Toshio Narimatsu

Disruption of Cell-Cell Junctions and Induction of Pathological Cytokines in the Retinal Pigment Epithelium of Light-Exposed Mice

PURPOSE To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flat-mount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-β-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL-6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPEs barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

Retinal Aging and Sirtuins

The process of aging involves the accumulating changes in the microenvironment that lead to cell senescence or apoptosis, and subsequent tissue or organ dysfunction. Multiple extrinsic and intrinsic events that cause DNA instability are associated with aging. Cells containing unstable DNA are biologically vulnerable, and if the DNA damage is too great for the cell to repair, it becomes senescent or dies by apoptosis. Thus, the cell’s capacity to repair its DNA determines the progress of aging, at least in part. Here, we focus on the sirtuins, the mammalian homologs of the yeast life-span-extending molecule, Sir2. Among the sirtuin family proteins in mammals, the one most similar to yeast Sir2 is SIRT1, which is involved in multiple pathways, including the repair of DNA double-strand breaks. Although the role of SIRT1 in mammalian longevity is not clear, it is expressed throughout the retina, where it may suppress aging. In fact, a mutant mouse model of retinal degeneration shows an abnormal subcellular localization of SIRT1 protein and accelerated retinal cell apoptosis. Further analyses are required to elucidate the mechanism of DNA damage and repair, including the contributions of the sirtuins, in the aged or diseased retinas, which will help us understand the mechanisms of retinal aging.

Biological effects of blocking blue and other visible light on the mouse retina.

To elucidate the biological effects of blocking fluorescent light on the retina using specific blocking materials.

Angiotensin II type 1 receptor blockade suppresses light-induced neural damage in the mouse retina

Exposure to light contributes to the development and progression of retinal degenerative diseases. However, the mechanisms underlying light-induced tissue damage are not fully understood. Here, we examined the role of angiotensin II type 1 receptor (AT1R) signaling, which is part of the renin-angiotensin system, in light-induced retinal damage. Light-exposed Balb/c mice that were treated with the AT1R blockers (angiotensin II receptor blockers; ARBs) valsartan, losartan, and candesartan before and after the light exposure exhibited attenuated visual function impairment, compared to vehicle-treated mice. This effect was dose-dependent and observed across the ARB class of inhibitors. Further evaluation of valsartan showed that it suppressed a number of light-induced retinal effects, including thinning of the photoreceptor cell layer caused by apoptosis, shortening of the photoreceptor cell outer segment, and increased levels of reactive oxygen species (ROS). The role of ROS in retinal pathogenesis was investigated further using the antioxidant N-acetyl-l-cysteine (NAC). Treatment of light-exposed mice with NAC before the light exposure suppressed the visual function impairment and photoreceptor cell histological changes due to apoptosis. Moreover, treatment with valsartan or NAC suppressed the induction of c-fos (a component of the AP-1 transcription factor) and the upregulation of fasl (a proapoptotic molecule whose transcript is regulated downstream of AP-1). Our results suggest that AT1R signaling mediates light-induced apoptosis, by increasing the levels of ROS and proapoptotic molecules in the retina. Thus, AT1R blockade may represent a new therapeutic approach for preventing light-induced retinal neural tissue damage.

Lutein acts via multiple antioxidant pathways in the photo-stressed retina

Lutein slows the progression of age-related macular degeneration (AMD), a leading cause of blindness in ageing societies. However, the underlying mechanisms remain elusive. Here, we evaluated lutein’s effects on light-induced AMD-related pathological events. Balb/c mice exposed to light (2000 lux, 3 h) showed tight junction disruption in the retinal pigment epithelium (RPE) at 12 h, as detected by zona occludens-1 immunostaining. Substantial disruption remained 48 h after light exposure in the vehicle-treated group; however, this was ameliorated in the mice treated with intraperitoneal lutein at 12 h, suggesting that lutein promoted tight junction repair. In the photo-stressed RPE and the neighbouring choroid tissue, lutein suppressed reactive oxygen species and increased superoxide dismutase (SOD) activity at 24 h, and produced sustained increases in sod1 and sod2 mRNA levels at 48 h. SOD activity was induced by lutein in an RPE cell line, ARPE19. We also found that lutein suppressed upregulation of macrophage-related markers, f4/80 and mcp-1, in the RPE-choroid tissue at 18 h. In ARPE19, lutein reduced mcp-1 mRNA levels. These findings indicated that lutein promoted tight junction repair and suppressed inflammation in photo-stressed mice, reducing local oxidative stress by direct scavenging and most likely by induction of endogenous antioxidant enzymes.

Neuroinflammation and Neurodegenerative Disorders of the Retina

The retina is composed of neural networks that are responsible for visual function, and vascular networks that support the tissue. Although vascular targeting therapies for retinal diseases have recently been developed, therapies directly targeting the neuronal component of these diseases have yet to be developed. Here, we review recent studies describing the pathological signaling that occurs within the neuronal cells of retinal disease models. The molecular changes caused by endogenous or exogenous factors in the retinal neural cells and the molecular events involved in neuroinflammation are illustrated. These underlying molecular mechanisms reveal promising targets for new therapeutic approaches for retinal neural disorders.