Development of a zebrafish assay for the identification of ALS drug targets

描述(申请人提供)：肌萎缩侧索硬化症是一种成人起病的运动神经元退行性疾病，一生的风险约为1/1000，在美国任何时候估计有30,000名成年人受到影响，每年新增约5,000例。约80%的病例在确诊后5年内死亡。目前还没有治愈方法，而且只有一种FDA批准的疗法对疾病的进展有轻微影响。确定ALS的药物靶点将促进我们对这种疾病的理解，并将揭示药物开发的相关靶点。肌萎缩侧索硬化症最具特征的遗传形式是由SOD1基因突变引起的。由于基于SOD1的肌萎缩侧索硬化症和散发性肌萎缩侧索硬化症都会导致相同的疾病，因此人们相信药物靶点将是相同的。SOD1的突变产生了一种活性未知的功能蛋白的显性获得。由于SOD1突变不会导致无脊椎动物出现类似肌萎缩侧索硬化症的表型，因此只存在这种疾病的啮齿动物模型，从而限制了可以进行的实验类型。例如，在ALS小鼠中进行遗传修饰物筛选是不可能的，这是识别药物干预的蛋白质目标的极好方法。此外，老鼠身上的药物筛查非常昂贵，需要大量的动物。为了产生另一个可用于遗传和药物筛选的ALS脊椎动物模型，我们产生了过度表达具有良好特征的SODG93A和G85R突变的转基因斑马鱼。当我们产生转基因斑马鱼时，我们加入了一个热休克启动子(HSP70)，驱动荧光蛋白DsRed来跟踪我们的转基因。在鉴定转基因品系后，我们发现携带SOD1突变的鱼开启了热休克反应，DsRed表达揭示了这一点，而不依赖于热休克(称为Sodmut HSP70诱导)。这表明含有突变SOD1的鱼在早期幼体阶段就表现出细胞压力。我们建议使用这种反应作为突变的SOD1功能获得毒性的读出。在这项建议中，我们提供了将苏打HSP70诱导与ALS表型联系起来的初步数据。然后，我们提出了开发和验证该读数作为筛查分析的目标。最后，我们将使用体内试验进行先导性遗传修饰筛选，以确定新的干预靶点。这种独特的体内脊椎动物SOD1突变毒性试验的开发和验证将使未来高通量筛选和合理的ALS药物设计的发展成为可能。 公共卫生相关性：肌萎缩侧索硬化症(ALS)是一种致命的神经退行性疾病，无法治愈。我们已经建立了SOD1ALS的斑马鱼模型，并发现它具有非常早的、很容易得分的表型。在这项提议中，我们将把这种表型发展成一种用于药物靶标识别和药物测试的检测方法。这是一种独特的方法，因为它是脊椎动物体内的快速筛查，并将作为遗传和药物筛查的基础，以确定ALS的治疗方法。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis is an adult onset motoneuron degenerative disease with a lifetime risk of ~1/1000 that affects an estimated 30, 000 adults in the US at any one time with ~5000 new cases/year. Approximately 80% of the cases are fatal 5 yrs after diagnosis. There is no cure and only one FDA approved therapy that has a minor effect on the progression of the disease. Identifying drug targets for ALS will advance both our understanding of this disease and will reveal relevant targets for drug development. The most well characterized genetic form of ALS is caused by mutations in the SOD1 gene. Because SOD1 based ALS and sporadic ALS both cause the same disease, it is believed that drug targets will be shared. Mutations in SOD1 produce a dominant gain of function protein with unknown activity. Since SOD1 mutants do not cause ALS-like phenotypes in invertebrates, only rodent models of this disease exist thus limiting the type of experiments that can be performed. For example it is not possible to do genetic modifier screens, which are an excellent way to identify protein targets for drug intervention, in ALS mice. In addition, drug screens in mice are very expensive and require large numbers of animals. To generate another vertebrate model of ALS that can be used for genetic and drug screens, we generated transgenic zebrafish over expressing the well-characterized SODG93A and G85R mutations. When we generated the transgenic zebrafish, we incorporated a heat shock promoter (hsp70) driving the fluorescent protein, DsRed, to track our transgene. Upon identifying the transgenic lines, we found that the fish carrying the sod1 mutations turned on the heat shock response, as revealed by DsRed expression, independent of heat shock (referred to as sodmut hsp70 induction). This suggests that fish containing mutant Sod1 exhibit cellular stress starting at early larval stages. We propose to use this response as a read-out of mutant Sod1 gain-of-function toxicity. In this proposal we present preliminary data linking the sodmut hsp70 induction to ALS phenotypes. We then present Aims to develop and validate this read-out as an assay for screening. Lastly, we will use this in vivo assay to perform a pilot genetic modifier screen to identify novel targets for intervention. Development and validation of this unique in vivo, vertebrate assay of Sod1 mutant toxicity will allow future development of high-throughput screens and rational drug design for ALS. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease that has no cure. We have generated a zebrafish model of SOD1 ALS and find that it has a very early, easily scored, phenotype. In this proposal we will develop this phenotype into an assay for drug target identification and drug testing. This is a unique approach as it is a rapid, in vivo screen in a vertebrate and will serve as a basis for both genetic and drug screens to identify therapeutics for ALS.