Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization.

OBJECTIVE: The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis. APPROACH AND RESULTS: We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. CONCLUSIONS: These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo.

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases.

A Docosahexaenoic Acid-Derived Pro-resolving Agent, Maresin 1, Protects Motor Neuron Cells Death.

Maresin 1 is a novel pro-resolving mediator derived from docosahexaenoic acid (DHA), with potent anti-inflammation effects against several animal models, including brain ischemia, sepsis, and lung fibrosis. However, its effect against motor neuron cell death is still not investigated. Therefore, we investigated the effects of maresin 1 on several stress-induced motor neuron cell death. Maresin 1 suppressed combinatorial stress which was evoked by superoxide dismutase 1 (SOD1)G93A and serum-free, -induced motor neuron cells death in a concentration-dependent manner, and had a stronger neuroprotective effective than DHA. Maresin 1 also had neuroprotective effects against transactivation response DNA-binding protein (TDP)-43A315T and serum-free stress, H2O2, and tunicamycin-induced cell death. Maresin 1 reduced the reactive oxygen species (ROS) production caused by SOD1G93A or TDP-43A315T. Moreover, maresin 1 suppressed the NF-κB activation induced by SOD1G93A and serum-free stress. These data indicate that maresin 1 has motor neuron protective effects against several stresses by reduction of ROS production or attenuation of the NF-κB activation. Maresin 1 also had neuroprotective effects against H2O2, and tunicamycin-induced cell death in a concentration-dependent manner. Finally, maresin 1 ameliorated the motor function deficits of spinal muscular atrophy model in which endoplasmic reticulum stress was upregulated. Thus, maresin 1 may be beneficial to protect against motor neuron diseases.

RS9, a novel Nrf2 activator, attenuates light-induced death of cells of photoreceptor cells and Müller glia cells.

The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration, a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661W cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element binding was increased in 661W cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes was increased in 661W cells after exposure to RS9. Furthermore, RS9 decreased the light-induced death of 661W cells (2500 lux, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer or the retina in the in vivo studies (8000 lux, 3 h). Heme oxygenase-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661W cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure.

Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc.

GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc.

The involvement of ATF4 and S-opsin in retinal photoreceptor cell damage induced by blue LED light.

PURPOSE: Blue light is a high-energy emitting light with a short wavelength in the visible light spectrum. Blue light induces photoreceptor apoptosis and causes age-related macular degeneration or retinitis pigmentosa. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress induced by blue light-emitting diode (LED) light exposure in murine photoreceptor cells. METHODS: The murine photoreceptor cell line was incubated and exposed to blue LED light (464 nm blue LED light, 450 lx, 3 to 24 h). The expression of the factors involved in the unfolded protein response pathway was examined using quantitative real-time reverse transcription (RT)-PCR and immunoblot analysis. The aggregation of short-wavelength opsin (S-opsin) in the murine photoreceptor cells was observed with immunostaining. The effect of S-opsin knockdown on ATF4 expression in the murine photoreceptor cell line was also investigated. RESULTS: Exposure to blue LED light increased the bip, atf4, and grp94 mRNA levels, induced the expression of ATF4 protein, and increased the levels of ubiquitinated proteins. Exposure to blue LED light in combination with ER stress inducers (tunicamycin and dithiothreitol) induced the aggregation of S-opsin. S-opsin mRNA knockdown prevented the induction of ATF4 expression in response to exposure to blue LED light. CONCLUSIONS: These findings indicate that the aggregation of S-opsin induced by exposure to blue LED light causes ER stress, and ATF4 activation in particular.

Toll-like receptor 4 inhibitor protects against retinal ganglion cell damage induced by optic nerve crush in mice.

