Aberrant VEGF RNA Stability in Amyotrophic Lateral Sclerosis

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种运动神经元的灾难性退行性疾病，不可避免地导致进行性虚弱和死亡。家族性ALS （fALS）与突变型超氧化物歧化酶（SOD）1之间的联系是ALS研究的一个重大突破。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增加，而不是酶功能障碍。在这一提议中，我们假设突变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） RNA稳定。最近的证据表明，VEGF是细胞应激时神经保护的关键因素，其在神经组织中的下调有助于加速运动神经元的死亡。在细胞应激反应早期促进VEGF上调的一种适应性途径是mRNA稳定。当细胞暴露于缺氧或细胞因子暴露（如神经炎症）引起的应激时，例如，由于转录物的3'非翻译区（UTR）中存在富含腺嘌呤和尿嘧啶的元素（are）， VEGF mRNA变得稳定。这种反应通常导致VEGF的增强和持续表达。然而，我们假设突变SOD1抑制RNA稳定并阻止VEGF上调。由此导致的神经保护功能丧失可能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VEGF RNA水平显著降低，再加上体外数据表明，当突变SOD1表达时，VEGF转录物显着不稳定。因此，本研究的长期目标是表征SOD1突变体对生长因子RNA稳定的下游毒性作用。我们提出了两个具体的目标来验证在体外SOD1突变模型中VEGF RNA稳定功能失调的假设。首先，我们将比较细胞因子、缺氧和氧化应激对表达野生型或突变型SOD1的神经和胶质细胞中VEGF 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sod1突变体诱导的VEGF RNA不稳定。其次，我们将通过检测蛋白质-蛋白质和VEGF RNA-蛋白质与RNA稳定关键细胞因子的相互作用，表征我们在SOD1突变细胞中观察到的异常VEGF 3'UTR核糖核蛋白复合物。这一建议的影响在于它有可能确定fALS中SOD1突变的一种新的“功能获得”机制。在更广泛的情况下，表征VEGF（和其他神经保护性生长因子）上调的适应性途径，如RNA稳定，也可能对散发性ALS和其他神经退行性疾病的发病机制有影响。

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.