Neuroprotective effects of 5-S-GAD against oxidative stress-induced apoptosis in RGC-5 cell.

金沢大学医薬保健研究域　医学系N-β-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, has been described as having multipotential biological activities toward various tissues. In a previous paper, we reported a novel neuroprotective action of 5-S-GAD on rat retinal ganglion cell apoptosis induced by optic nerve injury and intraocular N-methyl-d-aspartate treatment in vivo. In the present study, we further investigated the protective mechanism of this small peptide against other types of apoptosis in cultured cells of the established rat retinal ganglion cell line RGC-5. Hydrogen peroxide and serum deprivation treatments induced intracellular reactive oxygen species levels and lipid peroxidation, revealed by 4-hydroxy-2-nonenal production, in RGC-5 cells within 9-12 h. The treatments also induced cell death accompanied by nuclear condensation, DNA laddering and increases in apoptotic Bax and caspase-3 proteins in RGC-5 cells within 12-24 h. 5-S-GAD at 25-50 μM clearly suppressed the cell death and apoptotic features induced by these treatments. 5-S-GAD restored the nuclear condensation, DNA laddering and increases in apoptotic proteins. Furthermore, 5-S-GAD directly activated anti-apoptotic phospho-Akt and Bcl-2 proteins in RGC-5 cells. 5-S-GAD also quenched the reactive oxygen species production and inhibited the lipid peroxidation induced by oxidative stress. Therefore, 5-S-GAD may complementarily protect RGC-5 cells against apoptosis through dual actions as a radical scavenger and an inducer of anti-apoptotic phospho-Akt and Bcl-2. Taken together, 5-S-GAD is a high-potential tool for rescuing the retinal ganglion cell apoptosis induced by a variety of glaucomatous conditions. Crown Copyright © 2009

A possible role of neuroglobin in the retina after optic nerve injury: A comparative study of zebrafish and mouse retina

Neuroglobin (Ngb) is a new member of the family of heme proteins and is specifically expressed in neurons of the central and peripheral nervous systems in all vertebrates. In particular, the retina has a 100-fold higher concentration of Ngb than do other nervous tissues. The role of Ngb in the retina is yet to be clarified. Therefore, to understand the functional role of Ngb in the retina after optic nerve injury (ONI), we used two types of retina, from zebrafish and mice, which have permissible and non-permissible capacity for nerve regeneration after ONI, respectively. After ONI, the Ngb protein in zebrafish was upregulated in the amacrine cells within 3 days, whereas in the mouse retina, Ngb was downregulated in the retinal ganglion cells (RGCs) within 3 days. Zebrafish Ngb (z-Ngb) significantly enhanced neurite outgrowth in retinal explant culture. According to these results, we designed an overexpression experiment with the mouse Ngb (m-Ngb) gene in RGC-5 cells (retinal precursor cells). The excess of m-Ngb actually rescued RGC-5 cells under hypoxic conditions and significantly enhanced neurite outgrowth in cell culture. These data suggest that mammalian Ngb has positive neuroprotective and neuritogenic effects that induce nerve regeneration after ONI. © Springer International Publishing Switzerland 2016.[Book Chapter

A novel neuroprotective role of a small peptide from flesh fly, 5-S-GAD in the rat retina in vivo.

金沢大学医薬保健研究域　医学系N-β-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from flesh fly, has been described as having multipotential biological activities toward various tissues. However, there has been no report testing its action on neural cells. In the present study, we investigate whether 5-S-GAD is neurotoxic or neuroprotective to the rat retina. 5-S-GAD at high doses (more than 200 pmol) induced apoptosis of retinal neurons 7 days after intraocular injection. 5-S-GAD at low doses (2-20 pmol) significantly attenuated the loss of retinal ganglion cells (RGCs) and the thinning of inner retina induced by NMDA in a dose-dependent manner. To understand the protective mechanism of 5-S-GAD, we investigated the influence of 5-S-GAD on the cell survival molecules, phospho-Akt and Bcl-2. 5-S-GAD (2-20 pmol) rapidly increased phospho-Akt expression 1-7 days and Bcl-2 expression 3-7 days after injection. The cellular localization of this increase was both in bipolar cells and RGCs. This neurosurvival effect of 5-S-GAD was further tested using another model of optic nerve injury. 5-S-GAD significantly blocked the apoptosis of RGCs 7 days after optic nerve crush. These results show that 5-S-GAD (2-20 pmol) protects against the NMDA- and optic nerve crush-induced apoptosis of RGCs. The neuroprotective action of 5-S-GAD in the retina might be mediated by the cell survival phospho-Akt/Bcl-2 system and offers a therapeutic option to rescue RGCs from various types of excitotoxic disease, such as glaucoma. © 2008 Elsevier B.V. All rights reserved