Toll-like receptor 4 (TLR4) plays key roles in innate immune responses and inflammatory reactions. TAK-242 (resatorvid) is a small-molecule cyclohexene derivative that selectively inhibits TLR4 signaling pathways and suppresses inflammatory reactions. Here we investigated the protective effects of TAK-242 against optic nerve crush (ONC) which induces axonal injury like glaucoma in mice. TAK-242 was injected intravitreally immediately after ONC. The effect of TAK-242 was evaluated by measuring the number of fluorogold-labeled retinal ganglion cells (RGCs) at 10 days after ONC. Furthermore, the expression levels of phosphorylated-nuclear factor-kappa B (p-NF-κB) and phosphorylated-p38 (p-p38) were measured by Western blotting. In addition, we examined activated astrocytes by immunostaining. TAK-242 significantly abrogated the loss of RGCs associated with ONC. Moreover, the expression levels of p-NF-κB and p-p38 were significantly reduced by TAK-242 treatment. Furthermore, TAK-242 and C34, a TLR4 inhibitor, significantly reduced astrocyte activation in the ganglion cell and inner plexiform layers, compared with vehicle treatment. These findings indicate that TAK-242 inhibits not only the TLR4 signaling pathway but also astrocyte activation downstream of this pathway, suggesting that the inhibition of TLR4 signaling is a promising candidate for the treatment of glaucoma.

Astaxanthin analogs, adonixanthin and lycopene, activate Nrf2 to prevent light-induced photoreceptor degeneration.

Carotenoids, in particular astaxanthin, possess potent antioxidant capabilities. Astaxanthin also induces NF-E2-related factor 2 (Nrf2), which plays a major regulatory role in the antioxidative response. However, little is known whether the carotenoid, by-products of astaxanthin, activate Nrf2. Toward this end, we screened eight astaxanthin analogs for Nrf2 activation in murine photoreceptor cell line, 661 W, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In addition, we monitored cell death in 661 W cells pretreated with astaxanthin analogs or only pretreated for 6 h with astaxanthin analogs and then exposed to light. Furthermore, we quantified the reactive oxygen species (ROS) production. Cell death was quantified after light exposure by nuclear staining. Nrf2-controlled genes Ho-1, Nqo-1, and Gclm by qRT-PCR and Nrf2 in the nucleus were upregulated in 661 W cells exposed astaxanthin, adonixanthin, echinenone, and lycopene. Moreover, astaxanthin, adonixanthin, echinenone, ß-carotene, adonirubin, and lycopene, but not canthaxanthin, suppressed ROS production and protected cells against light-induced damage. Moreover, pretreatment with adonixanthin or lycopene only before light exposure protected against light-induced cell damage and Nrf2 silencing canceled these effects. These findings indicate that the more potent astaxanthin analogs, adonixanthin and lycopene, protect against light-induced cell damage through not only an anti-oxidative response but also through Nrf2 activation.

The Involvement of the Oxidative Stress in Murine Blue LED Light-Induced Retinal Damage Model.

The aim of study was to establish a mouse model of blue light emitting diode (LED) light-induced retinal damage and to evaluate the effects of the antioxidant N-acetylcysteine (NAC). Mice were exposed to 400 or 800 lx blue LED light for 2 h, and were evaluated for retinal damage 5 d later by electroretinogram amplitude and outer nuclear layer (ONL) thickness. Additionally, we investigated the effect of blue LED light exposure on shorts-wave-sensitive opsin (S-opsin), and rhodopsin expression by immunohistochemistry. Blue LED light induced light intensity dependent retinal damage and led to collapse of S-opsin and altered rhodopsin localization from inner and outer segments to ONL. Conversely, NAC administered at 100 or 250 mg/kg intraperitoneally twice a day, before dark adaptation and before light exposure. NAC protected the blue LED light-induced retinal damage in a dose-dependent manner. Further, blue LED light-induced decreasing of S-opsin levels and altered rhodopsin localization, which were suppressed by NAC. We established a mouse model of blue LED light-induced retinal damage and these findings indicated that oxidative stress was partially involved in blue LED light-induced retinal damage.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) promotes the progression of brain glioblastoma via Na+/K+-ATPase.

