Mechanism and neurodevelopmental role of DISC 1 and NRG/ErbB signaling cross-tal

DISC 1 和 NRG/ErbB 信号交叉的机制和神经发育作用

• 批准号: 7929559
• 负责人: Saurav Rao Seshadri
• 金额: $ 3.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929559
• 关键词: Affect; American; Binding; Cell model; Cerebral cortex; Co-Immunoprecipitations; Data; Development; Disease; ERBB2 gene; Electroporation; Embryo; ErbB4 gene; Fractionation; Functional disorder; Genetic Predisposition to Disease; Genetic Transcription; Goals; Immigration; Immunohistochemistry; In Situ Hybridization; Infusion procedures; Knock-out; Knockout Mice; Knowledge; Lead; Lentivirus Vector; Measurement; Mental disorders; Messenger RNA; Modeling; NRG2 gene; NRG3 gene; Neuregulin 1; Neuregulins; Neurons; Nuclear; Pathogenesis; Pathway interactions; Physiological; Play; Process; Promoter Regions; Proteins; Radial; Recombinants; Reverse Transcriptase Polymerase Chain Reaction; Role; Schizophrenia; Signal Pathway; Signal Transduction; Susceptibility Gene; Western Blotting; base; in utero; in vivo; mRNA Expression; migration; mouse model; neurodevelopment; overexpression; receptor; research study; small hairpin RNA; transcription factor

DESCRIPTION (provided by applicant): This project aims to study the cross-talk between DISCI and NRG/ErbB signaling and its impact on radial neuronal migration, towards better understanding the pathophysiology of Schizophrenia (SZ) during neurodevelopment. DISC1 and NRG1 are both major susceptibility genes for SZ, and preliminary data indicates that NRG1 signaling can affect expression of DISK1. We intend to treat primary cortical neuron cultures with recombinant NRG1, NRG2, and NRG3 to study the effects of NRG signaling on DISK1 expression at the protein and mRNA level by Western blot and RT-PCR respectively. In conjunction with this treatment, we will either infect primary neurons with lentiviral vectors to express shRNA to NRG receptors ErbB2, ErbB3, or ErbB4, or pharmacologically block the PI3K/Akt, MARK, or STAT secondary signaling cascades in order to determine which receptor and downstream signaling pathway transduces these effects. Preliminary results also indicate that DISK1 expression regulates transcription of ErbB4. We will confirm this result at the protein and mRNA level using a knockout mouse model of DISK1. We have also shown that DISK1 binds directly to ErbB4; we will determine whether this interaction is necessary for DISK1 control of ErbB4 expression, by overexpressing D1SC1 missing the binding domain to ErbB4 and measurement of ErbB4 expression in primary neurons. Based on the results obtained, we will either study DISK1 effects on nuclear localization of ErbB4 by immunohistochemistry and nuclear fractionation, or interaction of nuclear D1SC1 with transcription factors which may bind to the 5' promoter region of ErbB4. We will also confirm that DISK1 can bind to the intracellular domain of ErbB4 by exogenous co- immunoprecipitation. We will perform in situ hybridization for D1SC1 in an embryonic NRG 1-knockout mouse model and for ErbB4 in a DISC1 knockout-model to confirm the above expression results in vivo. Next, we will modulate DISK1 and NRG1 expression in vivo using in utero electroporation, to study the physiological impact of these mechanisms, observing radial neuronal migration as an indicator of the functional impact of the DISC1/NRG1 pathway. Finally, we will modulate ErbB4 expression using in utero electroporation to observe its role in radial migration, and perform rescue experiments with DISK1 to study whether the DISC1/ErbB4 cross-talk plays a role in this important neurodevelopmental process. Schizophrenia is a devastating psychiatric disorder affecting an estimated 1% of Americans. This project aims to study the convergent mechanisms by which major susceptibility genes for schizophrenia (D1SC1, Neuregulins, and ErbB receptors) affect development and lead to disease pathophysiology.

描述（由申请人提供）：该项目旨在研究 DISCI 和 NRG/ErbB 信号之间的串扰及其对径向神经元迁移的影响，以更好地了解精神分裂症（SZ）在神经发育过程中的病理生理学。 DISC1和NRG1都是SZ的主要易感基因，初步数据表明NRG1信号传导可以影响DISK1的表达。我们打算用重组NRG1、NRG2和NRG3处理原代皮质神经元培养物，通过Western blot和RT-PCR分别在蛋白质和mRNA水平上研究NRG信号对DISK1表达的影响。结合这种治疗，我们将用慢病毒载体感染原代神经元，以向 NRG 受体 ErbB2、ErbB3 或 ErbB4 表达 shRNA，或者通过药理学阻断 PI3K/Akt、MARK 或 STAT 二级信号级联，以确定哪个受体和下游信号传导途径转导这些效应。初步结果还表明 DISK1 表达调节 ErbB4 的转录。我们将使用 DISK1 敲除小鼠模型在蛋白质和 mRNA 水平上确认这一结果。我们还表明 DISK1 直接与 ErbB4 结合；我们将通过过表达缺失 ErbB4 结合域的 D1SC1 并测量原代神经元中 ErbB4 的表达来确定这种相互作用对于 DISK1 控制 ErbB4 表达是否是必需的。根据获得的结果，我们将通过免疫组织化学和核分级研究DISK1对ErbB4核定位的影响，或者核D1SC1与可能结合ErbB4 5'启动子区域的转录因子的相互作用。我们还将通过外源免疫共沉淀证实 DISK1 可以与 ErbB4 的胞内结构域结合。我们将在NRG 1敲除胚胎小鼠模型中对D1SC1进行原位杂交，在DISC1敲除模型中对ErbB4进行原位杂交，以证实上述体内表达结果。接下来，我们将使用子宫内电穿孔调节体内 DISK1 和 NRG1 表达，以研究这些机制的生理影响，观察径向神经元迁移作为 DISC1/NRG1 通路功能影响的指标。最后，我们将利用子宫内电穿孔调节ErbB4的表达，观察其在径向迁移中的作用，并用DISK1进行救援实验，以研究DISC1/ErbB4串扰是否在这一重要的神经发育过程中发挥作用。精神分裂症是一种毁灭性的精神疾病，影响着大约 1% 的美国人。该项目旨在研究精神分裂症主要易感基因（D1SC1、神经调节蛋白和 ErbB 受体）影响发育并导致疾病病理生理学的趋同机制。