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

描述(申请人提供)：本项目旨在研究DISCI和NRG/ErbB信号之间的串扰及其对放射状神经元迁移的影响，以更好地理解精神分裂症(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信号转导可以影响DISK1的表达。我们打算用重组NRG1、NRG2和NRG3处理原代培养的大脑皮层神经元，通过Western印迹和RT-PCR分别从蛋白质和mRNA水平研究NRG信号对DISK1表达的影响。在这种治疗的同时，我们将用慢病毒载体感染原代神经元，以表达针对NRG受体ErbB2、ErbB3或ErbB4的shRNA，或者从药物上阻断PI3K/Akt、Mark或STAT二级信号级联，以确定哪个受体和下游信号通路转导这些作用。初步结果还表明，DISK1的表达调控ErbB4的转录。我们将使用DISK1基因敲除的小鼠模型在蛋白质和mRNA水平上证实这一结果。我们还证明了DISK1直接与ErbB4结合；我们将通过过表达D1SC1缺失与ErbB4结合的结构域和测量原代神经元中ErbB4的表达来确定这种相互作用是否对于DISK1控制ErbB4的表达是必要的。在此基础上，我们将通过免疫组织化学和核分裂来研究DISK1对ErbB4核定位的影响，或者核D1SC1与可能与ErbB4 5‘启动子区域结合的转录因子的相互作用。我们还将证实DISK1可以通过外源免疫共沉淀与ErbB4的胞内结构域结合。我们将在胚胎NRG1基因敲除小鼠模型中进行D1SC1的原位杂交，并在DISC1基因敲除模型中进行ErbB4的原位杂交，以确认上述体内表达的结果。接下来，我们将利用宫内电穿孔技术在体内调节DISK1和NRG1的表达，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，我们将利用宫内电穿孔来调节ErbB4的表达，以观察其在径向迁移中的作用，并与DISK1进行抢救性实验，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，估计有1%的美国人受到影响。本项目旨在研究精神分裂症的主要易感基因(D1SC1、NeuRegins和ErbB受体)影响发育并导致疾病病理生理的趋同机制。

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.