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

具有和不具有精神病高危突变的人类诱导神经元细胞分析

• 批准号: 8925150
• 负责人: Thomas C. Sudhof
• 金额: $ 99.49万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8925150
• 关键词: 16p11.2; 22q11.2; Accounting; Axon; Biological; Biological Assay; Biological Models; Blood Cells; Brain; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Chemicals; Chronic; Coculture Techniques; Computing Methodologies; Data; Disease; Electrophysiology (science); Engineering; Equilibrium; Evaluation; Event; Excitatory Synapse; Functional Imaging; Functional disorder; Future; Gene Expression; Genes; Genetic Engineering; Genomics; Gold; Human; Image; Individual; Industry; Inhibitory Synapse; Kinetics; Laboratories; Measurement; Measures; Methods; Microfluidics; Modeling; Molecular; Mus; Mutate; Mutation; Neurons; Nuclear; Patients; Pharmaceutical Preparations; Phenotype; Primary Cell Cultures; Printing; Probability; Procedures; Property; Protocols documentation; Psychotic Disorders; Recommendation; Regulator Genes; Reproducibility; Research Personnel; Schizophrenia; Slice; Source; Staining method; Stains; Synapses; Synaptic Transmission; Testing; Tissues; Transfection; Validation; Work; base; cellular engineering; cost; foot; high risk; high throughput screening; human subject; induced pluripotent stem cell; interest; mouse model; mutant; neurotransmitter release; novel; novel therapeutics; postsynaptic; receptor; research study; transcriptome sequencing

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.

项目1将表征和定义与以下疾病相关的高风险突变的突触和细胞表型： 人类精神分裂症（SCZ）（NRXN 1外显子缺失、22 q11.2缺失和16 p11.2重复） 诱导的神经元（iN）细胞来源于iPS细胞。我们将确定最强大的表型变化， 突变的iN细胞（工程化的和天然存在的），以及电生理学、基因组学和形态测量学 最具成本效益地检测这些差异的检测方法。最有前途的模型和分析将是 选择用于未来的高通量筛选，基于鲁棒性（优先考虑那些 揭示在多于一个突变中观察到的表型）和跨实验室的交叉验证， 在种属内和种属间以及小鼠的源组织间（iN细胞、原代培养的神经元和mPFC）， 脑切片-项目2）。 该项目包括9个具体目标：（1）用每种高风险的基因工程形式产生iN细胞， 突变，用于突变和非突变细胞的功能评价。(2)为了从iPS细胞生成iN细胞， 携带高风险突变的患者和对照组。(3)为了表征这些突触表型， 使用电生理学和功能成像在人类iN细胞中进行突变。(4)鉴定新 形态学突触和细胞表型的这些突变在iN细胞中使用的形态计量分析， 高清图像。(5)确定与高风险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。(7)为了整合功能性、分子性和 来自小鼠和人、突变体和对照iN细胞的形态学数据。在这最后一个目标中， 在整个研究中，考虑到人iN细胞中目标3-6以及项目2的所有结果 （小鼠模型），将确定最稳健和可重现的病理生理学模型; 将评估哪些突触和细胞表型在突变之间重叠而不是不同;以及 将提出建议，以选择一个或多个模型系统和测定，用于未来的高- 新疗法的通量筛选。 这项工作将通过分析三种感兴趣的突变中的每一种进行，首先研究iN细胞， 将突变的工程形式与来自相同对照系的非突变细胞进行比较， 在SCZ患者与对照个体中观察到的自然发生的突变。这项工作将开发一个 或携带特定突变的iN细胞中突触和细胞功能障碍的更多病理生理学模型， 用于将来高通量筛选新疗法。