Investigating the role of DISC1 using iPSCs from patients with mental disorders

使用精神障碍患者的 iPSC 研究 DISC1 的作用

• 批准号: 8652075
• 负责人: Ha Nam Nguyen
• 金额: $ 4.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652075
• 关键词: Adult; Affect; American; Attention; Autistic Disorder; Balanced Chromosomal Translocation; Base Pairing; Biological; Bipolar Disorder; Brain; Cell Line; Cell Proliferation; Cell physiology; Cellular Morphology; Complex; DNA; Development; Diagnosis; Disease; Disease susceptibility; Embryo; Etiology; Evaluation; Family; Family member; Figs - dietary; Functional disorder; Gene Targeting; Generations; Genes; Genetic; Genome; Human; Human Development; In Vitro; Individual; Investigation; Knock-in Mouse; Knowledge; Lead; Major Depressive Disorder; Mental disorders; Molecular; Molecular Target; Mood Disorders; Morphogenesis; Morphology; Mus; Mutation; Nervous system structure; Neurons; Pathology; Pathway interactions; Patients; Physiological; Property; Proteins; Proto-Oncogene Proteins c-akt; Recording of previous events; Regulation; Risk; Role; Scaffolding Protein; Schizophrenia; Sequence Homology; Series; Signal Transduction; Somatic Cell; System; Techniques; Technology; Testing; Transcription Coactivator; Transplantation; adult neurogenesis; base; cell motility; gene function; human FRAP1 protein; human stem cells; in vivo; induced pluripotent stem cell; innovation; insight; mTOR Signaling Pathway; member; migration; nerve stem cell; neurodevelopment; neuron development; neuroregulation; nuclease; public health relevance; repaired; stem cells

DESCRIPTION (provided by applicant): The genetic complexity underlying the vast majority of mental disorders has made the study of these diseases exceptionally challenging. Many risk-associated genes have been identified but the biological role of these genes is largely unknown. Disrupted in Schizophrenia 1 (DISC1) is a gene in which mutations have been associated with increased risk for schizophrenia and affective disorders. The functional role of this gene has been investigated in mice and was shown to have a prominent influence on many aspects of neuronal development including cell morphology and migration. However, the role of this gene in human neuronal development has not been explored, primarily due to the limited access to human neurons. This constitutes a significant gap in our knowledge because of the relatively low homology between mouse Disc1 and human DISC1 genes (~60% sequence homology). Using an innovative human stem cell-based approach, this proposal is focused on induced pluripotent stem cells (iPSCs) generated from patients harboring a rare 4bp deletion in DISC1 who belong to an American family with a history of mental illness. Because iPSCs retain the genetic information of the donor individual, human neurons derived from these patient-specific iPSCs and those of healthy family members without the deletion will be evaluated to determine the functional role of DISC1. The hypothesis that this mutation in DISC1 will alter development of human neurons will be investigated through three specific aims. (1) Generate isogenic iPSC lines that differ exclusively at this DISC1 4bp locus using a gene targeting and editing technique called transcription activator-like effector nucleases (TALENs). This technique will allow for the generation of rescue lines in which the 4bp deletion has been corrected in patient-derived iPSCs, and deletion lines in which the 4bp deletion will be introduced to intact control-iPSCs. These complementary isogenic iPSCs will provide the basis for a functional evaluation of mutations in DISC1 with a controlled genetic background. (2) Characterize neuronal development in human neurons derived from deletion and intact DISC1 iPSCs. After differentiating iPSCs into neurons, the molecular, cellular, physiological and functional properties of the deletion lines and intact lines will be analyzed both in vitro and in vivo. (3) T determine the role of DISC1 in the AKT-mTOR pathway. In mice, Disc1 has been shown to interact with this pathway to regulate neuronal development. Determining the interaction between DISC1 and AKT-mTOR will provide critical information regarding the functional overlap between mouse Disc1 and human DISC1. Furthermore, components of the mTOR pathway have also been implicated in risk for mental disorders and investigation of key proteins in this pathway may lead to additional insight into the role of this pathway as a potential locus of convergence in genetic regulation of human neuronal development.

描述（由申请人提供）：绝大多数精神疾病的遗传复杂性使对这些疾病的研究异常具有挑战性。已经鉴定出许多与风险相关的基因，但是这些基因的生物学作用在很大程度上未知。精神分裂症1（DISC1）中的破坏是一个基因，其中突变与精神分裂症和情感障碍的风险增加有关。该基因的功能作用已在小鼠中研究，并被证明对神经元发育的许多方面具有显着影响，包括细胞形态和迁移。然而，尚未探索该基因在人类神经元发育中的作用，这主要是由于对人类神经元的访问有限。由于小鼠Disc1和人disc1基因之间的同源性相对较低（〜60％的序列同源性），这在我们的知识上构成了显着差距。使用创新的人类干细胞方法，该建议集中在诱发多能干细胞（IPSC）上，该干细胞（IPSC）是由属于患有精神疾病史的美国家庭中罕见的4BP缺失的患者产生的。由于IPSC保留了供体个体的遗传信息，因此将评估从这些患者特异性的IPSC和没有缺失的健康家庭成员的人类神经元的遗传信息，以确定DISC1的功能作用。 Disc1中这种突变将改变人类神经元的发展的假设将通过三个特定目标研究。 （1）生成的同基因IPSC线，使用称为转录激活剂样效应核酸酶（TALENS）的基因靶向和编辑技术在此Disc1 4bp基因座上仅差异。该技术将允许在患者衍生的IPSC中校正4BP缺失的救援线，并将4BP删除的删除线引入完整的控制-IPSC。这些互补的等源性IPSC将为具有受控遗传背景的DISC1的突变的功能评估提供基础。 （2）表征来自缺失和完整disc1 iPSC的人神经元中的神经元发育。将IPSC区分开为神经元后，将在体外和体内分析缺失线和完整线的分子，细胞，生理和功能特性。 （3）t确定Disc1在Akt-MTOR途径中的作用。在小鼠中，盘已证明与这种调节神经元发育的途径相互作用。确定Disc1和Akt-MTOR之间的相互作用将提供有关小鼠Disc1和Human Disc1之间功能重叠的关键信息。此外，MTOR途径的组成部分还与精神障碍的风险有关，对这种途径中关键蛋白的研究可能会导致对该途径作为人类神经元发育遗传调节的潜在融合基因座的作用。