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

描述（由申请人提供）：肌萎缩性侧索硬化症是一种成人发病的运动神经元退行性疾病，终生风险约为1/1000，在美国估计有30，000名成人在任何时候受累，每年约有5000例新发病例。大约80%的病例在诊断后5年内是致命的。没有治愈方法，只有一种FDA批准的疗法对疾病的进展有轻微的影响。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。ALS最好的特征性遗传形式是由SOD1基因突变引起的。由于基于SOD1的ALS和散发性ALS都引起相同的疾病，因此相信药物靶标将是共享的。SOD1的突变产生一种活性未知的显性功能蛋白。由于SOD1突变体不会在无脊椎动物中引起ALS样表型，因此仅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，不可能在ALS小鼠中进行遗传修饰筛选，这是识别药物干预的蛋白质靶点的绝佳方法。此外，在小鼠中进行药物筛选非常昂贵，需要大量的动物。为了产生另一种可用于遗传和药物筛选的ALS脊椎动物模型，我们产生了过表达充分表征的SODG93A和G85R突变的转基因斑马鱼。当我们产生转基因斑马鱼时，我们引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，如DsRed表达所揭示的，与热休克无关（称为sodmut hsp70诱导）。这表明含有突变体Sod 1的鱼在早期幼虫阶段就表现出细胞应激。我们建议使用这种反应作为读出的突变体Sod1获得的功能毒性。在这个建议中，我们提出了初步的数据连接sodmut热休克蛋白70诱导ALS表型。然后，我们提出的目的是开发和验证这种读出作为一种检测筛选。最后，我们将使用这种体内试验进行试点遗传修饰筛选，以确定新的干预目标。开发和验证这种独特的体内脊椎动物Sod 1突变毒性测定将允许未来开发ALS的高通量筛选和合理的药物设计。 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，目前尚无治愈方法。我们已经产生了一个斑马鱼模型的SOD 1 ALS，并发现它有一个非常早期的，容易得分，表型。在本提案中，我们将把这种表型开发成用于药物靶点鉴定和药物测试的测定。这是一种独特的方法，因为它是一种快速的脊椎动物体内筛选，并将作为遗传和药物筛选的基础，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.