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

基本信息

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
项目类别：
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

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

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项目摘要

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）：肌萎缩性侧面硬化症是一种成人发作运动神经元退行性疾病，终生风险〜1/1000，每次都会影响美国估计有30,000名成年人，每年有〜5000例新病例。诊断后约80％的病例是致命的5年。没有治愈方法，只有一种FDA批准的疗法对疾病的进展有很小的影响。识别ALS的药物靶标将既可以提高我们对这种疾病的理解，又将揭示药物开发的相关靶标。 ALS的遗传形式最有特征，是由SOD1基因突变引起的。由于基于SOD1的ALS和零星ALS都会引起相同的疾病，因此人们认为将共享药物靶标。 SOD1中的突变会产生具有未知活性的功能蛋白的显性增益。由于SOD1突变体不会在无脊椎动物中引起ALS样表型，因此仅存在该疾病的啮齿动物模型，因此限制了可以进行的实验类型。例如，不可能进行遗传修饰筛网，这是鉴定ALS小鼠药物干预蛋白靶标的绝佳方法。此外，小鼠的药物筛查非常昂贵，需要大量动物。为了生成可用于遗传和药物筛查的ALS的另一个脊椎动物模型，我们在表达良好的SODG93A和G85R突变的情况下产生了转基因斑马鱼。当我们生成转基因斑马鱼时，我们掺入了驱动荧光蛋白DSRED的热激启动子（HSP70）以跟踪我们的转基因。在识别转基因线后，我们发现携带SOD1突变的鱼会触发热激反应，如DSRED表达所揭示的那样，与热休克无关（称为Sodmut HSP70诱导）。这表明含有突变SOD1的鱼在早期幼虫阶段表现出细胞应激。我们建议将此反应用作突变SOD1功能获得毒性的读出。在此提案中，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，我们提出的目的是开发和验证此读出来，作为筛选的测定法。最后，我们将使用此体内测定法来执行试验遗传修饰符屏幕以识别干预的新目标。对SOD1突变毒性的这种独特的体内，脊椎动物测定的开发和验证将使未来的高通量筛选和ALS的合理药物设计的发展。公共卫生相关性：肌萎缩性横向硬化症（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，发现它具有非常早，易于评分的表型。在此提案中，我们将将这种表型开发为用于药物靶标识别和药物测试的测定法。这是一种独特的方法，因为它是脊椎动物中快速的体内筛选，它将作为遗传和药物筛查鉴定ALS治疗剂的基础。