Development of zebrafish as a screening tool for remyelination drugs

描述(申请人提供)：多发性硬化症(MS)是年轻人中最具破坏性的神经系统疾病，全球有超过250万人受到影响。目前治疗MS和其他脱髓鞘疾病的治疗方法主要集中在控制疾病中的免疫成分和炎症。这些治疗并不是为了通过髓鞘再生来修复中枢神经系统的损伤。目前，许多实验室正在进行研究，以确定能够促进髓鞘再生的小分子。其中一些研究集中在特定的可识别途径上，而另一些研究基本上是“黑匣子”筛选，分子靶标可能未知，但表型靶标髓鞘形成可以确定。一旦识别出这样的铅分子，它们的最优化 是必要的。优化包括药物化学，但如果不能在早期阶段确定先导化合物的体内影响，筛选先导化合物的衍生物可能是一个漫长的过程，可能不会成功。目前的项目重点是开发一种新的体内筛选将增强髓鞘形成的药物。斑马鱼模型正在成为体内药物筛选的重要工具。斑马鱼在一段快速的时间内发生髓鞘形成，受精后七天脊髓出现显著的髓鞘形成。考虑到胚胎的透明度，荧光蛋白标签是一种容易检测髓鞘形成增加的方法。为了实现我们的主要目标，我们将开发一种新的转基因斑马鱼品系，它本身将是髓鞘形成的极好读数。我们将用蛋白零(P0)启动子驱动荧光蛋白报告基因的表达，在斑马鱼中，它在早期和强烈地在少突胶质细胞中表达，并且只在较晚的时间在周围神经系统中表达。这将专门标记成熟的少突胶质细胞及其髓鞘，使荧光蛋白成像能够快速读出药物疗效。使用体内方法将使我们能够筛选出许多药物的毒性或其他负面影响。我们将优化成像技术，作为髓鞘形成本身的补充读数，研究体内时间推移成像，以建立最佳时间框架，并对增强髓鞘形成的药物进行区域评估。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is the most devastating neurological disease in young adults, affecting more than 2.5 million people worldwide. Current therapies to treat MS and other demyelinating diseases are primarily focused on control of the immune component and inflammation in the disease. These treatments are not directed at repairing the damage in the central nervous system through remyelination. Studies are currently underway in many laboratories to identify small molecules that will enhance remyelination. Some of these studies are focused on specific identifiable pathways, while others are essentially "black box" screens, for which the molecular target may not be known, but the phenotypic target, myelination, can be identified. Once such lead molecules are identified, their optimization is needed. Optimization includes medicinal chemistry, but the screening of derivatives of lead compounds can be a lengthy and possibly unsuccessful process, if the in vivo impact of these compounds is not established at an early stage. The current project focuses on developing a new in vivo screen for drugs that will enhance myelination. The zebrafish model is becoming an important in vivo screen for drugs. Myelination occurs in the zebrafish over a rapid time frame, with significant myelination of the spinal cord by seven days post fertilization. Given the transparency of the embryo, fluorescent protein tags are an easy screen for increases in myelination. In order to accomplish our main goal, we will develop a new transgenic zebrafish line that will be an excellent readout of myelination per se. We will drive fluorescent protein reporter expression with the Protein zero (P0) promoter, which in the zebrafish is expressed early and strongly in oligodendrocytes and only much later in the peripheral nervous system. This will specifically mark mature oligodendrocytes as well as their myelin sheaths, allowing fluorescent protein imaging as a rapid readout of drug efficacy. Using an in vivo approach will allow us to screen out numerous drugs for toxicity or other negative impacts. We will optimize imaging techniques as complementary readouts of myelination per se, studying in vivo time lapse imaging to establish optimal time frames and regional assessment of drugs enhancing myelination.