Optic Nerve Regeneration In Zebrafish Research Articles

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Specific Activation of Yamanaka Factors via HSF1 Signaling in the Early Stage of Zebrafish Optic Nerve Regeneration.

In contrast to the case in mammals, the fish optic nerve can spontaneously regenerate and visual function can be fully restored 3-4 months after optic nerve injury (ONI). However, the regenerative mechanism behind this has remained unknown. This long process is reminiscent of the normal development of the visual system from immature neural cells to mature neurons. Here, we focused on the expression of three Yamanaka factors (Oct4, Sox2, and Klf4: OSK), which are well-known inducers of induced pluripotent stem (iPS) cells in the zebrafish retina after ONI. mRNA expression of OSK was rapidly induced in the retinal ganglion cells (RGCs) 1-3 h after ONI. Heat shock factor 1 (HSF1) mRNA was most rapidly induced in the RGCs at 0.5 h. The activation of OSK mRNA was completely suppressed by the intraocular injection of HSF1 morpholino prior to ONI. Furthermore, the chromatin immunoprecipitation assay showed the enrichment of OSK genomic DNA bound to HSF1. The present study clearly showed that the rapid activation of Yamanaka factors in the zebrafish retina was regulated by HSF1, and this sequential activation of HSF1 and OSK might provide a key to unlocking the regenerative mechanism of injured RGCs in fish.

Optic nerve regeneration in larval zebrafish exhibits spontaneous capacity for retinotopic but not tectum specific axon targeting.

In contrast to mammals, retinal ganglion cells (RGC) axons of the optic nerve even in mature zebrafish exhibit a remarkable capacity for spontaneous regeneration. One constraint of using adult zebrafish is the limited ability to visualize the regeneration process in live animals. To dynamically visualize and trace the degree of target specific optic nerve regeneration, we took advantage of the optical transparency still preserved in post developmental larval zebrafish. We developed a rapid and robust assay to physically transect the larval optic nerve and find that by 96 hours post injury RGC axons have robustly regrown onto the optic tectum. We observe functional regeneration by 8 days post injury, and demonstrate that similar to adult zebrafish, optic nerve transection in larval zebrafish does not prominently induce cell death or proliferation of RGC neurons. Furthermore, we find that partial optic nerve transection results in axonal growth predominantly to the original, contralateral tectum, while complete transection results in innervation of both the correct contralateral and ‘incorrect’ ipsilateral tectum. Axonal tracing reveals that although regenerating axons innervate the ‘incorrect’ ipsilateral tectum, they successfully target their topographically appropriate synaptic areas. Combined, our results validate post developmental larval zebrafish as a powerful model for optic nerve regeneration, and reveal intricate mechanistic differences between axonal growth, midline guidance and synaptic targeting during zebrafish optic nerve regeneration.

Prior Exposure to Immunosuppressors Sensitizes Retinal Microglia and Accelerates Optic Nerve Regeneration in Zebrafish.

As adult mammals lack the capacity to replace or repair damaged neurons, degeneration and trauma (and subsequent dysfunction) of the central nervous system (CNS) seriously constrains the patient's life quality. Recent work has shown that appropriate modulation of acute neuroinflammation upon CNS injury can trigger a regenerative response; yet, the underlying cellular and molecular mechanisms remain largely elusive. In contrast to mammals, zebrafish retain high regenerative capacities into adulthood and thus form a powerful model to study the contribution of neuroinflammation to successful regeneration. Here, we used pharmacological immunosuppression methods to study the role of microglia/macrophages during optic nerve regeneration in adult zebrafish. We first demonstrated that systemic immunosuppression with dexamethasone (dex) impedes regeneration after optic nerve injury. Secondly, and strikingly, local intravitreal application of dex or clodronate liposomes prior to injury was found to sensitize retinal microglia. Consequently, we observed an exaggerated inflammatory response to subsequent optic nerve damage, along with enhanced tectal reinnervation. In conclusion, we found a strong positive correlation between the acute inflammatory response in the retina and the regenerative capacity of the optic nerve in adult zebrafish subjected to nerve injury.

