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

基本信息

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
项目类别：
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

项目摘要

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) 是一种常见且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% 不幸的是，对病因学和病理生理学的理解进展受到阻碍。缺乏适当的模型来充分捕捉事物的复杂性和异构性遗传风险和表型表现的多样性。诱导多能干细胞的出现。（hiPSC）技术为研究人类细胞和电路行为提供了一个重要的新平台含有个体完整的一级序列风险等位基因的细胞因此，现在大门就在眼前。愿意开发改进的体外模型，以增强我们对病因学的理解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。提议在两种高渗透性 SCZ 风险中生成携带患者特异性突变的新型 hIPSC 系 GRIN2A 和 SP4 基因编码 N-甲基-D-天冬氨酸受体 (NMDAR) 的亚基。是离子型谷氨酸受体的一个亚类，在发育、可塑性和 NMDAR 功能减退是 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。此外，由于对 NMDAR 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。属于 SP1 转录因子家族，可识别称为 GC 盒的 DNA 序列，经常在转录起始位点 (TSS) 的上游发现，已被证明具有功能性调节作用。 NMDAR 通过与 GRIN1、GRIN2A、TSS 上游的 GC 盒直接相互作用来表达此外，Sp4 低等态小鼠的海马长时程增强能力降低。 NMDAR 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 SCZ 风险基因，通过全基因组关联研究和罕见变异与 SCZ 相关我们建议使用 CRIPSR 编辑来生成 GRIN2A 和 SP4 中的患者特异性突变。考虑 hIPSC 系的遗传背景，这两个基因将在同一细胞中进行单独编辑。六个 SCZ 多基因风险评分升高的对照 hiPSC 系我们将进行质量控制实验。为了确认突变是正确的并且不存在脱靶突变，将验证功能突变。通过 qPCR、蛋白质印迹和电生理学进行确认。一旦验证，我们将量化其效果。使用多电极阵列对自发网络活动的这些突变及其对该提案的总体目标是开发新的细胞模型。 SCZ 可以让我们了解病理生理学并帮助识别 SCZ 中的趋同分子机制确定这种使人衰弱的疾病的治疗靶点的目标。