Upregulation of Leukemia Inhibitory Factor (LIF) during the Early Stage of Optic Nerve Regeneration in Zebrafish

Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3–5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture invitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury invivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

鱼类视网膜神经节细胞（retinal ganglion cells，RGCs）在视神经损伤后可实现轴突再生，而哺乳动物的RGCs通常无法完成该过程。白细胞介素6（interleukin 6, IL-6）型细胞因子参与细胞分化、增殖、存活及轴突再生过程，因此可能在斑马鱼RGCs的损伤后再生中发挥作用。本研究中，我们对IL-6型细胞因子的表达进行了检测，发现其中的白血病抑制因子（leukemia inhibitory factor, LIF）在损伤后3天（days post-injury, dpi）的斑马鱼RGCs中表达上调。随后我们证实，LIF的下游靶标信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）在损伤后3~5 dpi时被激活。为明确LIF的功能，我们采用LIF特异性反义吗啉代寡核苷酸（LIF-specific antisense morpholino oligonucleotides, LIF MOs）开展了LIF敲降实验。通过切断的视神经将LIF MOs导入斑马鱼RGCs后，可降低LIF的表达，并消除损伤后RGCs中STAT3的激活。上述结果表明，神经损伤后表达上调的LIF可激活斑马鱼RGCs中的贾纳斯激酶（Janus kinase, Jak）/STAT3信号通路。此外，LIF敲降会损害体外视网膜外植体培养中的轴突萌芽，降低再生相关分子生长相关蛋白43（growth-associated protein 43, GAP-43）的表达，并延缓视神经损伤后体内的功能恢复。本研究全面证实了LIF在成年斑马鱼视神经再生及功能恢复中的有益作用。