Chikako Harada

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration.

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.

Dock3 Stimulates Axonal Outgrowth via GSK-3β-Mediated Microtubule Assembly

Dock3, a new member of the guanine nucleotide exchange factors, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes axonal outgrowth downstream of brain-derived neurotrophic factor (BDNF) signaling. We previously showed that Dock3 forms a complex with Fyn and WASP (Wiskott–Aldrich syndrome protein) family verprolin-homologous (WAVE) proteins at the plasma membrane, and subsequent Rac1 activation promotes actin polymerization. Here we show that Dock3 binds to and inactivates glycogen synthase kinase-3β (GSK-3β) at the plasma membrane, thereby increasing the nonphosphorylated active form of collapsin response mediator protein-2 (CRMP-2), which promotes axon branching and microtubule assembly. Exogenously applied BDNF induced the phosphorylation of GSK-3β and dephosphorylation of CRMP-2 in hippocampal neurons. Moreover, increased phosphorylation of GSK-3β was detected in the regenerating axons of transgenic mice overexpressing Dock3 after optic nerve injury. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the CNS, where it regulates cell polarity and promotes axonal outgrowth by stimulating dual pathways: actin polymerization and microtubule assembly.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

Dock3 attenuates neural cell death due to NMDA neurotoxicity and oxidative stress in a mouse model of normal tension glaucoma

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.

N-acetylated-α-linked-acidic dipeptidase inhibitor has a neuroprotective effect on mouse retinal ganglion cells after pressure-induced ischemia

Excessive glutamate receptor activation is thought to be involved in the retinal ganglion cell (RGC) death after ischemic injury. In this study, we examined the effect of 2-PMPA (2-(phosphonomethyl)pentanedioic acid) on RGC survival in an ischemia-reperfusion model using C57BL/6 mouse eyes. 2-PMPA is a NAALADase (N-acetylated-alpha-linked-acidic dipeptidase) inhibitor, an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. 100mg/kg 2-PMPA were given with intraperitoneal injections 30 min before ischemia followed per hour injection for 3h. 2-PMPA increased surviving RGCs as well as retinal thickness after pressure-induced retinal ischemia. In addition, neuroprotection afforded by 2-PMPA was greater than that of N-methyl-D-aspartate receptor blocker. These data indicate that NAALADase inhibition may be useful in retinal disorders in which excessive amino acid transmission is pathogenic.

Spermidine Alleviates Severity of Murine Experimental Autoimmune Encephalomyelitis

PURPOSEnTo assess the effects of spermidine on the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), with a focus on optic neuritis often associated with MS and EAE.nnnMETHODSnMyelin oligodendrocyte glycoprotein-induced EAE mice were administered with or without spermidine at 30 mM in drinking water for 25 days. Clinical signs of EAE were scored daily, and visual functions were measured by multifocal electroretinograms. Histopathology analysis of the spinal cord and optic nerve was performed after mice were killed on day 25. Hydrogen peroxide (H(2)O(2)) was detected using the probe 2-7 dichlorofluorescein diacetate (DCFDA) in the optic nerve. The effect of spermidine on H(2)O(2)-induced retinal ganglion cell apoptosis was investigated by lactate dehydrogenase assay.nnnRESULTSnDaily clinical scoring revealed that the severity of EAE was significantly attenuated in the spermidine-treated group, which was confirmed by milder demyelination and improved axon survival in the spinal cord of spermidine-treated mice. Visual functions were significantly improved in spermidine-treated mice compared with vehicle-treated mice. Spermidine treatment ameliorated the extent of demyelination in the optic nerve and prevented cell loss in the retinal ganglion cell layer. Furthermore, fewer DCFDA-labeled cells were found in the optic nerve in the spermidine-treated EAE mice, and in vitro analysis revealed that spermidine reduced H(2)O(2)-induced retinal ganglion cell apoptosis, suggesting that spermidine alleviated the severities of EAE, particularly of optic neuritis, by acting as an antioxidant.nnnCONCLUSIONSnThe results from this study suggest that oral spermidine administration could be a useful treatment for MS.

Dock3 regulates BDNF-TrkB signaling for neurite outgrowth by forming a ternary complex with Elmo and RhoG

Dock3, a new member of the guanine nucleotide exchange factor family, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes neurite outgrowth through the formation of a protein complex with Fyn and WAVE downstream of brain‐derived neurotrophic factor (BDNF) signaling. Here, we report a novel Dock3‐mediated BDNF pathway for neurite outgrowth. We show that Dock3 forms a complex with Elmo and activated RhoG downstream of BDNF‐TrkB signaling and induces neurite outgrowth via Rac1 activation in PC12 cells. We also show the importance of Dock3 phosphorylation in Rac1 activation and show two key events that are necessary for efficient Dock3 phosphorylation: membrane recruitment of Dock3 and interaction of Dock3 with Elmo. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the central nervous system where it stimulates actin polymerization by multiple pathways.

