Is There an Optimal Combination of AREDS2 Antioxidants Zeaxanthin, Vitamin E and Vitamin C on Light-Induced Toxicity of Vitamin A Aldehyde to the Retina?

Vitamins C and E and zeaxanthin are components of a supplement tested in a large clinical trial-Age-Related Eye Disease Study 2 (AREDS2)-and it has been demonstrated that they can inhibit the progression of age-related macular degeneration. The aim of this study was to determine the optimal combinations of these antioxidants to prevent the phototoxicity mediated by vitamin A aldehyde (ATR), which can accumulate in photoreceptor outer segments (POS) upon exposure to light. We used cultured retinal pigment epithelial cells ARPE-19 and liposomes containing unsaturated lipids and ATR as a model of POS. Cells and/or liposomes were enriched with lipophilic antioxidants, whereas ascorbate was added just before the exposure to light. Supplementing the cells and/or liposomes with single lipophilic antioxidants had only a minor effect on phototoxicity, but the protection substantially increased in the presence of both ways of supplementation. Combinations of zeaxanthin with α-tocopherol in liposomes and cells provided substantial protection, enhancing cell viability from ~26% in the absence of antioxidants to ~63% in the presence of 4 µM zeaxanthin and 80 µM α-tocopherol, and this protective effect was further increased to ~69% in the presence of 0.5 mM ascorbate. The protective effect of ascorbate disappeared at a concentration of 1 mM, whereas 2 mM of ascorbate exacerbated the phototoxicity. Zeaxanthin or α-tocopherol partly ameliorated the cytotoxic effects. Altogether, our results suggest that the optimal combination includes upper levels of zeaxanthin and α-tocopherol achievable by diet and/or supplementations, whereas ascorbate needs to be at a four-fold smaller concentration than that in the vitreous. The physiological relevance of the results is discussed.

Red Light Irradiation In Vivo Upregulates DJ-1 in the Retinal Ganglion Cell Layer and Protects against Axotomy-Related Dendritic Pruning.

Retinal ganglion cells (RGCs) undergo dendritic pruning in a variety of neurodegenerative diseases, including glaucoma and autosomal dominant optic atrophy (ADOA). Axotomising RGCs by severing the optic nerve generates an acute model of RGC dendropathy, which can be utilized to assess the therapeutic potential of treatments for RGC degeneration. Photobiomodulation (PBM) with red light provided neuroprotection to RGCs when administered ex vivo to wild-type retinal explants. In the current study, we used aged (13-15-month-old) wild-type and heterozygous B6;C3-Opa1Q285STOP (Opa1+/-) mice, a model of ADOA exhibiting RGC dendropathy. These mice were pre-treated with 4 J/cm2 of 670 nm light for five consecutive days before the eyes were enucleated and the retinas flat-mounted into explant cultures for 0-, 8- or 16-h ex vivo. RGCs were imaged by confocal microscopy, and their dendritic architecture was quantified by Sholl analysis. In vivo 670 nm light pretreatment inhibited the RGC dendropathy observed in untreated wild-type retinas over 16 h ex vivo and inhibited dendropathy in ON-center RGCs in wild-type but not Opa1+/- retinas. Immunohistochemistry revealed that aged Opa1+/- RGCs exhibited increased nitrosative damage alongside significantly lower activation of NF-κB and upregulation of DJ-1. PBM restored NF-κB activation in Opa1+/- RGCs and enhanced DJ-1 expression in both genotypes, indicating a potential molecular mechanism priming the retina to resist future oxidative insult. These data support the potential of PBM as a treatment for diseases involving RGC degeneration.

Computational Studies towards the Identification of Novel Rhodopsin-Binding Compounds as Chemical Chaperones for Misfolded Opsins.

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber's congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber's congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.

Photostimulation of mitochondria as a treatment for retinal neurodegeneration.

Absorption of photon energy by neuronal mitochondria leads to numerous downstream neuroprotective effects. Red and near infrared (NIR) light are associated with significantly less safety concerns than light of shorter wavelengths and they are therefore, the optimal choice for irradiating the retina. Potent neuroprotective effects have been demonstrated in various models of retinal damage, by red/NIR light, with limited data from human studies showing its ability to improve visual function. Improved neuronal mitochondrial function, increased blood flow to neural tissue, upregulation of cell survival mediators and restoration of normal microglial function have all been proposed as potential underlying mechanisms of red/NIR light.

Zeaxanthin in combination with ascorbic acid or alpha-tocopherol protects ARPE-19 cells against photosensitized peroxidation of lipids.

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.