A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish.
A small molecule screen identifies in vivo modulators of peripheral nerve regeneration in zebrafish.
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DOI：
10.1371/journal.pone.0178854
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Granato M
中科院分区：
文献类型：
--
作者：
Bremer J;Skinner J;Granato M
成年脊椎动物保留了损伤后再生外周神经的能力，尽管再生经常是不完全的，经常导致功能障碍。使用整个生物体的小分子筛选具有鉴定生物学相关靶标的高潜力，但目前可用的用于体内外周神经再生的测定要么非常费力和/或需要复杂的技术。在这里，我们利用斑马鱼幼鱼的光学透明性，开发了一种简单快速的胸鳍去除试验，测量周围神经再生在体内。切断鳍后24小时，我们观察到强大的和刻板的神经再生鳍的基础。类似于激光介导的神经横断，鳍切断后的神经再生需要雪旺细胞和FGF信号传导，证实鳍切断测定鉴定了与外周神经再生相关的途径。从一个具有已知靶点的小分子库中，我们确定了21种损害周围神经再生的化合物。这些化合物中的几种靶向已知的神经再生调节剂，进一步验证了鳍去除测定。12种已鉴定的化合物影响以前不知道的控制外周神经再生的靶点。使用激光介导的神经横断试验，我们测试了这些化合物中的10种，并确认其中6种化合物损害外周神经再生：EGFR抑制剂，糖皮质激素，前列腺素D2，视黄酸激动剂，钙通道抑制剂和拓扑异构酶I抑制剂。因此，我们建立了一个技术上简单的分析，以快速确定有价值的切入点到脊椎动物周围神经再生的关键途径。
Adult vertebrates have retained the ability to regenerate peripheral nerves after injury, although regeneration is frequently incomplete, often leading to functional impairments. Small molecule screens using whole organisms have high potential to identify biologically relevant targets, yet currently available assays for in vivo peripheral nerve regeneration are either very laborious and/or require complex technology. Here we take advantage of the optical transparency of larval zebrafish to develop a simple and fast pectoral fin removal assay that measures peripheral nerve regeneration in vivo. Twenty-four hours after fin amputation we observe robust and stereotyped nerve regrowth at the fin base. Similar to laser mediated nerve transection, nerve regrowth after fin amputation requires Schwann cells and FGF signaling, confirming that the fin amputation assay identifies pathways relevant for peripheral nerve regeneration. From a library of small molecules with known targets, we identified 21 compounds that impair peripheral nerve regeneration. Several of these compounds target known regulators of nerve regeneration, further validating the fin removal assay. Twelve of the identified compounds affect targets not previously known to control peripheral nerve regeneration. Using a laser-mediated nerve transection assay we tested ten of those compounds and confirmed six of these compounds to impair peripheral nerve regeneration: an EGFR inhibitor, a glucocorticoid, prostaglandin D2, a retinoic acid agonist, an inhibitor of calcium channels and a topoisomerase I inhibitor. Thus, we established a technically simple assay to rapidly identify valuable entry points into pathways critical for vertebrate peripheral nerve regeneration.