Mikiko Nagashima

Changes of phospho-growth-associated protein 43 (phospho-GAP43) in the zebrafish retina after optic nerve injury: A long-term observation

The major model animal of optic nerve regeneration in fish is goldfish. A closely related zebrafish is the most popular model system for genetic and developmental studies of vertebrate central nervous system. A few challenging works of optic nerve regeneration have been done with zebrafish. However, knowledge concerning the long term of optic nerve regeneration apparently lacks in zebrafish. In the present study, therefore, we followed changes of zebrafish behavior and phosphorylated form of growth-associated protein 43 (phospho-GAP43) expression in the zebrafish retina over 100 days after optic nerve transection. Optomotor response was fast recovered by 20-25 days after axotomy whereas chasing behavior (a schooling behavior) was slowly recovered by 80-100 days after axotomy. The temporal pattern of phospho-GAP43 expression showed a biphasic increase, a short-peak (12 folds) at 1-2 weeks and a long-plateau (4 folds) at 1-2 months after axotomy. The recovery of optomotor response well correlated with projection of growing axons to the tectum, whereas the recovery of chasing behavior well correlated with synaptic refinement of retinotectal topography. The present data strongly suggest that phospho-GAP43 plays an active role in both the early and late stages of optic nerve regeneration in fish.

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo.

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish (Carassius auratus). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up‐regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose‐dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra‐ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo. None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO‐cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.

Involvement of retinoic acid signaling in goldfish optic nerve regeneration.

Recently, we identified a retina-specific retinol-binding protein, purpurin, as a trigger molecule in the early stage of goldfish optic nerve regeneration. Purpurin protein was secreted by photoreceptors to injured ganglion cells, at 2-5 days after optic nerve injury. Purpurin bound to retinol induced neurite outgrowth in retinal explant cultures and retinoic acid (RA) had a comparable effect on neurite outgrowth. These results indicate that purpurin acts as a retinol transporter and facilitates conversion of retinol to RA. Intracellularly, RA is transported into the nucleus with cellular retinoic acid-binding protein IIb (CRABPIIb) and binds with retinoic acid receptor alpha (RARalpha) as a transcriptional regulator of target genes. Here, we investigated the RA signaling through RA synthesis to RARalpha in the goldfish retina during optic nerve regeneration by RT-PCR. Retinaldehyde dehydrogenase 2 (RALDH2; an RA synthetic enzyme) mRNA was increased by 2.7-fold in the retina at 7-10 days and then gradually decreased until 40 days after nerve injury. In contrast, cytochrome P450 26a1 (CYP26a1; an RA degradative enzyme) mRNA was decreased to less than half in the retina at 5-20 days and then gradually returned to the control level by 40 days after nerve injury. CRABPIIb mRNA was increased by 1.5-fold in the retina at 10 days after axotomy, RARalphaa mRNA was increased by 1.8-fold in the retina at 10 days after axotomy. The cellular changes in the RA signaling molecules after optic nerve injury were almost all located in the ganglion cells, as evaluated by in situ hybridization. The present data described for the first time that RA signaling through RALDH2 and CRABPIIb to RARalpha was serially upregulated in the ganglion cells at 7-10 days just after the purpurin induction. Therefore, we conclude that the triggering action of purpurin on optic nerve regeneration is mediated by RA signaling pathway.

HSP70, the earliest-induced gene in the zebrafish retina during optic nerve regeneration: Its role in cell survival

Fish retinal ganglion cells (RGCs) can survive and regrow their axons after optic nerve injury. Injured RGCs express anti-apoptotic proteins, such as Bcl-2, after nerve injury; however, upstream effectors of this anti-apoptotic protein are not yet fully understood. Heat shock proteins (HSPs) play a crucial role in cell survival against various stress conditions. In this study, we focused on HSP70 expression in the zebrafish retina after optic nerve injury. HSP70 mRNA and protein levels increased rapidly 2.3-fold in RGCs by 1-6 h after injury and returned to control levels by 1-3 days. HSP70 transcription is regulated by heat shock factor 1 (HSF1). HSF1 mRNA and phosphorylated-HSF1 protein rapidly increased by 2.2-fold in RGCs 0.5-6 h after injury. Intraocular injection of HSP inhibitor I significantly suppressed the induction of HSP70 expression after nerve injury. It also suppressed Bcl-2 protein induction and resulted in TUNEL-positive cell death of RGCs at 5 days post-injury. Zebrafish treated with HSP inhibitor I retarded axonal elongation or visual function after injury, as analyzed by GAP43 expression and behavioral analysis of optomotor response, respectively. These results strongly indicate that HSP70, the earliest induced gene in the zebrafish retina after optic nerve injury, is a crucial factor for RGCs survival and optic nerve regeneration in fish.

