突变，提供人类发现的跨物种验证，以及确定这些突变的突触表型在iN细胞，培养的原代神经元和脑切片（医学前额叶皮层）中是否相同的新尝试。项目3将开发和优化本项目所需的干细胞方法（iN细胞方案的大规模实施;大CNV的新靶向突变策略;纯抑制性iN细胞的衍生;无小鼠iN细胞方案的开发），并且还需要开发基于本研究中开发的病理生理学模型的未来高通量筛选测定。这些实验将提供新的见解的特点，神经元衍生的重编程方法，到突触表型所产生的每一个这些突变，并最终到病理生理易感性的风险，包括SCZ的精神疾病。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。这两种观察结果对未来SCZ的研究都有深远的影响。为了完成这项工作，我们召集了一支来自斯坦福大学的优秀科学团队（分子神经科学领域的Sudhof博士，干细胞生物学领域的Wernig博士和精神分裂症遗传学领域的Levinson博士）;罗格斯大学（神经科学和干细胞生物学的庞博士）;辛辛那提儿童医院医疗中心（生物信息学和高内涵成像领域的Aronow博士）; Eli Lilly and Company（Isaac和Ursu博士从事电生理学，Merchant博士从事药物开发，Dage博士从事高含量成像和分析开发，科利尔在基因组学和系统生物学，和伊斯特伍德在生物统计学）;和细胞动力学公司。(Dr. Swanson，代表一家领先的干细胞生物技术公司）。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障碍的病理生理学。

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.