Crocetin protects against mitochondrial damage induced by UV-A irradiation in corneal epithelial cell line HCE-T cells.

The corneal epithelium is located at the outermost layer of the ocular surface and continuously exposed to environmental factors, such as ultraviolet (UV) radiation from sunlight. UV irradiation causes excessive production of reactive oxygen species (ROS) in cells, which results in oxidative damage to membrane-bound organelles such as mitochondria, eventually leading to cell death. Crocetin, a natural carotenoid found in plants, has various biological properties including antioxidant activity. In this study, we investigated the effects of crocetin on UV-A-induced cell injury in the corneal epithelium. Using an in vitro system with the human corneal epithelial cell-transformed (HCE-T) cell line, pretreatment with 10 µM crocetin suppressed the reduction of cell viability induced by UV-A exposure. Crocetin ameliorated the decrease in oxygen consumption rates and the mitochondrial fragmentation that occurred following UV-A irradiation. Crocetin inhibited both ROS production and the activation of the apoptosis pathway; it also preserved the defects of epithelial cell polarity and barrier function in UV-A-irradiated HCE-T cells. The reduction in apical Mucin-16 expression was partially recovered in the presence of crocetin. Taking these findings together, we conclude that crocetin has a protective effect against UV-A irradiation-induced mitochondrial injury in corneal epithelial cells.

Retinal damage alters gene expression profile in lacrimal glands of mice.

Early detection of such retinal diseases as glaucoma and age-related macular degeneration (AMD) is important to prevent blindness. There have been reports of changes in some components in the tears of glaucoma and AMD patients, suggesting tears' potential usefulness in screening for retinal diseases. We hypothesized that retinal damage might alter gene expression in the lacrimal gland, leading to those changes in tear components. We caused retinal damage in mice by intravitreal injection of N-methyl-d-aspartate (NMDA) or excessive light exposure. Hematoxylin and eosin staining showed no histological changes in the lacrimal glands of animals whose retinas had been damaged. However, RNA sequencing of lacrimal glands on the 3rd day after NMDA injection or light exposure revealed changes in the expression of 491 genes (268 up-regulated; 223 down-regulated) in the NMDA group and 531 genes (311 up-regulated; 220 down-regulated) in the light group. Further gene-set enrichment analysis indicated that both types of retinal damage activated the immune system in the lacrimal glands. This is the first demonstration that retinal damage can alter gene expression in the lacrimal glands, and it might lead to a novel non-invasive screening method for early detection of retinal diseases.

Lipid Droplet Accumulation Promotes RPE Dysfunction.

Non-exudative age-related macular degeneration (AMD) is an irreversibly progressive retinal degenerative disease characterized by dysfunction and loss of retinal pigment epithelium (RPE). It has been suggested that impaired phagocytosis of the RPE is involved in the progression of non-exudative AMD, but the mechanism is not fully clear. In this study, we investigated the effect of lipid droplet accumulation on RPE function. Compared to young mice, the expression of lipid droplet-associated proteins increased in the RPE-choroidal complex, and lipid droplet in the RPE was observed in aged pigmented mice (12-month-old). Repeated treatment of the photoreceptor outer segment against ARPE-19 resulted in lipid droplets in ARPE-19 cells in vitro. Oleic acid treatment for ARPE-19 cells to form intracellular lipid droplet reduced the POS uptake into the ARPE-19 cells without causing a decrease in cell viability. The suppression of the POS uptake by lipid droplet formation improved by inhibiting lipid droplet formation using triacsin C. Moreover, the amount of intracellular reactive oxygen species was suppressed by the triacsin C treatment. These results indicate that lipid droplet is involved in the RPE dysfunction, and inhibiting lipid droplet formation may be a target for preventing and treating non-exudative AMD.

Reduced mitochondrial complex II activity enhances cell death via intracellular reactive oxygen species in STHdhQ111 striatal neurons with mutant huntingtin.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HTT) gene. Here, we examined the effects of antioxidants on 3-nitropropionic acid (3-NP; a mitochondrial complex II inhibitor)-induced mitochondrial dysfunction and cell death in STHdhQ111 striatal cells carrying homozygous mutant HTT with extended CAG repeats compared with those in STHdhQ7 striatal cells. 3-NP reduced cell viability and increased cell death both in STHdhQ111 and STHdhQ7, and the cytotoxicity was markedly attenuated by antioxidants (N-acetyl-l-cysteine and edaravone). Furthermore, 3-NP increased intracellular reactive oxygen species (ROS) production in both cell lines, and this increase was inhibited by antioxidants. Mitochondrial ROS was also increased by 3-NP in STHdhQ111 but not in STHdhQ7, and this increase was significantly inhibited by edaravone. Mitochondrial membrane potential (MMP) was lower in STHdhQ111 than that in STHdhQ7, and antioxidants prevented 3-NP-induced MMP decrease in STHdhQ111.3-NP enhanced oligomerization of dynamin-related protein 1 (Drp1), a protein that promotes mitochondrial fission in both cells, and both antioxidants prevented the increase in oligomerization. These results suggest that reduced mitochondrial complex II activity enhances cell death via intracellular ROS production and Drp1 oligomerization in striatal cells with mutant HTT and antioxidants may reduce striatal cell death.