Cell fate of müller cells during photoreceptor regeneration in an N-methyl-N-nitrosourea- induced retinal degeneration model of zebrafish

Zebrafish can regenerate several organs such as the tail fin, heart, central nervous system, and photoreceptors. Very recently, a study has demonstrated the photoreceptor regeneration in the alkylating agent N-methyl-N-nitrosourea (MNU)- induced retinal degeneration (RD) zebrafish model, in which whole photoreceptors are lost within a week after MNU treatment and then regenerated within a month. The research has also shown massive proliferation of Müller cells within a week. To address the question of whether proliferating Müller cells are the source of regenerating photoreceptors, which remains unknown in the MNU-induced zebrafish RD model, we employed a BrdU pulse-chase technique to label the proliferating cells within a week after MNU treatment. As a result of the BrdU pulse-chase technique, a number of BrdU+ cells were observed in the outer nuclear layer as well as the inner nuclear layer. This implies that regenerating photoreceptors are derived from proliferating Müller cells in the zebrafish MNU-induced RD model. © Springer International Publishing Switzerland 2016.[Book Chapter

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 5 dpl. The results of this study suggest a role of Sox2 as one of the proliferation initiators in ependymal cells after SCI. © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society.12 months Embargo Perio

Long-acting genipin derivative protects retinal ganglion cells from oxidative stress models in vitro and in vivo through the Nrf2/antioxidant response element signaling pathway

金沢大学医薬保健研究域医学系金沢大学理工研究域電子情報学系Previously, we reported that genipin, a herbal iridoid, had neuritogenic and neuroprotective actions on PC12 cells. Although nitric oxide (NO)-activated signalings were proposed to be neuritogenic, the neuroprotective action of genipin remains to be elucidated. From the standpoint of NO activation, we tested a possible protective mechanism through the nitrosative Kelch-like ECH-associated protein (Keap1)/NF-E2-related factor 2 (Nrf2)-antioxidant response element pathway in rat retinal ganglion cells (RGC-5 cells) in culture, and in vivo, against hydrogen peroxide and optic nerve injury (ONI), respectively, using a long-acting (1R)-isoPropyloxygenipin (IPRG001). IPRG001 induced NO generation and the expressions of antioxidative enzymes, such as heme oxygenase-1 (HO-1), in RGC-5 cells. The protective action of IPRG001 depended on HO-1 and NO induction. We found that S-nitrosylation of Keap1 by IPRG001 may contribute to translocation of Nrf2 to the nucleus and triggered transcriptional activation of antioxidative enzymes. Furthermore, apoptotic cells were increased and 4-hydroxy-2-nonenal was accumulated in rat retina following ONI. Pre-treatment with IPRG001 almost completely suppressed apoptosis and accumulation of 4-hydroxy-2-nonenal in RGCs following ONI accompanied by HO-1 induction. These data demonstrate for the first time that IPRG001 exerts neuroprotective action in RGCs in vitro and in vivo, through the Nrf2/antioxidant response element pathway by S-nitrosylation against oxidative stress. © 2010 International Society for Neurochemistry

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. © 2012 Elsevier Ltd. All rights reserved

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. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

金沢大学医薬保健研究域　医学系Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury. © 2007 Elsevier Ltd. All rights reserved

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

Unlike mammals, fish motor function can recover within 6-8 weeks 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 1-15 days 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 1-6 days 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 10-15 days and then crossed at 4-6 weeks after SCI. These long-distance descending axons from originally axotomized neurons have a major role in restoration of motor function after SCI. © 2012 Elsevier Ltd. All rights reserved.Thesis of Kazuhiro Ogai / 大貝 和裕 博士論文 金沢大学医薬保健学総合研究科（保健学専攻