Kazuhiro Ogai

A molecular mechanism of optic nerve regeneration in fish: the retinoid signaling pathway.

The fish optic nerve regeneration process takes more than 100 days after axotomy and comprises four stages: neurite sprouting (1-4 days), axonal elongation (5-30 days), synaptic refinement (35-80 days) and functional recovery (100-120 days). We screened genes specifically upregulated in each stage from axotomized fish retina. The mRNAs for heat shock protein 70 and insulin-like growth factor-1 rapidly increased in the retinal ganglion cells soon after axotomy and function as cell-survival factors. Purpurin mRNA rapidly and transiently increased in the photoreceptors and purpurin protein diffusely increased in all nuclear layers at 1-4 days after injury. The purpurin gene has an active retinol-binding site and a signal peptide. Purpurin with retinol functions as a sprouting factor for thin neurites. This neurite-sprouting effect was closely mimicked by retinoic acid and blocked by its inhibitor. We propose that purpurin works as a retinol transporter to supply retinoic acid to damaged RGCs which in turn activates target genes. We also searched for genes involved in the second stage of regeneration. The mRNA of retinoid-signaling molecules increased in retinal ganglion cells at 7-14 days after injury and tissue transglutaminase and neuronal nitric oxide synthase mRNAs, RA-target genes, increased in retinal ganglion cells at 10-30 days after injury. They function as factors for the outgrowth of thick, long neurites. Here we present a retinoid-signaling hypothesis to explain molecular events during the early stages of optic nerve regeneration in fish.

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration.

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.

Anti-inflammatory effects of lipoic acid through inhibition of GSK-3β in lipopolysaccharide-induced BV-2 microglial cells

Activated microglial cells play an important role in immune and inflammatory responses in CNS and play a role in neurodegenerative diseases. We examined the effects of lipoic acid (LA) on inflammatory responses of BV-2 microglial cells activated by lipopolysaccharide (LPS), and explored the underlying mechanisms of action of LA. BV-2 cells treated with LPS showed an up-regulation of mRNA of the pro-inflammatory molecules, inducible nitric oxide synthase (iNOS). LA suppressed the expression of iNOS and furthermore, LPS-induced production of nitrite. Moreover, LA suppressed the nuclear translocation of RelA, a component of nuclear factor-kappa B (NF-κB) that contains transcriptional activator domain for LPS. The mechanisms of LA-mediated anti-inflammatory effects on microglia remain unknown, and we suggested an involvement of Akt/glycogen synthase kinase-3β (GSK-3β) phosphorylation. The results showed that inhibitor of phosphatidylinositol 3-kinase prevented LA-mediated suppression of LPS induction of RelA and expression of iNOS. Furthermore, these inflammatory actions were prevented by GSK-3β inhibitors. These data demonstrate a role for LA as a chemical modulator of inflammatory responses by microglia, and thus may be a therapeutic strategy for treating neurodegenerative diseases with an inflammatory component.

Protective action of nipradilol mediated through S-nitrosylation of Keap1 and HO-1 induction in retinal ganglion cells.

Nipradilol (Nip), which has α1- and β-adrenoceptor antagonist and nitric oxide (NO)-donating properties, has clinically been used as an anti-glaucomatous agent in Japan. NO mediates cellular signaling pathways that regulate physiological functions. The major signaling mechanisms mediated by NO are cGMP-dependent signaling and protein S-nitrosylation-dependent signalings. Nip has been described as having neuroprotective effects through cGMP-dependent pathway in retinal ganglion cells (RGCs). However, the effect seems to be partial. On the other hand, whether Nip can prevent cell death through S-nitrosylation is not yet clarified. In this study, we therefore focused on the neuroprotective mechanism of Nip through S-nitrosylation. Nip showed a dramatic neuroprotective effect against oxidative stress-induced death of RGC-5 cells. However, denitro-nipradilol, which does not have NO-donating properties, was not protective against oxidative stress. Furthermore, an NO scavenger significantly reversed the protective action of Nip against oxidative stress. In addition, we demonstrated that α1- or β-adrenoceptor antagonists (prazosin or timolol) did not show any neuroprotective effect against oxidative stress in RGC-5 cells. We also demonstrated that Nip induced the expression of the NO-dependent antioxidant enzyme, heme oxygenase-1 (HO-1). S-nitrosylation of Kelch-like ECH-associated protein by Nip was shown to contribute to the translocation of NF-E2-related factor 2 to the nucleus, and triggered transcriptional activation of HO-1. Furthermore, RGC death and levels of 4-hydroxy-2-nonenal (4HNE) were increased after optic nerve injury in vivo. Pretreatment with Nip significantly suppressed RGC death and accumulation of 4HNE after injury through an HO-1 activity-dependent mechanism. These data demonstrate a novel neuroprotective action of Nip against oxidative stress-induced RGC death in vitro and in vivo.

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.