Mitochondria dynamics in the aged mice eye and the role in the RPE phagocytosis.

Aging is a predominant risk factor for various eye diseases. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, and its etiology remains unclear. Fragmented and dysfunctional mitochondria are associated with age-related diseases. The retinal pigment epithelium (RPE), a polarized cell layer that functions in visual pigment recycling and degeneration, is suspected as the primary region site of AMD. In the present study, we investigated the relationship between mitochondrial dysfunction and RPE aging. Compared to young mice, aged pigmented mice (C57BL/6J, 12-month-old) exhibit decreased visual function without retinal thinning. Consistently, the rhodopsin expression level decreased in the outer segment of aged mice. Moreover, the cell volume of the RPE increased in aged animals. Interestingly, the expression of mitochondria dynamics-related proteins, including Drp1, was altered in the RPE-choroid complex but not in the neural retina after aging. Electron microscopy revealed that mitochondrial size decreased and cristae width increased in aged RPE. The photoreceptor outer segment (POS) treatment of ARPE-19 cells causes Drp1 activation. Furthermore, pharmacological suppression of mitochondrial fission improved the phagocytosis of the POS. These findings indicate that mitochondrial dysfunction and fission in RPE impede phagocytosis and cause retardation of the visual cycle, which can be one of the age-related defects in the retina that may contribute to the onset of AMD.

Free-Radical Scavenger NSP-116 Protects the Corneal Epithelium against UV-A and Blue LED Light Exposure.

The corneal epithelium is continuously exposed to oxygen, light, and environmental substances. Excessive exposure to those stresses is thought to be a risk factor for eye diseases. Photokeratitis is damage to the corneal epithelium resulting in a painful eye condition caused by unprotected exposure to UV rays, usually from sunlight, and is often found in people who spend a long time outdoors. In modern life, human eyes are exposed to artificial light from light-emitting diode (LED) displays of computers and smartphones, and it has been shown that short-wavelength (blue) LED light can damage eyes, especially photoreceptors. However, the effect of blue LED light on the cornea is less understood. In addition, it is important to develop new treatments for preserving human eyesight and eye health from light stress. Here, we used human corneal epithelial cells-transformed (HCE-T) cells as an in-vitro model to investigate the protective effect of NSP-116, an imidazolyl aniline derivative, against the oxidative stress induced by light in the corneal epithelium. Treatment with 10 µM NSP-116 significantly increased the cell viability and reduced the death ratio following UV or blue LED light exposure. Furthermore, NSP-116 treatment decreased light-induced reactive oxygen species production and preserved the mitochondrial membrane potential. Immunoblotting data showed that NSP-116 suppressed the stress response pathway. Finally, NSP-116 treatment prevented corneal epithelial apoptosis induced by blue LED light in an in-vivo mouse model. In conclusion, NSP-116 has a protective effect against oxidative stress and corneal cell death from both UV and blue LED light exposure.

Pharmacological inhibition of mitochondrial fission attenuates oxidative stress-induced damage of retinal pigmented epithelial cells.

Mitochondria maintain their function by the process of mitochondrial dynamics, which involves repeated fusion and fission. It is thought that the failure of mitochondrial dynamics, especially excessive fission, is related to the progression of several diseases. A previous study demonstrated that mitochondrial fragmentation occurs in the retinal pigmented epithelial (RPE) cells of patients with non-exudative age-related macular degeneration (AMD). We predicted that the suppression of mitochondrial fragmentation offers a novel therapeutic strategy for non-exudative AMD. We investigated whether the inhibition of mitochondrial fission was effective against the oxidative stress-induced damage of ARPE-19 cells. The treatment of ARPE-19 cells with H2O2 caused mitochondrial fragmentation, but treatment with mitochondrial division inhibitor 1 (Mdivi-1) suppressed fragmentation. Additionally, Mdivi-1 protected ARPE-19 cells against H2O2-induced damage, and suppressed the release of cytochrome c from the mitochondria. Mitochondrial function was evaluated by staining with JC-1 and measuring the production of reactive oxygen species (ROS), which revealed that mitochondrial function improved in the Mdivi-1-treated group. These findings indicated that the inhibition of mitochondrial fission would be a novel therapeutic target for non-exudative AMD.

Antitumour Effects of Astaxanthin and Adonixanthin on Glioblastoma.

Several antitumour drugs have been isolated from natural products and many clinical trials are underway to evaluate their potential. There have been numerous reports about the antitumour effects of astaxanthin against several tumours but no studies into its effects against glioblastoma. Astaxanthin is a red pigment found in crustaceans and fish and is also synthesized in Haematococcus pluvialis; adonixanthin is an intermediate product of astaxanthin. It is known that both astaxanthin and adonixanthin possess radical scavenging activity and can confer a protective effect on several damages. In this study, we clarified the antitumour effects of astaxanthin and adonixanthin using glioblastoma models. Specifically, astaxanthin and adonixanthin showed an ability to suppress cell proliferation and migration in three types of glioblastoma cells. Furthermore, these compounds were confirmed to transfer to the brain in a murine model. In the murine orthotopic glioblastoma model, glioblastoma progression was suppressed by the oral administration of astaxanthin and adonixanthin at 10 and 30 mg/kg, respectively, for 10 days. These results suggest that both astaxanthin and adonixanthin have potential as treatments for glioblastoma.