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 in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

Growth-Associated Protein43 (GAP43) Is a Biochemical Marker for the Whole Period of Fish Optic Nerve Regeneration

In adult visual system, goldfish can regrow their axons and fully restore their visual function even after optic nerve transection. The optic nerve regeneration process in goldfish is very long and it takes about a half year to fully recover visual function via synaptic refinement. Therefore, we investigated time course of growth-associated protein 43 (GAP43) expression in the goldfish retina for over 6 months after axotomy. In the control retina, very weak immunoreactivity could be seen in the retinal ganglion cells (RGCs). The immunoreactivity of GAP43 started to increase in the RGCs at 5 days, peaked at 7-20 days and then gradually decreased at 30-40 days after axotomy. The weak but significant immunoreactivity of GAP43 in the RGCs continued during 50-90 days and slowly returned to the control level by 180 days after lesion. The levels of GAP43 mRNA showed a biphasic pattern; a short-peak increase (9-folds) at 1-3 weeks and a long plateau increase (5-folds) at 50-120 days after axotomy. Thereafter, the levels declined to the control value by 180 days after axotomy. The changes of chasing behavior of pair of goldfish with bilaterally axotomized optic nerve also showed a slow biphasic recovery pattern in time course. Although further experiment is needed to elucidate the role of GAP43 in the regrowing axon terminals, the GAP43 is a good biochemical marker for monitoring the whole period of optic nerve regeneration in fish.

Purpurin expression in the zebrafish retina during early development and after optic nerve lesion in adults

Purpurin, a retina-specific protein, is known to play a role in cell adhesion during development of the chicken retina. Although purpurin has been significantly detected in adult chicken retina, its function in the matured retina is not well understood. Therefore, to determine the expression pattern of purpurin in the retina, we simultaneously investigated expression patterns of purpurin in the zebrafish retina during development in larvae and optic nerve regeneration after nerve transection in adults. In early development, levels of purpurin suddenly increased in the zebrafish retina 3 to 5 days after fertilization, and purpurin-positive immunoreactivity was diffusely located in all retinal layers. In contrast, levels of purpurin mRNA rapidly increased in the adult retina 1-3 days after optic nerve transection, and rapidly declined by 10 days after injury. Signal for purpurin mRNA was seen only in photoreceptors. Immunohistochemistry showed that levels of purpurin protein were also increased in the retina 1-3 days after nerve injury, but positive staining was located in photoreceptors and ganglion cells, and the staining in ganglion cells was stronger than that in photoreceptors. Thus, the transient expression of purpurin protein was greatly different during development and optic nerve regeneration. In the former, purpurin may be required in all retinal layers, whereas in the latter, purpurin may be required for injured ganglion cells.

Purpurin is a key molecule for cell differentiation during the early development of zebrafish retina

Recently, we cloned purpurin cDNA as an upregulated gene in the axotomized fish retina. The retina-specific protein was secreted from photoreceptors to ganglion cell layer during an early stage of optic nerve regeneration in zebrafish retina. The purpurin worked as a trigger molecule for axonal regrowth in adult injured fish retina. During zebrafish development, purpurin mRNA first appeared in ventral retina at 2 days post-fertilization (dpf) and spread out to the outer nuclear layer at 3 dpf. Here, we investigated the role of purpurin for zebrafish retinal development using morpholino gene knockdown technique. Injection of purpurin morpholino into the 1-2 cell stage of embryos significantly inhibited the transcriptional and translational expression of purpurin at 3 dpf. In the purpurin morphant, the eyeball was significantly smaller and retinal lamination of nuclear and plexiform layers was not formed at 3 dpf. Retinal cells of purpurin morphants were still proliferative and undifferentiated at 3 dpf. The visual function of purpurin morphant estimated by optomotor response was also suppressed at 5 dpf. By contrast, the control morphants with random sequence morpholino showed retinal lamination with distinct layers and differentiated cells at 3 dpf. These results strongly suggest that purpurin is a key molecule for not only optic nerve regeneration in adult but also cell differentiation during early development in embryo.

HSP70 gene expression in the zebrafish retina after optic nerve injury: a comparative study under heat shock stresses.

Unlike mammals, fish retinal ganglion cells (RGCs) can regrow their axon and restore visual function after optic nerve injury (ONI), which is the best model for CNS repair. Heat shock protein 70 (HSP70) plays an important role for cell protection by various environmental stresses. HSP70 is transactivated by phosphorylated Heat shock factor-1 (pHSF-1). In this study, we investigate the expression of HSP70 and pHSF-1 after ONI and heat shock condition (at 37°C for 30 min) in zebrafish (ZF) retina. After ONI, HSP70 was increased for a long time in the RGCs during 0.5–24 h and then returned to the control level by 72 h. In contrast, HSP70 increased for a short time in all nuclear layers at 0.5–1 h, and then returned to control level by 3 h after heat shock. The level of HSF-1 mRNA was significantly increased in ZF retina 0.5–24 h after ONI, but not after heat shock. pHSF-1 was detected after both heat shock and ONI. These results indicate that the increased expression of HSP70 mRNA after ONI is caused by both activations of translocation of pHSF-1 and of transcription of HSF-1 mRNA in the nucleus, whereas the increased expression of HSP70 mRNA after heat shock is caused only by activation of pHSF-1 translocation into the nucleus.

The expression of KLF4 in zebrafish retina during optic nerve regeneration

large part of CFP positive cells were differentiated into myelinating mature oligodendrocytes. Nevertheless the number of CFP-labeled mature oligodendrocytes was not significantly increased, which would not be insufficient to repair the injury. Taken together, these results indicate that OPCs have a potential capacity as an endogenous cell source to regenerate oligodendrocytes after WMI. Development of interventions that promote this process are needed for achieving complete restoration from the injury.