Analysis of human induced neuronal cells with and without psychosis high-risk mut

项目1将描述和定义与以下相关的高危突变的突触和细胞表型 精神分裂症(SCZ)(NRXN1外显子缺失，22q11.2缺失和16p11.2重复)，人类 诱导性神经(IN)细胞来源于iPS细胞。我们将确定最显著的表型变化 在细胞中突变(工程和自然发生)，以及电生理、基因组和形态计量学 最具成本效益的检测这些差异的方法。最有希望的模型和分析方法将是 选择用于未来的高通量筛选，基于稳健性(优先处理那些符合以下条件的模型和分析 揭示在不止一个突变中观察到的表型)和跨实验室的交叉验证， 小鼠体内、跨物种和跨来源的组织(细胞、原代培养的神经元和mPFC 脑片--项目2)。 该项目包括9个具体目标：(1)在细胞中产生每种高风险的工程形式 突变用于突变和非突变细胞的功能评价。(2)从iPS细胞中产生In细胞 携带高危突变的患者和对照。(3)确定这些神经元的突触表型(S) 使用电生理学和功能成像技术研究人类细胞内的突变。(4)识别小说 细胞内这些突变的形态、突触和细胞表型的形态计量学分析 高清图像。(5)识别与高危突变相关的基因调控网络。(6)测试 所有程序和结果的跨实验室重现性。(七)把功能、分子和 小鼠和人的形态数据，细胞中的突变和对照。在这最后一个目标中，它正在进行中 在整个研究过程中，考虑到人类细胞内AIMS 3-6以及项目2的所有结果 (小鼠模型)，将确定最健壮和可重现的病理生理模型； 将评估哪些突触和细胞表型在突变中重叠或不同；以及 将为选择一个或多个模型系统和分析提出建议，以用于未来的高 新疗法的通过量筛选。 这项工作将通过对感兴趣的三个突变的每一个进行分析，首先在细胞中研究 突变的工程化形式与来自同一控制线的非突变细胞进行比较，然后在 在SCZ患者和对照组中观察到的自然发生的突变。这项工作将开发出一种 或更多携带特定突变的细胞内突触和细胞功能障碍的病理生理模型， 用于未来对新疗法的高通量筛选。

Project 1 will characterize and define synaptic and cellular phenotypes of high-risk mutations associated with schizophrenia (SCZ) (NRXN1 exonic deletions, 22q11.2 deletions and 16p11.2 duplications), in human induced neuronal (iN) cells derived from iPS cells. We will identify the most robust phenotypic changes in mutated iN cells (engineered and naturally-occurring), and the electrophysiological, genomic and morphometric assays that detect those differences most cost-effectively. The most promising models and assays will be selected for future high-throughput screening, based on robustness (prioritizing those models and assays that reveal phenotypes that are observed in more than one mutation) and cross-validation across laboratories, within and across species, and across source tissues in mice (iN cells, primary cultured neurons and mPFC brain slices -- Project 2). The project includes 9 specific aims: (1) To generate iN cells with an engineered form of each high-risk mutation for functional evaluation of mutant and non-mutant cells. (2) To generate iN cells from iPS cells from patients carrying high-risk mutations and controls. (3) To characterize the synaptic phenotype(s) of these mutations in human iN cells using electrophysiology and functional imaging. (4) To identify novel morphological synaptic and cellular phenotypes of these mutations in iN cells using morphometric analysis of high-definition images. (5) To identify gene regulatory networks associated with high-risk mutations. (6) To test cross-lab reproducibility of all procedures and findings. (7) To integrate functional, molecular and morphological data from mouse and human, mutant and control iN cells. In this last Aim, which is ongoing throughout the study, taking into account all results from Aims 3-6 in human iN cells as well as from Project 2 (mouse models), the most robust and reproducible pathophysiological models will be identified; the extent to which synaptic and cellular phenotypes are overlapping across mutations vs. distinct will be evaluated; and recommendations will be made for the selection of one or more model systems and assays for future high- throughput screening of novel therapeutics. This work will proceed through the analysis of each of the three mutations of interest, studying iN cells first in the engineered form of the mutation compared with non-mutant cells from the same control line, and then in the naturally-occurring mutations observed in SCZ patients vs. control individuals. This work will develop one or more pathophysiological models of synaptic and cellular dysfunction in iN cells carrying specific mutations, for future use in high-throughput screening of novel therapeutics.