Yoshiki Koriyama

NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1−/−; Ngr2−/−; Ngr3−/−; which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1−/−; Ngr3−/−), but not Ngr1 and Ngr2 (Ngr1−/−; Ngr2−/−), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1−/−; Ngr3−/− mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.

The mature optic nerve cannot regenerate when injured, leaving victims of traumatic nerve damage or diseases such as glaucoma with irreversible visual losses. Recent studies have identified ways to stimulate retinal ganglion cells to regenerate axons part-way through the optic nerve, but it remains unknown whether mature axons can reenter the brain, navigate to appropriate target areas, or restore vision. We show here that with adequate stimulation, retinal ganglion cells are able to regenerate axons the full length of the visual pathway and on into the lateral geniculate nucleus, superior colliculus, and other visual centers. Regeneration partially restores the optomotor response, depth perception, and circadian photoentrainment, demonstrating the feasibility of reconstructing central circuitry for vision after optic nerve damage in mature mammals.

Protective effect of lipoic acid against oxidative stress is mediated by Keap1/Nrf2-dependent heme oxygenase-1 induction in the RGC-5 cellline

Oxidative stress plays a key role in neurodegeneration of CNS neurons such as in Alzheimer disease, Parkinsons disease and glaucoma. R-α-lipoic acid (R-LA) has been shown to have a neuroprotective effect through its antioxidant activity. However, the mechanism underlying its neuroprotection is totally unknown in retinal neurons. In this study, we show that R-LA has a dramatic neuroprotective effect against oxidative stress-induced death of the retinal neuronal RGC-5 cell line. We observed that R-LA induces the expression of heme oxygenase-1 (HO-1) by promoting the translocation of NF-E2-related factor 2 (Nrf2) to the nucleus. We examined the mechanism underlying HO-1 induction by R-LA by focusing on downstream signaling pathways. We found that R-LA activates Akt, and HO-1 induction by R-LA (involving Nrf2 translocation to the nucleus) was suppressed by phosphoinositide 3-kinase (PI3K) inhibitors. In addition, R-LA produced reactive oxygen species (ROS), including hydrogen peroxide. Pretreatment with a ROS scavenger or a NADPH oxidase inhibitor suppressed R-LA-induced Nrf2 translocation to the nucleus and HO-1 induction. These results suggest that ROS production triggered by R-LA might modify Kelch-like ECH-associated protein (Keap1), which in turn induces HO-1 expression through the PI3K signaling pathway. Furthermore, R-LA significantly attenuated cell death and accumulation of 4-hydroxy-2-nonenal (4HNE) in the retina induced by optic nerve injury in vivo through an HO-1 activity-dependent mechanism. These data demonstrate for the first time that R-LA exerts a neuroprotective effect against oxidative stress in retinal neurons in vitro and in vivo by inducing HO-1 through Keap1/Nrf2 signaling.

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

J. Neurochem. (2010) 115, 79–91.

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.

Role of Purpurin as a Retinol-Binding Protein in Goldfish Retina during the Early Stage of Optic Nerve Regeneration: Its Priming Action on Neurite Outgrowth

Unlike mammals, the fish optic nerve can regenerate after injury. So far, many growth or trophic factors have been shown as an axon-regenerating molecule. However, it is totally unknown what substance regulates or triggers the activity of these factors on axonal elongation. Therefore, we constructed a goldfish retina cDNA library prepared from the retina treated with optic nerve transection 5 d previously, when it was just before regrowing optic axons after injury. A cDNA clone for goldfish purpurin for which expression was upregulated during the early stage of optic nerve regeneration was isolated from the retina cDNA library. Purpurin was discovered as a secretory retinol-binding protein in developing chicken retinas. Levels of purpurin mRNA and protein transiently increased and rapidly decreased 2–5 d and 10 d after axotomy, respectively. Purpurin mRNA was localized to the photoreceptor cells, whereas the protein was diffusely found in all of the retinal layers. A recombinant purpurin alone did not affect any change of neurite outgrowth in explant culture of the control retina, whereas a concomitant addition of the recombinant purpurin and retinol first induced a drastic enhancement of neurite outgrowth. Furthermore, the action of retinol-bound purpurin was effective only in the control (untreated) retinas but not in those primed (treated) with a previous optic nerve transection. Thus, purpurin with retinol is the first candidate molecule of priming neurite outgrowth in the early stage 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.

Upregulation of retinal transglutaminase during the axonal elongation stage of goldfish optic nerve regeneration

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1-2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG(R)) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG(R) was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG(R) mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG(R) protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG(R) inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG(R) protein decreased in rat RGCs within 1-3 days after nerve injury. Furthermore, the addition of recombinant TG(R) to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG(R) promotes axonal elongation at the surface of injured RGCs after optic nerve injury.

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

N-beta-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 9-12h. 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 12-24h. 5-S-GAD at 25-50 microM 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.

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.