Yoshiki Kuse

Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm(2)). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light.

Nrf2 protects photoreceptor cells from photo-oxidative stress induced by blue light

ABSTRACT Oxidative stress plays a key role in age‐related macular degeneration and hereditary retinal degenerations. Light damage in rodents has been used extensively to model oxidative stress‐induced photoreceptor degeneration, and photo‐oxidative injury from blue light is particularly damaging to photoreceptors. The endogenous factors protecting photoreceptors from oxidative stress, including photo‐oxidative stress, are continuing to be elucidated. In this study, we evaluated the effect of blue light exposure on photoreceptors and its relationship to Nrf2 using cultured murine photoreceptor (661W) cells. 661W cells were exposed to blue light at 2500 lux. Exposure to blue light for 6–24 h resulted in a significant increase in intracellular reactive oxygen species (ROS) and death of 661W cells in a time‐dependent fashion. Blue light exposure resulted in activation of Nrf2, as indicated by an increase in nuclear translocation of Nrf2. This was associated with a significant induction of expression of Nrf2 as well as an array of Nrf2 target genes, including antioxidant genes, as indicated by quantitative reverse transcription PCR (qRT‐PCR). In order to determine the functional role of Nrf2, siRNA‐mediated knockdown studies were performed. Nrf2‐knockdown in 661W cells resulted in significant exacerbation of blue light‐induced reactive oxygen species levels as well as cell death. Taken together, these findings indicate that Nrf2 is an important endogenous protective factor against oxidative stress in photoreceptor cells. This suggests that drugs targeting Nrf2 could be considered as a neuroprotective strategy for photoreceptors in AMD and other retinal conditions. Graphical abstract Figure. No Caption available. HighlightsBlue light increases ROS levels and cell death in the 661W photoreceptor cell line.Blue light induces nuclear translocation of Nrf2.Blue light increases expression of Nrf2 and Nrf2‐target genes.Nrf2 knockdown in 661W cells exacerbates blue light‐induced photo‐oxidative stress, including both ROS and cell death.

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.

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 enzymes 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 promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina

Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.

CCR3 Is Associated with the Death of a Photoreceptor Cell-line Induced by Light Exposure

The C-C chemokine receptor type 3 (CCR3) is the receptor for eotaxins (CCL-11, 24, 26), RANTES (CCL-5) and MCP-3 (CCL-7). It was reported that an inhibition of CCR3 by antagonists or antibodies reduces the degree of laser-induced choroidal neovascularization in mice, a model for wet age-related macular degeneration (AMD). Although several chemokine receptors have the potential of reducing the degree of the chronic inflammation in experimental dry AMD, the association of CCR3 remains unknown. The purpose of this study was to determine the role played by CCR3 in the death of 661W cells which are cells of a murine photoreceptor-derived cell line as an in vitro model of dry AMD. The expression of CCR3 was increased in the 661W cells after light exposure. Inhibition of CCR3 reduced the rate of cell death induced by light exposure. A blockade of CCR3 signaling by CCR3 silencing and two kinds of CCR3 antagonists, SB 328437 and SB 297006, reduced the rate of light-induced cell death. In addition, CCR3 inhibition decreased the level of reactive oxygen species and the activation of caspase-3/7 induced by light exposure. These findings indicated that the CCR3 blockade should be considered for the treatment of the dry AMD.

Colored lenses suppress blue light-emitting diode light-induced damage in photoreceptor-derived cells

Abstract. Blue light-emitting diodes (LEDs) in liquid crystal displays emit high levels of blue light, exposure to which is harmful to the retina. Here, we investigated the protective effects of colored lenses in blue LED light-induced damage to 661W photoreceptor-derived cells. We used eight kinds of colored lenses and one lens that reflects blue light. Moreover, we evaluated the relationship between the protective effects of the lens and the transmittance of lens at 464 nm. Lenses of six colors, except for the SY, PN, and reflective coating lenses, strongly decreased the reduction in cell damage induced by blue LED light exposure. The deep yellow lens showed the most protective effect from all the lenses, but the reflective coating lens and pink lens did not show any effects on photoreceptor-derived cell damage. Moreover, these results were correlated with the lens transmittance of blue LED light (464 nm). These results suggest that lenses of various colors, especially deep yellow lenses, may protect retinal photoreceptor cells from blue LED light in proportion to the transmittance for the wavelength of blue LED and the suppression of reactive oxygen species production and cell damage.

Transient acceleration of autophagic degradation by pharmacological Nrf2 activation is important for retinal pigment epithelium cell survival

Non-exudative age-related macular degeneration (AMD) is mainly caused by the accumulation of lipofuscin and drusen on the retinal pigment epithelium (RPE). Both oxidative stress and autophagic dysfunction accelerate the deposition of lipofuscin at the RPE. One of the key regulators in the response against oxidative stress is the NF-E2-Related Factor 2 (Nrf2)-kelch like ECH associated protein 1 (Keap1) axis, which is also closely associated with the autophagy pathway. Nrf2 activation upregulates the expression levels of certain anti-oxidative enzymes [e.g. Heme oxygenase-1 (HO-1)], which attenuates oxidative damage. However, until now, the relationship between cytoprotective effects of Nrf2 activation and autophagic degradation remain unclear. To address these questions, we investigated the effects of a novel Nrf2 activator, RS9, on RPE damage. We found that RS9 protected ARPE-19 cells against NaIO3-induced oxidative damage, and that the protective effects of RS9 were inhibited by co-treatment with zinc protoporphyrin, an HO-1 inhibitor. Next, we examined the involvement of autophagic degradation in the protective effects of RS9. Co-treatment with RS9 and chloroquine, a lysosomal acidification inhibitor, inhibited the protective effect. Furthermore, western blotting and immunostaining showed that RS9 accelerated autophagy flux and induced transient upregulation of p62 [also known as sequestosome 1 (SQSTM1)]. Co-treatment with chloroquine and RS9 also inhibited the degradation of autophagosomes. Transient upregulation of SQSTM1 by RS9 was unaltered by HO-1 knockdown using siRNA. RS9 and chloroquine had the same actions in light damaged adult zebrafish retina as those in vitro. In conclusion, we clarified the relationship between acceleration of the autophagy pathway and the cytoprotective effects of Nrf2 activation in RPE cells and zebrafish retina. These findings indicated that Nrf2 activation could be a promising therapeutic approach for non-exudative AMD by supporting RPE maintenance.

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

&NA; 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.

Progranulin deficiency causes the retinal ganglion cell loss during development

Astrocytes are glial cells that support and protect neurons in the central nervous systems including the retina. Retinal ganglion cells (RGCs) are in contact with the astrocytes and our earlier findings showed the reduction of the number of cells in the ganglion cell layer in adult progranulin deficient mice. In the present study, we focused on the time of activation of the astrocytes and the alterations in the number of RGCs in the retina and optic nerve in progranulin deficient mice. Our findings showed that the number of Brn3a-positive cells was reduced and the expression of glial fibrillary acidic protein (GFAP) was increased in progranulin deficient mice. The progranulin deficient mice had a high expression of GFAP on postnatal day 9 (P9) but not on postnatal day 1. These mice also had a decrease in the number of the Brn3a-positive cells on P9. Taken together, these findings indicate that the absence of progranulin can affect the survival of RGCs subsequent the activation of astrocytes during retinal development.