Mechanisms of RhoA inactivation and CDC42 and Rac1 activation during zebrafish optic nerve regeneration

When axons of the mammalian central nervous system (CNS) are injured, they fail to regenerate, while those of lower vertebrates undergo regeneration after injury. Wingless-type MMTV integration site family (Wnt) proteins play important roles in the CNS, and are reported to be activated after mammalian spinal cord or brain injury. Moreover, for axon growth to proceed, it is thought that small G-proteins, such as CDC42 and Rac1, need to be activated, whereas RhoA must be inactivated. However, the cell and molecular mechanisms involved in optic nerve regeneration remain unclear. In this study, we investigated axonal regeneration after injury using the zebrafish optic nerve as a model system. We sought to clarify the role of Wnt proteins and the mechanisms involved in the activation and inactivation of small G-proteins in nerve regeneration. After optic nerve injury, mRNA levels of Wnt5b, TAX1BP3 and ICAT increased in the retina, while those of Wnt10a decreased. These changes were associated with a reduction in β-catenin in nuclei. We found that Wnt5b activated CDC42 and Rac1, leading to the inactivation of RhoA, which appeared to be dependent on increased TAX1BP3 mRNA levels. Furthermore, we found that mRNA levels of Daam1a and ARHGEF16 decreased. We speculate that the decrease in β-catenin levels, which also further reduces levels of active RhoA, might contribute to regeneration in the zebrafish. Collectively, our novel results suggest that Wnt5b, Wnt10a, ICAT and TAX1BP3 participate in the activation and inactivation of small G-proteins, such as CDC42, Rac1 and RhoA, during the early stage of optic nerve regeneration in the zebrafish.

Activating transcription factor 3 promotes spinal cord regeneration of adult zebrafish

Zebrafish is an excellent model to study the mechanisms underlying successful central nervous system (CNS) regeneration. Previous study shows that activating transcription factor 3 (ATF3) promotes neurite outgrowth and is involved in optic nerve regeneration in zebrafish. Here, we used zebrafish model to investigate the role of ATF3 in regeneration following spinal cord injury (SCI). Quantitative polymerase chain reaction (qPCR) and in situ hybridization revealed that ATF3 mRNA levels increased at 12 h and 6 d following SCI. Double labeled immunofluorescence showed that ATF3 expressed in motoneurons. Treatment of anti-sense ATF3 morpholino (MO) inhibited locomotor recovery and decreased axon regeneration of spinal cord injured zebrafish. Further, inhibition of ATF3 up-regulated the expression of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These data suggest that ATF3 could promote locomotor recovery and axon regrowth in zebrafish SCI model possibly by regulating inflammatory response.

Complementary research in mammals and fish indicates MMP-2 as a pleiotropic contributor to optic nerve regeneration.

Complementary research in mammals and fish indicates MMP-2 as a pleiotropic contributor to optic nerve regeneration.

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.

FRO 2014: MMP‐2 as a potential benefactor in zebrafish optic nerve regeneration

AbstractThe zebrafish retinotectal system proves to be a valuable research model to investigate regenerative strategies of damaged retinal ganglion cells (RGCs) and thus holds great promise towards identifying new regenerative molecules. Recent research performed in our lab shows that retinal broad‐spectrum MMP inhibition seriously reduces retinotectal regeneration after optic nerve crush (ONC) in the adult zebrafish. Based on preliminary results, we now intend to determine the exact role of MMP‐2 in zebrafish RGC axonal regrowth and identify underlying molecules, which might ultimately serve as new therapeutic targets for the injured mammalian CNS. Hereto, we will pursue the following goals: (1) Determination of the regenerative potential of RGCs after retinal MMP‐2 inhibition/knockdown in zebrafish subjected to ONC. (2) Proteomic identification of MMP‐2 regulated pathways underlying a successful regeneration in the zebrafish visual system.

Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.