A triterpenoid Nrf2 activator, RS9, promotes LC3-associated phagocytosis of photoreceptor outer segments in a p62-independent manner.

Daily phagocytosis of shed photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is required to sustain the visual function. Recent reports revealed that POS phagocytosis is progressed with LC3-associated manner. Patients with age-related macular degeneration (AMD) had impaired autophagic degradation in the RPE. Nrf2 is a key antioxidant transcriptional regulator that ameliorates oxidative stress which is another contributor to AMD pathogenesis. Nrf2 activation also induces the autophagy receptor protein, p62. However, the role of the Nrf2-p62 pathway in LC3-associated phagocytosis of POS is poorly understood. Here, we investigated the relationships between Nrf2 activation and POS phagocytosis progression. A triterpenoid Nrf2 activator, RS9, facilitated POS uptake into phagolysosomes in RPE cells. RS9 also induced the expression of the autophagy-related proteins, LC3-II and p62, as well as phase-2 antioxidant enzymes. The effect of RS9 on POS phagocytosis was abolished by autophagy inhibition. Unexpectedly, p62 knockdown did not inhibit the effect of RS9 on POS phagocytosis, although, RS9-mediated LC3-II induction by RS9 was inhibited in p62 knockdown RPE cells. We also found that RS9 activated the AMPKα-mTOR signaling pathway earlier than p62 induction. Knockdown of AMPKα1, but not α2, inhibited the RS9-mediated activation of LC3-associated phagocytosis and RS9-mediated induction of LC3-II. Furthermore, intravitreal treatment of RS9 to adult mice decreased the size of POS phagolysosomes after light exposure. Collectively, these results showed that RS9-mediated activation of POS phagocytosis was mainly ascribed to the enhancement of autophagy via AMPKα1 activation. Our findings reveal novel effects of Nrf2 and AMPK α1 activation that contribute to the maintenance of the RPE function via LC3-associated POS phagocytosis.

Bilberry extract and anthocyanins suppress unfolded protein response induced by exposure to blue LED light of cells in photoreceptor cell line.

Purpose: The purpose of this study was to investigate the effects of bilberry extract with its anthocyanins on retinal photoreceptor cell damage and on the endoplasmic reticulum (ER) stress induced by exposure to blue light-emitting diode (LED) light. Methods: Cultured murine photoreceptor cells (661W) were exposed to blue LED light with or without bilberry extract or its anthocyanins in the culture media. Aggregated short-wavelength opsin (S-opsin) in murine photoreceptor cells was observed with immunostaining. The expression of factors involved in the unfolded protein response was examined with immunoblot analysis and quantitative real-time reverse transcription (RT)-PCR. Furthermore, cell death was observed with double staining with Hoechst 33342 and propidium iodide after dithiothreitol (DTT) treatment. Results: Bilberry extract and anthocyanins suppressed the aggregation of S-opsin, activation of ATF4, and expression of the mRNA of the factors associated with the unfolded protein response (UPR). In addition, bilberry extract and the anthocyanins inhibited the death of photoreceptor cells induced by DTT, an ER stress inducer. Conclusions: These findings suggest that bilberry extract containing anthocyanins can alter the effects of blue LED light and DTT-induced retinal photoreceptor cell damage. These effects were achieved by modulating the activation of ATF4 and through the suppression of the abnormal aggregation of S-opsin.

Exposure to excessive blue LED light damages retinal pigment epithelium and photoreceptors of pigmented mice.

To determine the characteristics of the damages of the retinal pigment epithelium (RPE) and photoreceptors of pigmented mice induced by exposure to blue light emitting diode (LED) light, and to determine the mechanisms causing the damages. Exposure to blue LED light for 3 days induced retinal damage, and the characteristics of the damage differed from that induced by white fluorescent light exposure. Ophthalmoscopy showed that blue LED exposure for 3 days induced white spots on the retina, and histological examinations showed materials accumulated at the IS/OS junction of the photoreceptors. The accumulated materials were stained by ionized calcium binding adapter molecule-1 (Iba-1), a marker for macrophages. The debris was also positive for periodic acid-Schiff (PAS). An enlarging the area of RPE was detected just after the blue LED exposure especially around the optic nerve, and this led to a secondary degeneration of the photoreceptors. Exposure of pigmented mice to 3 consecutive days of blue LED light will cause RPE and photoreceptor damage. The damage led to an accumulation of macrophages and drusen-like materials around the outer segments of the photoreceptors. This blue light exposed model may be useful for investigating the pathogenesis of non-exudative age-related macular degeneration.