Modeling schizophrenia with patient-specific mutations in GRIN2A and SP4

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、阴性症状和认知受损，在10个国家中， 美国的不幸的是，了解病因学和病理生理学的进展受到以下因素的阻碍： 缺乏适当的模式，充分捕捉复杂和多样的性质， 遗传风险和表型表现的多样性。诱导多能干细胞的出现 hiPSCs技术为研究人体细胞和回路行为提供了一个重要的新平台 含有个体的全部一级序列风险等位基因的细胞。因此，门现在 开放开发改进的体外模型，可以提高我们对病因学的理解， 综合背景下的病理生理学，将患者基因组与其细胞表型相关联。这里我们 提出在两个高度渗透的SCZ风险中产生携带患者特异性突变的新型hIPSC系， GRIN 2A和SP 4基因。GRIN 2A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 中枢神经系统的病理生理学NMDAR功能减退是SCZ的主要因果假设 因为亚麻醉剂量的NMDAR拮抗剂在健康受试者中产生急性精神病。在 此外，由于对NMDAR的自身免疫反应性导致的自身免疫性脑炎导致严重的精神病。SP4 属于转录因子的SP1家族，其识别称为GC盒的DNA序列， 通常发现于转录起始位点（TSS）的上游。SP 4已被证明在功能上调节 通过与GRIN 1，GRIN 2A， GRIN2B。此外，Sp 4亚纯型小鼠海马长时程增强减少， NMDAR表达和SCZ相关行为异常。重要的是，GRIN 2A和SP 4都很高， 优先SCZ风险基因，通过全基因组关联研究与SCZ相关， 分析.我们建议使用CRIPSR编辑在GRIN 2A和SP 4中产生患者特异性突变， 考虑hIPSC系的遗传背景。这两个基因将在同一个基因中单独编辑。 六个对照hiPSC系具有升高的SCZ多基因风险评分。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脱靶突变。功能突变的验证将 通过qPCR、Western blot和电生理学进行确认。一旦得到验证，我们将量化 这些突变对使用多电极阵列的自发网络活动及其对 转录组与RNA测序。该提案的总体目标是开发新的细胞模型， SCZ，可以告诉我们病理生理学，并帮助确定收敛的分子机制SCZ与 目的是确定这种使人衰弱的疾病的治疗靶点。

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.