Glycoprotein nonmetastatic melanoma protein B (GPNMB), which is involved in invasion and metastasis, was found to be overexpressed in various cancers. High levels of GPNMB and Na+/K+-ATPase α subunits are associated with a poor prognosis in glioblastoma patients. We showed that GPNMB interacts with Na+/K+-ATPase α subunits to activate PI3K/Akt and MEK/ERK pathways. However, it remains unclear whether the interaction of GPNMB and Na+/K+-ATPase α subunits is involves in progression of glioma. The tumor size induced by the injection of glioma GL261 cells was larger in transgenic mice overexpressing GPNMB when compared with wild-type mice. Additionally, the interaction of GPNMB and Na+/K+-ATPase α subunits was identified in the murine glioma model and in the tumors of glioblastoma patients. Ouabain, a Na+/K+-ATPase inhibitor, suppressed the glioma growth induced by the injection of glioma cells in the transgenic mice overexpressing GPNMB and blocked the GPNMB-induced migration of glioma cells. These findings indicate that GPNMB promotes glioma growth via Na+/K+-ATPase α subunits. Thus, the interaction between GPNMB and Na+, K+-ATPase α subunits represents a novel therapeutic target for the treatment of brain glioblastomas.

Edaravone is a free radical scavenger that protects against laser-induced choroidal neovascularization in mice and common marmosets.

Choroidal neovascularization (CNV) is a main characteristic in exudative type of age-related macular degeneration (AMD). Our study aimed to evaluate the effects of edaravone, a free radical scavenger on laser-induced CNV. CNV was induced by laser photocoagulation to the subretinal choroidal area of mice and common marmosets. Edaravone was administered either intraperitoneally twice a day for 2 weeks or intravenously just once after laser photocoagulation. The effects of edaravone on laser-induced CNV were evaluated by fundus fluorescein angiography, CNV area measurements, and the expression of 4-hydroxy-2-nonenal (4-HNE) modified proteins, a marker of oxidative stress. Furthermore, the effects of edaravone on the production of H2O2-induced reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF)-induced cell proliferation were evaluated using human retinal pigment epithelium cells (ARPE-19) and human retinal microvascular endothelial cells, respectively. CNV areas in the edaravone-treated group were significantly smaller in mice and common marmosets. The expression of 4-HNE modified proteins was upregulated 3 h after laser photocoagulation, and intravenously administered edaravone decreased it. In in vitro studies, edaravone inhibited H2O2-induced ROS production and VEGF-induced cell proliferation. These findings suggest that edaravone may protect against laser-induced CNV by inhibiting oxidative stress and endothelial cell proliferation.

Establishment of a drug evaluation model against light-induced retinal degeneration using adult pigmented zebrafish.

Age-related macular degeneration (AMD) is a major cause of irreversible loss of central vision in the elderly. Zebrafish is an attractive animal model in some respects, lower cost, smaller housing facilities and easier genetic manipulation compared to rodents. The present study aimed to establish a drug evaluation method against light irradiation, as a dry AMD disease model, using adult pigmented zebrafish. Intravitreal administration of an antioxidant, N-acetylcysteine, protected against light-induced retinal degeneration in a concentration-dependent manner. We established a new drug evaluation model against light-induced retinal degeneration that can provide new knowledge about dry AMD pathology and therapy.

Neuroprotective Effect of Ocular Hypotensive Drugs: Latanoprost/Timolol in Combination Are More Effective than Each as Monotherapy in RGC-5.

