Development of zebrafish as a screening tool for remyelination drugs

多发性硬化症（MS）是年轻人中最具破坏性的神经系统疾病，影响全球超过250万人。 目前治疗MS和其他脱髓鞘疾病的疗法主要集中在控制疾病中的免疫成分和炎症。这些治疗并不针对通过髓鞘再生修复中枢神经系统的损伤。目前许多实验室正在进行研究，以确定将增强髓鞘再生的小分子。这些研究中的一些集中在特定的可识别的途径，而另一些基本上是“黑匣子”屏幕，其分子靶点可能是未知的，但表型靶点，髓鞘形成，可以确定。一旦这种先导分子被识别出来， 是必要的。优化包括药物化学，但是如果这些化合物的体内影响在早期阶段没有建立，则先导化合物的衍生物的筛选可能是漫长的并且可能不成功的过程。目前的项目重点是开发一种新的体内筛选药物，以增强髓鞘形成。斑马鱼模型正在成为一种重要的体内药物筛选方法。在斑马鱼中，髓鞘形成在一个快速的时间框架内发生，在受精后7天脊髓的髓鞘形成显著。鉴于胚胎的透明性，荧光蛋白标签是一种简单的髓鞘形成增加的筛选方法。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系，这将是髓鞘形成本身的极好读数。我们将用蛋白零（P0）启动子驱动荧光蛋白报告基因表达，该启动子在斑马鱼中在少突胶质细胞中早期且强烈地表达，并且仅在外周神经系统中晚得多地表达。这将特异性地标记成熟的少突胶质细胞以及它们的髓鞘，允许荧光蛋白成像作为药物功效的快速读出。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，以建立最佳的时间框架和增强髓鞘形成的药物的区域评估。

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.