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

开发用于鉴定 ALS 药物靶标的斑马鱼测定法

• 批准号: 7826954
• 负责人: CHRISTINE E BEATTIE
• 金额: $ 18.78万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7826954
• 关键词: Address; Adolescent; Adult; Affect; Amyotrophic Lateral Sclerosis; Animals; Automobile Driving; Biological Assay; Cellular Stress; Data; Degenerative Disorder; Development; Diagnosis; Disease; Disease Progression; Drug Delivery Systems; Drug Design; DsRed; Embryo; Exhibits; FDA approved; Female; Fertilization; Fishes; Funding Mechanisms; Future; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genetic Screening; Goals; Heat-Shock Response; Hour; Intervention; Invertebrates; Investigational Drugs; Knowledge; Larva; Link; Mediating; Minor; Modeling; Molecular Chaperones; Molecular Target; Monitor; Motor Neurons; Mus; Mutation; National Institute of Neurological Disorders and Stroke; Neurodegenerative Disorders; Nucleic Acids; Pharmaceutical Preparations; Phase; Phenotype; Preclinical Drug Evaluation; Process; Proteins; RNA; Reading; Rodent Model; Route; SOD1 gene; Screening procedure; Staging; System; Tertiary Protein Structure; Testing; Therapeutic; Time; Toxic effect; Transgenes; Transgenic Organisms; Translational Research; Validation; Zebrafish; Zebrafish Proteins; assay development; base; drug development; drug discovery; drug testing; gain of function; high standard; high throughput screening; in vivo; lifetime risk; male; mutant; novel; pre-clinical; programs; promoter; public health relevance; research study; response; tool; zebrafish development

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.

描述（由申请人提供）：肌萎缩性脊髓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，估计在任何时间都会影响美国 30, 000 名成年人，每年约有 5000 例新病例。大约 80% 的病例在诊断后 5 年内死亡。目前尚无治愈方法，只有一种 FDA 批准的疗法对疾病的进展影响较小。确定 ALS 的药物靶点将增进我们对这种疾病的了解，并揭示药物开发的相关靶点。 ALS 最明确的遗传形式是由 SOD1 基因突变引起的。由于基于 SOD1 的 ALS 和散发性 ALS 都会引起相同的疾病，因此相信药物靶点将是共享的。 SOD1 突变导致功能蛋白显着增加，但活性未知。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 小鼠中进行基因修饰筛选，而基因修饰筛选是识别药物干预的蛋白质靶点的极好方法。此外，小鼠药物筛选非常昂贵并且需要大量动物。为了生成另一种可用于遗传和药物筛选的 ALS 脊椎动物模型，我们生成了过度表达已明确表征的 SODG93A 和 G85R 突变的转基因斑马鱼。当我们生成转基因斑马鱼时，我们掺入了驱动荧光蛋白 DsRed 的热休克启动子 (hsp70)，以跟踪我们的转基因。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 表达所示，与热休克无关（称为 sodmut hsp70 诱导）。这表明含有突变体 Sod1 的鱼从早期幼虫阶段就开始表现出细胞应激。我们建议使用这种反应作为突变体 Sod1 功能获得毒性的读数。在本提案中，我们提供了将 sodmut hsp70 诱导与 ALS 表型联系起来的初步数据。然后，我们提出开发和验证该读数作为筛选测定的目标。最后，我们将使用这种体内测定进行试点遗传修饰筛选，以确定新的干预靶标。这种独特的体内脊椎动物 Sod1 突变体毒性测定的开发和验证将有助于未来开发 ALS 的高通量筛选和合理的药物设计。 公众健康相关性：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。我们已经生成了 SOD1 ALS 的斑马鱼模型，并发现它具有非常早期、易于评分的表型。在本提案中，我们将把这种表型开发成药物靶标识别和药物测试的测定方法。这是一种独特的方法，因为它是一种在脊椎动物体内进行的快速体内筛选，并将作为遗传和药物筛选的基础来确定 ALS 的治疗方法。