The Role of HuR in mutant SOD1 dysregulation of VEGF mRNA Processing

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。血管内皮生长因子（VEGF）基因缺失导致运动神经元选择性变性，提供了VEGF与ALS之间的直接联系。此外，VEGF还能减弱表达铜锌超氧化物歧化酶(SOD)-1突变的ALS小鼠的表型。转录后调控在VEGF表达中起关键作用，特别是在细胞应激条件下，允许适应性反应促进细胞存活。我们最近发现突变SOD1破坏VEGF转录后调控，导致RNA和蛋白质表达显著下降。这一发现支持sod1相关ALS的功能毒性增加假说。这些最初的观察由一项探索性的R21基金资助，最近发表（Lu et al., 2007），导致了三个重要的发现，开始描绘这种毒性作用的机制。首先，我们发现突变体SOD1能够直接结合VEGF 3‘非翻译区（3’ utr）的富au元件（ARE）。这些顺式元件通过与细胞因子的相互作用对VEGF的转录后调控至关重要。其次，我们发现突变型而非野生型SOD1与HuR相关，HuR是通过ARE调节VEGF mRNA稳定性的主要因子。第三，突变体SOD1导致细胞质易位和HuR的翻译后修饰。基于这些发现，我们在本研究中假设突变SOD1通过破坏HuR的正常调节功能对VEGF mRNA加工产生负面影响。我们提出了三个具体目标：1)研究突变体SOD1的新型RNA结合特性及其对HuR的负面影响。2)研究突变体SOD1是否通过蛋白-蛋白或蛋白-RNA相互作用破坏了HuR的RNA稳定功能，或增加了VEGF mRNA与翻译沉默子或RNA不稳定子的关联。3)研究突变体SOD1如何诱导HuR易位到细胞质及其翻译后修饰。该提案的长期目标是：a)确定突变SOD1如何破坏VEGF转录后调节和b)这种新功能对ALS表型的影响。公共卫生相关性：肌萎缩性侧索硬化症是一种无情的运动神经元疾病，可导致肌肉进行性瘫痪并最终死亡。这种疾病无法治愈，也没有任何缓解疗法。这一建议将解决生长因子调控的一个新领域，可能有助于导致这种疾病，因此可能最终导致新的治疗方法。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a catastrophic degenerative disease of motor neurons that inexorably leads to progressive weakness and death. Genetic depletion of vascular endothelial growth factor (VEGF) leads to selective degeneration of motor neurons, providing a direct link between VEGF and ALS. Furthermore, VEGF attenuates the phenotype of ALS mice expressing the copper-zinc superoxide dismutase (SOD)-1 mutation. Posttranscriptional regulation plays a critical role in VEGF expression, particularly under conditions of cellular stress, allowing an adaptive response to promote cell survival. We recently showed that mutant SOD1 disrupts VEGF posttranscriptional regulation, leading to a significant decline in RNA and protein expression. This finding supports the toxic gain of function hypothesis for SOD1-associated ALS. These initial observations, funded by an exploratory R21 grant and recently published (Lu et al., 2007), have led to three important findings that begin to delineate the mechanism for this toxic effect. First, we discovered that mutant SOD1 is capable of directly binding AU-rich elements (ARE) in the 3' untranslated region (3'UTR) of VEGF. These cis elements are critical for posttranscriptional regulation of VEGF through interaction with cellular factors. Second, we have found that mutant but not wild-type SOD1 associates with HuR, a major regulator of VEGF mRNA stability via the ARE. Third, mutant SOD1 leads to cytoplasmic translocation and posttranslational modification of HuR. Based on these findings, we hypothesize in this proposal that mutant SOD1 exerts its negative effect on VEGF mRNA processing by disrupting the normal regulatory function of HuR. We propose three specific aims: 1) To investigate the novel RNA binding property of mutant SOD1 and its negative effect on HuR. 2) To investigate whether mutant SOD1, through its protein-protein or protein-RNA interaction, disrupts the RNA stabilizing function of HuR, or increases the association of VEGF mRNA with translational silencers or RNA destabilizers. 3) To investigate how mutant SOD1 induces HuR translocation to the cytoplasm and its post-translational modification. The long term objective of this proposal is: a) to determine how mutant SOD1 disrupts VEGF posttranscriptional regulation and b) the impact of this novel function on the ALS phenotype. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis is a relentless disease of motor neurons that leads to progressive paralysis of the muscles and ultimately death. There is no cure for this disease or any mitigating therapy. This proposal will address a novel area of growth factor regulation that may contribute to the cause of this disease, and thus may ultimately lead to novel therapies.