Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish
复制标题
成簇规则间隔短回文重复序列 (CRISPR)/Cas9 介导的 kif15 突变加速斑马鱼神经元发育和再生过程中的轴突生长
DOI：
10.1111/tra.12621
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发表时间：
2019-01-01
期刊：
影响因子：
4.5
通讯作者：
Liu, Mei
中科院分区：
文献类型：
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作者：
Dong, Zhangji;Wu, Shuwen;Liu, Mei
KIF 15，脊椎动物驱动蛋白-12，最为人所知的是有丝分裂运动蛋白，但继续在神经元中表达。与KIF 11（脊椎动物驱动蛋白-5）一样，KIF 15与轴突中的微管相互作用，以限制它们相对于彼此的滑动。与KIF 11不同，KIF 15还调节轴突分支形成位点和生长锥中微管和肌动蛋白丝之间的相互作用。我们最初对这些马达的研究是在培养的大鼠神经元上进行的，但我们现在正在使用斑马鱼将这些研究扩展到体内模型。我们先前通过将剪接阻断的吗啉注射到胚胎中来研究斑马鱼的kif 15。与细胞培养工作一致，这些研究表明，当KIF 15水平降低时，轴突生长得更快，更长。在本研究中，我们应用基于CRISPR/Cas9的敲除技术来创建kif 15突变体，并用Tg（mnx 1：GFP）转基因或瞬时表达elavl 3：EGFP-alpha微管蛋白来标记神经元。然后，我们通过实时成像的纯合子，杂合子突变体，以确定在尾初级运动神经元和Rohon-Beard（R-B）感觉神经元的发展过程中的kif 15的耗竭的影响，他们的野生型兄弟姐妹进行了比较。结果表明，与野生型相比，kif 15纯合突变体和杂合突变体的分支数量减少，而轴突生长加快。在R-B感觉神经元，激光照射后，损伤的轴突与损失的kif 15显示出显着更大的再生速度。考虑到这些结果和kif 15药物目前正在开发的事实，我们将kif 15作为一个新的靶点，用于治疗性增强受损轴突的再生。
KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.