Modeling schizophrenia with patient-specific mutations in GRIN2A and SP4

利用 GRIN2A 和 SP4 患者特异性突变模拟精神分裂症

• 批准号: 10741466
• 负责人: BRADY J MAHER
• 金额: $ 49.61万
• 依托单位: LIEBER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741466
• 关键词: Acute; Anesthetics; Autoimmune encephalitis; Behavior; Behavioral; CRISPR/Cas technology; Cell Line; Cell model; Cells; Cellular biology; Central Nervous System; Complement; Complex; DNA Sequence; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Dose; Electrophysiology (science); Elements; Etiology; Family; Functional disorder; Future; Genes; Genetic; Genetic Risk; Genetic Transcription; Genome; Glutamate Receptor; Goals; Heterozygote; Hippocampus; Human; Image; Impaired cognition; Individual; Investigation; Link; Long-Term Potentiation; Mental disorders; Modeling; Molecular; Molecular Analysis; Mus; Mutation; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Nature; Neurites; Neurons; Pathway interactions; Patients; Pattern; Penetrance; Phenotype; Play; Prevalence; Process; Protein Truncation; Psychiatry; Psychoses; Public Health; Quality Control; Resources; Risk; Role; SP1 gene; Schizophrenia; Symptoms; Technology; Transcription Initiation Site; United States; Validation; Variant; Western Blotting; antagonist; confocal imaging; differentiation protocol; directed differentiation; exome; exome sequencing; experimental study; gene function; genome wide association study; genome-wide analysis; genomic locus; homologous recombination; immunoreactivity; improved; in vitro Model; induced pluripotent stem cell; insight; knock-down; loss of function mutation; multi-electrode arrays; mutant; mutation correction; nerve stem cell; neuron development; novel; novel therapeutics; off-target mutation; polygenic risk score; public health relevance; rare variant; receptor expression; receptor function; risk variant; schizophrenia risk; stem cells; therapeutic target; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY: Schizophrenia (SCZ) is a common and debilitating mental illness characterized by positive symptoms, negative symptoms, and impaired cognition, with a lifetime prevalence approaching 1% in the United States. Unfortunately, progress in understanding the etiology and pathophysiology is hindered by the lack of appropriate models that adequately capture both the complex and heterogeneous nature of the genetic risk and the diversity of the phenotypic manifestations. The advent of induced pluripotent stem cell (hiPSCs) technology provides an important new platform to study the cellular and circuit behavior of human cells that contain an individual’s full complement of primary sequence risk alleles. Thus, the door is now opened to developing improved in vitro models that can enhance our understanding of etiology and pathophysiology in an integrated context, relating patient genomes to their cellular phenotypes. Here, we propose to generate novel hIPSC lines harboring patient-specific mutations in two highly penetrant SCZ risk genes, GRIN2A and SP4. GRIN2A encodes a subunit of the N-methyl-D-aspartate receptor (NMDAR) which are a subclass of ionotropic glutamate receptors that play a pivotal role in the development, plasticity, and pathophysiology in the central nervous system. NMDAR hypofunction is a leading causal hypothesis for SCZ because subanesthetic doses of the NMDAR antagonists produces acute psychosis in healthy subjects. In addition, autoimmune encephalitis due auto-immunoreactivity to NMDARs leads to severe psychosis. SP4 belongs to the SP1 family of transcription factors that recognizes DNA sequences termed GC-boxes that are often found upstream of transcription start sites (TSS). SP4 has been shown to functionally regulate the expression of NMDARs through direct interactions with GC-boxes upstream of the TSS of GRIN1, GRIN2A, and GRIN2B. In addition, Sp4 hypomorphic mice have reduced hippocampal long-term potentiation, reduced NMDAR expression, and behavioral abnormalities related to SCZ. Importantly, both GRIN2A and SP4 are high priority SCZ risk genes, being associated with SCZ through genome-wide association studies and rare variant analysis. We propose to use CRIPSR editing to generate patient-specific mutations in GRIN2A and SP4 with consideration of the genetic background of the hIPSC lines. Both genes will be individually edited in the same six control hiPSCs lines with elevated SCZ polygenic risk scores. We will perform quality control experiments to confirm mutations are correct and no off-target mutations are present. Validation of functional mutations will be confirmed with qPCR, Western blot and electrophysiology. Once validated, we will quantify the effect of these mutations on spontaneous network activity using multi-electrode arrays and their effect on the transcriptome with RNA sequencing. The overall goal of this proposal is to develop novel cellular models of SCZ that can inform us about pathophysiology and help identify convergent molecular mechanism in SCZ with the goal of identifying therapeutic targets for this debilitating disorder.

项目摘要：精神分裂症(SCZ)是一种常见的、使人衰弱的精神疾病，其特征是 阳性症状、阴性症状和认知障碍，终生患病率接近1% 美国。不幸的是，在理解病因和病理生理学方面的进展受到以下因素的阻碍 缺乏适当的模型来充分反映 遗传风险和表型表现的多样性。诱导多能干细胞的出现 (HiPSCs)技术为研究人类细胞和电路行为提供了重要的新平台 包含个体全部主要序列风险等位基因的细胞。因此，门现在是 对开发改进的体外模型持开放态度，可以增强我们对病因学和 综合背景下的病理生理学，将患者基因组与其细胞表型联系起来。在这里，我们 建议在两个高渗透性SCZ风险中产生含有患者特异性突变的新的HiPSC系 基因，GRIN2A和SP4。GRIN2A编码N-甲基-D-天冬氨酸受体(NMDAR)的一个亚单位，该亚单位 是离子型谷氨酸受体的一个亚类，在发育、可塑性和 中枢神经系统的病理生理学。NMDAR功能低下是SCZ的主要因果假说 因为亚麻醉剂量的NMDAR拮抗剂会在健康受试者中产生急性精神病。在……里面 此外，由于对NMDARs的自身免疫反应而导致的自身免疫性脑炎会导致严重的精神病。SP4 属于转录因子SP1家族，可识别称为GC-box的DNA序列 通常发现转录起始点(TS)的上游。SP4已被证明在功能上调节 通过与GRIN1、GRIN2A、GRIN2、GRIN2、GRIN1、GRIN1、GRIN2、GRIN1、GRIN1、GRIN2、GRIN1、GRIN 和GRIN2B。此外，SP4缺陷症小鼠海马区长时程增强降低， NMDAR的表达，以及与SCZ相关的行为异常。重要的是，GRIN2A和SP4都很高 优先的SCZ风险基因，通过全基因组关联研究和罕见的变异与SCZ相关 分析。我们建议使用CRIPSR编辑来产生患者特有的GRIN2A和SP4突变 考虑HiPSC系的遗传背景。这两个基因将在同一个 6个SCZ多基因风险评分升高的对照HiPSCs系。我们将进行质量控制实验 以确认突变是正确的，并且不存在非靶标突变。功能突变的验证将 经定量聚合酶链式反应、蛋白质印迹和电生理证实。一旦得到验证，我们将量化 利用多电极阵列对自发网络活动的这些突变及其对 转录组和RNA测序。这项提案的总体目标是开发新的细胞模型 SCZ可以为我们提供有关SCZ的病理生理信息，并帮助确定SCZ的收敛分子机制 确定这种衰弱障碍的治疗靶点的目标。