Bodrikov Vsevolod, Welte Cornelia, Wiechers Marianne, Weschenfelder Markus, Kaur Gurjot, Shypitsyna Aleksandra, Pinzon-Olejua Alejandro, Bastmeyer Martin, Stuermer Claudia A O
Department of Biology, University of Konstanz, Konstanz, Germany.
Zoological Institute, Cell and Neurobiology Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
J Comp Neurol. 2017 Oct 1;525(14):2991-3009. doi: 10.1002/cne.24253. Epub 2017 Jun 23.
This study explored why lesioned retinal ganglion cell (RGC) axons regenerate successfully in the zebrafish optic nerve despite the presence of Rtn4b, the homologue of the rat neurite growth inhibitor RTN4-A/Nogo-A. Rat Nogo-A and zebrafish Rtn4b possess characteristic motifs (M1-4) in the Nogo-A-specific region, which contains delta20, the most inhibitory region of rat Nogo-A. To determine whether zebrafish M1-4 is inhibitory as rat M1-4 and Nogo-A delta20, proteins were recombinantly expressed and used as substrates for zebrafish single cell RGCs, mouse hippocampal neurons and goldfish, zebrafish and chick retinal explants. When offered as homogenous substrates, neurites of hippocampal neurons and of zebrafish single cell RGCs were inhibited by zebrafish M1-4, rat M1-4, and Nogo-A delta20. Neurite length increased when zebrafish single cell RGCs were treated with receptor-type-specific antagonists and, respectively, with morpholinos (MO) against S1PR2 and S1PR5a-which represent candidate zebrafish Nogo-A receptors. In a stripe assay, however, where M1-4 lanes alternate with polylysine-(Plys)-only lanes, RGC axons from goldfish, zebrafish, and chick retinal explants avoided rat M1-4 but freely crossed zebrafish M1-4 lanes-suggesting that zebrafish M1-4 is growth permissive and less inhibitory than rat M1-4. Moreover, immunostainings and dot blots of optic nerve and myelin showed that expression of Rtn4b is very low in tissue and myelin at 3-5 days after lesion when axons regenerate. Thus, Rtn4b seems to represent no major obstacle for axon regeneration in vivo because it is less inhibitory for RGC axons from retina explants, and because of its low abundance.
本研究探讨了为何尽管存在大鼠神经突生长抑制因子RTN4 - A/Nogo - A的同源物Rtn4b，受损的视网膜神经节细胞（RGC）轴突仍能在斑马鱼视神经中成功再生。大鼠Nogo - A和斑马鱼Rtn4b在Nogo - A特异性区域具有特征基序（M1 - 4），该区域包含大鼠Nogo - A的最具抑制性的区域delta20。为了确定斑马鱼的M1 - 4是否像大鼠的M1 - 4和Nogo - A delta20一样具有抑制作用，将蛋白质进行重组表达，并用作斑马鱼单细胞RGC、小鼠海马神经元以及金鱼、斑马鱼和鸡视网膜外植体的底物。当作为均匀底物提供时，海马神经元和斑马鱼单细胞RGC的神经突受到斑马鱼M1 - 4、大鼠M1 - 4和Nogo - A delta20的抑制。当用受体类型特异性拮抗剂以及分别用针对S1PR2和S1PR5a的吗啉代（MO）处理斑马鱼单细胞RGC时，神经突长度增加，S1PR2和S1PR5a代表候选的斑马鱼Nogo - A受体。然而，在条纹试验中，M1 - 4泳道与仅含聚赖氨酸（Plys）的泳道交替排列，来自金鱼、斑马鱼和鸡视网膜外植体的RGC轴突避开大鼠M1 - 4泳道，但能自由穿过斑马鱼M1 - 4泳道，这表明斑马鱼M1 - 4允许生长，且抑制作用比大鼠M1 - 4小。此外，对视神经和髓磷脂的免疫染色和斑点印迹显示，损伤后3 - 5天，当轴突再生时，Rtn4b在组织和髓磷脂中的表达非常低。因此，Rtn4b似乎并不是体内轴突再生的主要障碍，因为它对视网膜外植体的RGC轴突抑制作用较小，且丰度较低。
