A role ofIransglutaminase during optic nerve regeneration in the goldfish

金鱼视神经损伤后可再生。视神经横断后7-10天，视神经纤维再生开始延长。它们在摘除术后3-5周到达视神经顶盖。最后，视神经横断后4-6个月视力恢复。为了了解视神经再生的分子机制，我们鉴定了在视神经再生早期表达特异性上调的基因。筛选了金鱼视网膜横断5 d后构建的cDNA文库，通过序列分析鉴定出两个克隆分别为Na、k - atp酶催化亚单胞菌3亚型和转谷氨酰胺酶（TG）神经型。在northern杂交中，这两种mrna的表达水平在第2天显著升高，在第5-10天达到峰值，然后在视神经横断后45-60天逐渐降低并恢复到对照水平。原位杂交显示了肛切开术后短暂性视网膜改变的位置。在视神经横断后10-20天，只在神经节细胞层中观察到表达增加，将TG cDNA插入质粒DNA中，得到TG融合蛋白（约70kDa）。免疫制备抗重组TG的血清。免疫组织化学染色进一步证实了原位杂交的数据。在外植体培养系统中，视神经横断后7天视网膜神经突的生长自然促进。低浓度的瓦巴因（Na，K atp酶α 3亚基的抑制剂）或抗TG的中性抗体完全阻断了受损视网膜的自发神经突生长。综上所述，这些数据表明Na、k - atp酶α 3亚基或TG的上调参与了轴突切除后神经节细胞轴突的再生。这种分子技术将成为未来神经再生基因研究的有用工具。

The goldfish optic nerve can regenerate after injury. Regrowth of optic nerve fibers starts to extend 7-10 days after optic nerve transection. They arrive at the optic tectum 3-5 weeks after axtfomy. Finally, visual function is restored 4-6 months after optic nerve transection. To understand the molecular mechanism of optic nerve regeneration, we identified genes whose expression is specifically upregulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened Two clones were identified as the Na, K-ATPase catalytic subunitalpha 3 isoform and the neural type of transglutaminase (TG) by sequence analysis. In northern hybridization, the expression level of the both mRNAs was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45-60 days after optic nerve transection. In situ hybridization have revealed the location of this transient retinal change after axotomy. The increased expression was observed only in the ganglion cell layer at 10-20 days after optic nerve transection, The cDNA of TG was inserted into the plasmid DNA and thus a fused protein of TG (ca. 70kDa) was obtained. Antiserum against the recombinant TG was made by immunization. Immunohistochemical staining further confirmed the data of in situ hybridization. In an explant culture system, neurite outgrowth fkm the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (an inhibitor of Na,K ATPase alpha 3 subunit) or neutral antibody against TG, completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that upregulation of the Na, K-ATPase alpha 3 subunit or TG is involved in the regrowth of ganglion cell axons after axotomy. Such molecular techniques will become useful tool for future genetic studies of nerve regeneration.