The combination of timolol and latanoprost, which are ocular hypotensive agents, has a greater ocular hypotensive effect than each as monotherapy. However, the protective effect of the combination is not well understood. In the present study, we investigated whether latanoprost/timolol in combination has an additive or synergistic cytoprotective effect on neuro retinal cells (RGC-5). To investigate the protective effects of timolol/latanoprost in combination, cultured RGC-5 were treated with various concentrations of these two agents, singly or together, after which the cells were exposed to oxidative stress, serum deprivation, or endoplasmic reticulum (ER) stress in vitro. Cells were also treated with an Akt inhibitor, LY294002, to examine the mechanism of the protective effect. Latanoprost, timolol, and the two in combination reduced cell death induced by oxidative stress, serum deprivation, or ER stress. The latanoprost/timolol combination reduced cell death to a greater extent than monotherapy with latanoprost or timolol on serum deprivation only, and LY294002 inhibited the protective effect of their combination. These findings suggest that timolol/latanoprost in combination have a protective effect against serum deprivation only by activation of Akt signaling. Furthermore, this combination has not only an ocular hypotensive effect but also a neuroprotective effect.

Analysis and characterization of anthocyanins and carotenoids in Japanese blue tomato.

Tomato (Solanum lycopersicum) is rich in anthocyanins, which are polyphenolic pigments. This study aimed to analyze and characterize the anthocyanin composition in cultivated blue tomato in Japan. The extracts of peel, seed, and pulp of tomatoes were purified following which anthocyanins and lycopene contents were analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry. Eleven types of anthocyanins were identified, including delphinidin, petunidin, and malvidin. Further, the antioxidant activity of anthocyanins was evaluated using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt radical quenching assays and electron spin resonance. "Blue tomato" extracts exert antioxidant activity. Thus, we showed that petunidin was present in the "blue tomato" peel while lycopene was present in the peel and pulp. Additionally, the blue tomato peel extract was found to significantly inhibit H2O2-induced cell death in vitro. This is the first study on cell protective effects of Japanese blue tomato extract and petunidin in murine photoreceptor cells.

The extracellular fragment of GPNMB (Glycoprotein nonmelanosoma protein B, osteoactivin) improves memory and increases hippocampal GluA1 levels in mice.

Glycoprotein nonmelanoma protein B (GPNMB, alias osteoactivin), a type I transmembrane glycoprotein, is cleaved by extracellular proteases, resulting in release of an extracellular fragment (ECF). GPNMB is widely expressed by neurons within the CNS, including the hippocampus; however, its function in the brain remains unknown. Here, we investigated the role of GPNMB in memory and learning by using transgenic (Tg) mice over-expressing GPNMB (Tg mice on a BDF-1 background) and ECF-treated mice. In the hippocampus of both wild-type and Tg mice, GPNMB was highly expressed in neurons and astrocytes. Tg mice exhibited memory improvements in two types of learning tasks but were impaired in a passive-avoidance test. In Tg mice, the hippocampus displayed increased levels of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluA1. Intracerebroventricular administration of ECF (50 ng) to Institute of Cancer Research (ICR) mice also improved memory in a passive-avoidance test and increased hippocampal GluA1 levels 24 h after treatment. In Tg mice and ECF (0.25 µg/mL)-treated hippocampal slices, long-term potentiation was promoted. These findings suggest that GPNMB may be a novel target for research on higher order brain functions.

Involvement of endoplasmic reticulum stress in optic nerve degeneration after chronic high intraocular pressure in DBA/2J mice.

DBA/2J mice are one of several animal strains used for experimental models of both intraocular hypertension and glaucoma. This study investigates the relationship between endoplasmic reticulum (ER) stress and optic nerve degeneration in DBA/2J mice. Intraocular pressure (IOP) was measured in DBA/2J mice between the ages of 6 and 15 months. Optic nerve damage was assessed at 15 months of age. The nerve was immunostained with antibodies to either neurofilament heavy chain (NFH) or phosphorylated NFH (pNFH), and optic nerve damage was assessed by performing NFH- and pNFH-positive axon counts. Expression levels of the ER stress proteins 78-kDa glucose-regulated protein, also known as binding immunoglobulin protein, and C/EBP homologous protein were assayed with Western blotting. We also investigated ER stress localization in the optic nerve by double immunostaining with antibodies to ionized calcium-binding adaptor molecule 1, myelin basic protein, and glial fibrillary acidic protein (GFAP). In DBA/2J mice, IOP began to rise at 8 months of age, and retinal degeneration was detected at 15 months of age. DBA/2J mice had fewer axons than controls at 15 months of age. ER stress-related protein levels were higher in the optic nerves of DBA/2J mice and were colocalized with GFAP-positive astrocytes. Our findings suggest that ER stress plays a role in optic nerve degeneration during chronic ocular hypertension. Furthermore, ER stress may be related in some way to astrocyte activation.

