Aberrant VEGF RNA Stability in Amyotrophic Lateral Sclerosis

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种灾难性的运动神经元退行性疾病，不可抗拒地导致进行性虚弱和死亡。家族性ALS(FALS)和突变的超氧化物歧化酶(SOD)1之间的联系是ALS研究中的一个重大突破。令人惊讶的是，这种突变蛋白的致病基础很可能是功能的毒性获得，而不是酶功能障碍。在这个建议中，我们假设突变的SOD1的毒性功能是通过破坏适当的RNA-蛋白质相互作用来抑制血管内皮生长因子(VEGF)的RNA稳定。最近的证据表明，在细胞应激过程中，血管内皮生长因子是神经保护的关键因素，其在神经组织中的下调导致运动神经元加速死亡。在细胞应激反应早期促进血管内皮生长因子上调的一个适应性途径是mRNA稳定。例如，当细胞暴露在由缺氧或细胞因子暴露(如神经炎症)引起的应激中时，由于转录本的3‘非翻译区(UTR)中存在富含腺嘌呤和尿苷的元件(ARE)，VEGF mRNA变得稳定。这种反应通常会导致血管内皮生长因子表达的增强和持续。然而，我们推测突变的SOD1抑制了RNA的稳定并阻止了VEGF的上调。随之而来的神经保护的丧失可能会加速运动神经元的死亡。在我们的初步研究中，我们观察到SOD1突变小鼠脊髓中的VEGF RNA水平显著降低，同时体外数据显示，当突变的SOD1表达时，VEGF转录本明显不稳定。因此，这项提议的广泛的长期目标是表征突变的SOD1对生长因子的RNA稳定的下游毒性效应。我们提出了两个特定的目的来检验我们的体外SOD1突变模型中血管内皮生长因子RNA稳定功能障碍的假设。首先，我们将比较细胞因子、低氧和氧化应激对表达野生型和突变型SOD1的神经和神经胶质来源细胞中血管内皮生长因子mRNA稳定和表达的影响。我们还将测试RNA稳定剂HUR是否可以逆转突变的SOD1诱导的VEGFRNA失稳。其次，我们将通过检测蛋白质-蛋白质和VEGFRNA-蛋白质与对RNA稳定至关重要的细胞因子的相互作用来表征我们在SOD1突变细胞中观察到的异常的VEGF3‘UTR型核糖核蛋白复合体。这一建议的影响是它有可能为FALS中的SOD1突变确定一种新的“功能获得”机制。在更广泛的图景中，表征上调血管内皮生长因子(和其他神经保护性生长因子)的适应途径，如RNA稳定，也可能在散发性ALS和其他神经退行性疾病的发病机制中有意义。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a catastrophic degenerative disease of motor neurons that inexorably leads to progressive weakness and death. A major breakthrough in ALS research occurred with the link between familial ALS (fALS) and mutant superoxide dismutase (SOD)1. Surprisingly, the pathogenic basis of this mutant protein is most likely a toxic gain of function rather than enzymatic dysfunction. In this proposal, we hypothesize that a toxic function of mutant SOD1 is the inhibition of vascular endothelial growth factor (VEGF) RNA stabilization through disruption of proper RNA-protein interactions. Recent evidence indicates that VEGF is a critical element for neuroprotection during cellular stress, and that its down-regulation in neural tissues contributes to accelerated motor neuron death. One adaptative pathway that promotes VEGF upregulation early in the response to cellular stress is mRNA stabilization. When cells are exposed to stress induced by hypoxia or cytokine exposure (as with neuroinflammation), for example, VEGF mRNA becomes stabilized due to the presence of adenine- and uridine-rich elements (ARE) in the 3' untranslated region (UTR) of the transcript. This response normally results in enhanced and sustained expression of VEGF. However, we postulate that mutant SOD1 inhibits RNA stabilization and prevents the upregulation of VEGF. The resultant loss of neuroprotection may then accelerate motor neuron death. In our preliminary studies, we have observed a significant diminution of VEGF RNA levels in the spinal cords of SOD1 mutant mice, coupled with in vitro data indicating a marked destabilization of the VEGF transcript when mutant SOD1 is expressed. The broad long-term objective of this proposal, therefore, is to characterize the downstream toxic effects of mutant SOD1 on RNA stabilization of growth factors. We propose two specific aims to test the hypothesis that VEGF RNA stabilization is dysfunctional in our in vitro SOD1 mutant models. First, we will compare the effects of cytokine, hypoxic and oxidative stress on VEGF mRNA stabilization and expression in cells of neural- and glial-origin expressing either wild-type or mutant SOD1. We will also test whether the RNA stabilizer, HuR, can reverse the mutant SOD1-induced destabilization of VEGF RNA. Second, we will characterize the aberrant VEGF 3'UTR ribonucleoprotein complex that we have observed in SOD1 mutant cells by examining protein-protein and VEGF RNA-protein interactions with cellular factors critical to RNA stabilization. The impact of this proposal is its potential for identifying a novel "gain of function" mechanism for the SOD1 mutation in fALS. In the broader picture, characterization of adaptative pathways for the upregulation of VEGF (and other neuroprotective growth factors), such as RNA stabilization, may also have implications in the pathogenesis of sporadic ALS and other neurodegenerative disease.