NF-κB in epiretinal membranes after human diabetic retinopathy

Aims/hypothesisFormation of epiretinal membranes (ERMs) in the posterior fundus results in progressive deterioration of vision. ERMs have been associated with numerous clinical conditions including proliferative diabetic retinopathy (PDR), but its pathogenic mechanisms are still unknown. This study was conducted to examine whether or not nuclear factor kappa B (NF-κB), a transcription factor that can be activated by various pathological conditions, is involved in the formation of ERMs after PDR.MethodsERM samples were obtained by vitrectomy from 22 cases with PDR aged 56±11 years with 18±10 years of diabetes and 15 cases with idiopathic ERM. They were processed for reverse transcription-polymerase chain reaction (RT-PCR) analysis. In addition, 5 ERM samples from PDR patients aged 51±16 years with 15±6 years of diabetes were processed for immunohistochemical analysis.ResultsNF-κB mRNA expression levels were higher (20 out of 22 cases vs. 9 out of 15 subjects in idiopathic ERM, p <0.05) in PDR subjects. Immunohistochemical analysis showed NF-κB protein expression in all the 5 ERMs derived from PDR patients, and that region was partially double-labelled with interleukin-8 (IL-8) and von Willebrand factor (vWF).Conclusions/interpretationThese results suggest a possibility that NF-κB is involved in the formation of ERMs after PDR, especially for the development of vascular endothelial cell component.

Activation of nuclear factor-kappa B in the conjunctiva with the epithelial scraping of the mouse cornea and human epidemic keratoconjunctivitis

Aim: To examine the expression of p65, one of nuclear factor-kappa B (NF-κB), in the conjunctival epithelium of the C57Bl6 mouse and a patient with epidemic keratoconjunctivitis (EKC). Methods: Normal and epithelial scraped cornea obtained 6 hours after the injury were processed for paraffin section. Samples of a normal and an EKC conjunctival epithelium were obtained using impression cytology. Both samples were analysed by immunocytochemistry using anti-p65 antibody. Results: Immunocytochemistry with the anti-NF-κB p65 antibody revealed that p65 was localised in the cytoplasm of the conjunctival epithelium in the C57Bl6 mouse without the treatment. Six hours after the scraping of the cornea, p65 protein was expressed in the nuclei of the conjunctival epithelium. p65 was localised in the cytoplasm of the conjunctival epithelium in the human normal eye. p65 protein was expressed in the nuclei of the conjunctival epithelial cells in the EKC patient. Conclusion: These findings suggest that NF-κB was activated in the conjunctiva in the epithelial scraping of the mouse cornea and in human EKC.

Distribution of p27(KIP1), cyclin D1, and proliferating cell nuclear antigen after retinal detachment

PurposeTo examine the expression of the p27(KIP1), cyclin D1, and proliferating cell nuclear antigen (PCNA) in the retina and retinal pigment epithelium (RPE) after retinal detachment.MethodsNormal eyes and eyes at 2 or 4xa0days after retinal detachment with the C57B16 mouse were analyzed by immunocytochemistry using anti-p27(KIP1), anti-cyclin D1, and anti-proliferating cell nuclear antigen (PCNA) antibodies as well as anti-glutamate synthetase (GS) antibody.ResultsThe p27(KIP1) positive nuclei were distributed in the inner nuclear layer (INL) and the RPE of the normal mice eye. In the INL, p27(KIP1) was detected in the middle sublayer, where the nuclei of glutamate synthetase positive Müller cells were situated. In contrast, cyclin D1 was not detected either in the retina or in the RPE. At 2 and 4xa0days after the retinal detachment, RPE cells under the detached retina were negative for p27(KIP1) and positive for cyclin D1 and PCNA. In the INL of the detached retina, p27(KIP1) was detected after 2xa0days, but was not detected after 4xa0days. In contrast, PCNA was not detected in the INL after 2xa0days, but was detected after 4xa0days. Cyclin D1 was detected in the middle sublayer of the INL at both 2 and 4xa0days after the retinal detachment.ConclusionThese results suggested that degradation of p27(KIP1) and expression of cyclin D1 was involved in the proliferation of the Müller cells as well as RPE cells after retinal detachment.