Glycoprotein nonmetastatic melanoma protein B ameliorates skeletal muscle lesions in a SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons and subsequent muscular atrophy. The quality of life of patients with ALS is significantly improved by ameliorating muscular symptoms. We previously reported that glycoprotein nonmetastatic melanoma protein B (GPNMB; osteoactivin) might serve as a target for ALS therapy. In the present study, superoxide dismutase 1/glycine residue 93 changed to alanine (SOD1(G93A) ) transgenic mice were used as a model of ALS. Expression of the C-terminal fragment of GPNMB was increased in the skeletal muscles of SOD1(G93A) mice and patients with sporadic ALS. SOD1(G93A) /GPNMB transgenic mice were generated to determine whether GPNMB expression ameliorates muscular symptoms. The weight and cross-sectional area of the gastrocnemius muscle, number and cross-sectional area of myofibers, and denervation of neuromuscular junctions were ameliorated in SOD1(G93A) /GPNMB vs. SOD1(G93A) mice. Furthermore, direct injection of a GPNMB expression plasmid into the gastrocnemius muscle of SOD1(G93A) mice increased the numbers of myofibers and prevented myofiber atrophy. These findings suggest that GPNMB directly affects skeletal muscle and prevents muscular pathology in SOD1(G93A) mice and may therefore serve as a target for therapy of ALS.

Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells.

PURPOSE: Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line. METHODS: ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK). RESULTS: Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation. CONCLUSIONS: These findings indicate that near-infrared light photobiomodulation (670 nm) may be a noninvasive, inexpensive, and easy adjunctive therapy to help inhibit the development of ocular diseases induced by the activation of phagocytosis.

Effect of a sigma-1 receptor agonist, cutamesine dihydrochloride (SA4503), on photoreceptor cell death against light-induced damage.

Cutamesine dihydrochloride is an agonist of sigma-1 receptor, which is a ligand-operated receptor chaperone at the mitochondrion-associated endoplasmic reticulum (ER) membrane. ER stress plays a pivotal role in light irradiation-induced retinal damage. In the present study, we examined whether cutamesine is effective against experimental degenerative retinal damages in vitro and in vivo. The effects of cutamesine against white light-induced retinal photoreceptor damage were evaluated in vitro by measuring cell death. The expression of sigma-1 receptor after the light exposure was examined by immunoblot analysis. The disruption of the mitochondrial membrane potential and caspase-3/7 activation after excessive light exposure were also examined. In addition, retinal damage in mice induced by irradiation to white light was evaluated using histological staining and electroretinography. Cutamesine reduced the cell death rate induced by light exposure, and the protective effect was prevented by N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD-1047) dihydrobromide, a sigma-1 receptor antagonist. Sigma-1 receptor expression was decreased by light exposure, and cutamesine suppressed the decreased expression of sigma-1 receptor protein. Cutamesine also reduced the mitochondrial damage and reduced the elevated level of caspase 3/7 activity; this effect was attenuated by BD-1047. In in vivo studies, cutamesine suppressed the light-induced retinal dysfunction and thinning of the outer nuclear layer in the mouse retina. These findings indicate that cutamesine protects against retinal cell death in vitro and in vivo by the agonistic effect of sigma-1 receptor. Therefore, sigma-1 receptor may have a potential as a therapeutic target in retinal diseases mediated by photoreceptor degeneration.