Heat shock protein 70 induction by valproic acid delays photoreceptor cell death by N‐methyl‐N‐nitrosourea in mice

Retinal degenerative diseases (RDs) are a group of inherited diseases characterized by the loss of photoreceptor cells. Selective photoreceptor loss can be induced in mice by an intraperitoneal injection of N‐methyl‐N‐nitrosourea (MNU) and, because of its selectivity, this model is widely used to study the mechanism of RDs. Although it is known that calcium‐calpain activation and lipid peroxidation are involved in the initiation of cell death, the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule to protect cells against environmental and physiological stresses. In this study, we investigated the role of HSP70 on photoreceptor cell death in mice. HSP70 induction by valproic acid, a histone deacetylase inhibitor, attenuated the photoreceptor cell death by MNU through inhibition of apoptotic caspase signals. Furthermore, HSP70 itself was rapidly and calpain‐dependently cleaved after MNU treatment. Therefore, HSP70 induction by valproic acid was dually effective against MNU‐induced photoreceptor cell loss as a result of its anti‐apoptotic actions and its ability to prevent HSP70 degradation. These findings might help lead us to a better understanding of the pathogenic mechanism of RDs.

Upregulation of anti-apoptotic factors in upper motor neurons after spinal cord injury in adult zebrafish

Unlike mammals, fish motor function can recover within 6-8weeks after spinal cord injury (SCI). The motor function of zebrafish is regulated by dual control; the upper motor neurons of the brainstem and motor neurons of the spinal cord. In this study, we aimed to investigate the framework behind the regeneration of upper motor neurons in adult zebrafish after SCI. In particular, we investigated the cell survival of axotomized upper motor neurons and its molecular machinery in zebrafish brain. As representative nuclei of upper motor neurons, we retrogradely labeled neurons in the nucleus of medial longitudinal fasciculus (NMLF) and the intermediate reticular formation (IMRF) using a tracer injected into the lesion site of the spinal cord. Four to eight neurons in each thin sections of the area of NMLF and IMRF were successfully traced at least 1-15days after SCI. TUNEL staining and BrdU labeling assay revealed that there was no apoptosis or cell proliferation in the axotomized neurons of the brainstem at various time points after SCI. In contrast, axotomized neurons labeled with a neurotracer showed increased expression of anti-apoptotic factors, such as Bcl-2 and phospho-Akt (p-Akt), at 1-6days after SCI. Such a rapid increase of Bcl-2 and p-Akt protein levels after SCI was quantitatively confirmed by western blot analysis. These data strongly indicate that upper motor neurons in the NMLF and IMRF can survive and regrow their axons into the spinal cord through the rapid activation of anti-apoptotic molecules after SCI. The regrowing axons from upper motor neurons reached the lesion site at 10-15days and then crossed at 4-6weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI.

Function of Sox2 in ependymal cells of lesioned spinal cords in adult zebrafish.

The sex-determining region Y-box 2 (Sox2) is related not only to pluripotency, but also to cell proliferation. Zebrafish can regain their motor function after spinal cord injury (SCI). Following SCI, new motor neurons are produced from proliferating ependymal cells. Here, we investigated the expression and function of Sox2 after SCI in zebrafish. Sox2 was upregulated as early as 1 day post-lesion (dpl) in ependymal cells, which was followed by cell proliferation. Sox2 knockdown significantly decreased the number of proliferating cells at 5dpl. The results of this study suggest a role of Sox2 as one of the proliferation initiators in ependymal cells after SCI.

Zebrafish scales respond differently to in vitro dynamic and static acceleration: Analysis of interaction between osteoblasts and osteoclasts

Zebrafish scales consist of bone-forming osteoblasts, bone-resorbing osteoclasts, and calcified bone matrix. To elucidate the underlying molecular mechanism of the effects induced by dynamic and static acceleration, we investigated the scale osteoblast- and osteoclast-specific marker gene expression involving osteoblast-osteoclast communication molecules. Osteoblasts express RANKL, which binds to the osteoclast surface receptor, RANK, and stimulates bone resorption. OPG, on the other hand, is secreted by osteoblast as a decoy receptor for RANKL, prevents RANKL from binding to RANK and thus prevents bone resorption. Therefore, the RANK-RANKL-OPG pathway contributes to the regulation of osteoclastogenesis by osteoblasts. Semaphorin 4D, in contrast, is expressed on osteoclasts, and binding to its receptor Plexin-B1 on osteoblasts results in suppression of bone formation. In the present study, we found that both dynamic and static acceleration at 3.0×g decreased RANKL/OPG ratio and increased osteoblast-specific functional mRNA such as alkaline phosphatase, while static acceleration increased and dynamic acceleration decreased osteoclast-specific mRNA such as cathepsin K. Static acceleration increased semaphorin 4D mRNA expression, while dynamic acceleration had no effect. The results of the present study indicated that osteoclasts have predominant control over bone metabolism via semaphorin 4D expression induced by static acceleration at 3.0×g.

Functional characterization of fish neuroglobin: zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.