The regenerative failure of mammalian optic axons is partly mediated by Socs3-dependent inhibition of Jak/Stat signaling (Smith et al., 2009, 2011). Whether Jak/Stat signaling is part of the normal regenerative response observed in animals that exhibit an intrinsic capacity for optic nerve regeneration, such as zebrafish, remains unknown. Nor is it known whether the repression of regenerative inhibitors, such as Socs3, contributes to the robust regenerative response of zebrafish to optic nerve damage. Here we report that Jak/Stat signaling stimulates optic nerve regeneration in zebrafish. We found that IL-6 family cytokines, acting via Gp130-coupled receptors, stimulate Jak/Stat3 signaling in retinal ganglion cells after optic nerve injury. Among these cytokines, we found that CNTF, IL-11, and Clcf1/Crlf1a can stimulate optic axon regrowth. Surprisingly, optic nerve injury stimulated the expression of Socs3 and Sfpq (splicing factor, proline/glutamine rich) that attenuate optic nerve regeneration. These proteins were induced in a Jak/Stat-dependent manner, stimulated each other's expression and suppressed the expression of regeneration-associated genes. In vivo, the injury-dependent induction of Socs3 and Sfpq inhibits optic nerve regeneration but does not block it. We identified a robust induction of multiple cytokine genes in zebrafish retinal ganglion cells that may contribute to their ability to overcome these inhibitory factors. These studies not only identified mechanisms underlying optic nerve regeneration in fish but also suggest new molecular targets for enhancing optic nerve regeneration in mammals.

ZRICH, a protein induced during optic nerve regeneration in zebrafish, promotes neuritogenesis and interacts with tubulin

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.

Intermediate filaments of zebrafish retinal and optic nerve astrocytes and Müller glia: differential distribution of cytokeratin and GFAP

BackgroundOptic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.FindingsGFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.ConclusionsStudies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.

Changing Pax6 expression correlates with axon outgrowth and restoration of topography during optic nerve regeneration

Pax6, a member of the highly conserved developmental Pax gene family, plays a crucial role in early eye development and continues to be expressed in adult retinal ganglion cells (RGCs). Here we have used Western blots and immunohistochemistry to investigate the expression of Pax6 in the formation and refinement of topographic projections during optic nerve regeneration in zebrafish and lizard. In zebrafish with natural (12-h light/dark cycle) illumination, Pax6 expression in RGCs was decreased during axon outgrowth and increased during the restoration of the retinotectal map. Rearing fish in stroboscopic illumination to prevent retinotopic refinement resulted in a prolonged decrease in Pax6 levels; return to natural light conditions resulted in map refinement and restoration of normal Pax6 levels. In lizard, RGC axons spontaneously regenerate but remain in a persistent state of regrowth and do not restore topography; visual training during regeneration, however, allows a stabilization of connections and return of topography. Pax6 was persistently decreased in untrained animals but remained increased in trained ones. In both species, changes in expression were not due to cell division or cell death. The results suggest that decreased Pax6 expression is permissive for axon regeneration and extensive searching, while higher levels of Pax6 are associated with restoration of topography.

Characterization of the domains of zRICH, a protein induced during optic nerve regeneration in zebrafish

Teleost fish show a remarkable capability of nerve regeneration in their CNS, while injuries to axon fibers in the CNS of mammals result in degeneration and loss of function. Understanding this difference has biomedical consequences to humans. Both extrinsic factors from the neuronal environment and intrinsic neuronal factors seem to play a role in successful nerve regeneration. Among the intrinsic factors, a number of proteins termed axonal growth associated proteins (GAPs) are strongly induced during axon regeneration. RICH proteins are axonal GAPs that show homology to mammalian myelin marker proteins termed CNPases. Sequence analysis distinguishes three domains in these proteins. In this report, mutant versions of zebrafish RICH proteins were generated to study the roles of the domains of the protein at biochemical and cellular levels. The central CNPase homology domain was sufficient for catalytic activity. The amino terminal acidic domain causes the anomalous electrophoretic migration observed for RICH proteins. The small C-terminal domain bears an isoprenylation motif and is necessary for the interaction of zRICH with cellular membranes. At the cellular level, expression of wild-type zRICH protein in PC12 cells did not induce neurite generation. Additionally, neither the expression of wild-type zRICH nor the expression of mutant versions of the protein interfered with the levels of differentiation of PC12 cells induced by nerve growth factor, suggesting that, at least in this model of neuronal differentiation, zRICH proteins do not participate in the process of generation of neurites.

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration.

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.