Generation of a mouse model to monitor ERAD in neurons

摘要 正确的蛋白质折叠对生物功能至关重要。不幸的是，许多蛋白质，特别是膜 蛋白质，往往会因为合成过程中的错误、突变、平衡的变化而错误折叠 蛋白质折叠/伴侣系统，或由于环境变化。错误折叠的蛋白质必须是 否则它们可能会干扰细胞功能，最终导致疾病。此外， 在生物体中积累错误折叠的蛋白质会缩短寿命。因此，理解如何错误折叠 从细胞中去除蛋白质具有重要的意义，这两个方面都是我们对 这一过程的生物学以及设计治疗与蛋白质有关的疾病的方法 折叠错误。一个特别的挑战是了解错误折叠的蛋白质是如何从内质中消除的 网状结构(ER)，制造大多数膜蛋白和分泌蛋白的地方。一种欣赏的方式 内质网蛋白质降解的重要性，称为内质网相关降解(ERAD)，强调了 系统中有时出现的缺陷，可导致激活内质网应激，延长激活时间 这会导致细胞死亡和组织功能障碍。不幸的是，目前很少有记者 可用于直接监测和/或可视化活体中的ERAD。在本申请中，我们建议填写 通过产生可用于体内研究ERAD的转基因小鼠来填补这一空白。因此，在目标1中，我们 建议建立一种转基因小鼠模型，表达一种基于荧光的报告，可用于 生化和目视测量神经元中ERAD的活性(由Thy1.2启动子驱动)。ERAD 我们选择的是CD3δ，用光开关荧光蛋白Dendra2标记。我们列举了 为什么这位记者特别适合测量神经元中的ERAD活动的许多优点。一次 生成的老鼠将被表征，以确保报告器可以可靠地用于测量缺陷中的 埃拉德。在验证后，我们将在AIM 2中与野生型(WT)和P497S UBQLN2杂交 我们最近建立的ALS转基因小鼠模型。P497S品系会出现认知缺陷和 运动神经元疾病，而UBQLN2WT小鼠没有表现出类似的疾病。免疫印迹显示有体型- 与WT和非WT相比，P497S动物脑和脊髓中泛素化蛋白的表达 转基因动物。此外，内质网应激标志物(PDI和磷酸化eIF2α)在脊髓中也升高 终末期P497S动物。内质网应激增加与突变体P497S的表达一致 干扰ERAD的蛋白，这是UBQLN2蛋白的已知功能。动物十字架将允许我们 评估是否为这种情况，以及任何干扰的时间进程。生成和验证Thy1.2 本文提出的表达CD3Dendra2基因的δ-Dendra2小鼠为评估 UBQLN2和其他与神经退行性疾病相关的突变蛋白对ERAD的干扰。

Summary Proper protein folding is vital for biological function. Unfortunately, many proteins, especially membrane proteins, tend to misfold either because of errors during synthesis, mutations, changes in the balance of protein folding/chaperone systems, or because of environmental changes. Misfolded proteins must be eliminated otherwise they could interfere with cellular function, eventually causing disease. Furthermore, accumulation of misfolded proteins in organisms can shorten lifespan. Therefore, understanding how misfolded proteins are removed from cells has important implications both in terms of our basic understanding of the biology of the process as well as for devising therapeutic methods to treat diseases linked to protein misfolding. A particular challenge is to understand how misfolded proteins are eliminated from the endoplasmic reticulum (ER), the site where most membrane and secretory proteins are made. An appreciation of the importance of protein degradation from the ER, called ER-associated degradation (ERAD), is underscored by defects that sometimes occur in the system, which can lead to activation of ER stress, prolonged activation of which leads to cell death and tissue malfunction. Unfortunately, at present there are few, if any, reporters that can be used to directly monitor and/or visualize ERAD in living organisms. In this application we propose to fill this void by generating transgenic mice that could be used for studying ERAD in vivo. Accordingly, in Aim 1 we propose to generate a transgenic mouse model that expresses a fluorescent-based reporter that can be used to biochemically and visually measure ERAD activity in neurons (driven by the Thy1.2 promoter). The ERAD reporter we have chosen is CD3δ tagged with the photoswitchable fluorescent protein Dendra2. We enumerate the many advantages why this reporter is particularly well suited for measuring ERAD activity in neurons. Once generated the mice will be characterized to ensure that the reporter can be reliably used to measure defects in ERAD. Upon validation we will cross the reporter mice in Aim 2 with wild type (WT) and a P497S UBQLN2 transgenic mouse model of ALS, which we recently generated. The P497S line develops cognitive deficits and motor neuron disease, whereas UBQLN2 WT mice do not show similar disease. Immunoblots indicate a build- up of ubiquitinated proteins in the brain and spinal cord of the P497S animals compared to WT and non- transgenic animals. In addition, ER stress markers (PDI and phosphorylated eIF2α) are elevated in spinal cord of end-stage P497S animals. The increase in ER stress is consistent with expression of the mutant P497S protein interfering with ERAD, which is a known function of UBQLN2 protein. The animal cross will allow us to evaluate if this is the case, and the time course of any interference. The generation and validation Thy1.2 expressing CD3δ-Dendra2 mice as proposed here should provide an extremely powerful tool for evaluating interference in ERAD caused by UBQLN2 and other mutant proteins linked to neurodegenerative diseases.