Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity.

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

Treatment of dry age-related macular degeneration.

A number of different approaches are under development for treating nonexudative manifestations of age-related macular degeneration (AMD). Some interventions target specific pathways that are believed to play a role in AMD pathogenesis, e.g. oxidative damage, lipofuscin accumulation, chronic inflammation (including complement activation), extracellular matrix changes (e.g. ß-amyloid accumulation), impaired choroidal blood flow, and apoptosis. In principle, these therapies can be combined ('combination therapy'), which may lead to synergistic effects that include better visual outcome, less likelihood for 'escape' (i.e. drug resistance), and less frequent treatment.

Natto (fermented soybean) extract extends the adult lifespan of Caenorhabditis elegans.

We investigated the effects of a water extract of natto on the aging of the nematode Caenorhabditis elegans. The water extract significantly prolonged the adult lifespan of the wild-type worms and rendered them resistant to oxidative and thermal stress. In addition, treatment with natto extract significantly delayed the accumulation of lipofuscin, a characteristic of aging cells. Our findings suggest that components of natto have a beneficial anti-aging effect in vivo.

Antioxidant and antitumor activities of selenium- and zinc-enriched oyster mushroom in mice.

Selenium and zinc are well-known essential trace elements with potent biological functions. However, the possible health benefits of the combined administration of dietary selenium and zinc have not been studied extensively. In this study, we prepared selenium- and zinc-enriched mushrooms (SZMs) containing increased levels of selenium and zinc. The effects of SZMs on antioxidant and antitumor activities were evaluated. Mice were fed with either a control diet or a diet supplemented with SZMs or sodium selenite and zinc sulfate for 6 weeks. Antioxidant capacity was investigated by measuring the activities of antioxidant enzymes and the levels of lipid peroxide products. Results showed that treatment with SZMs significantly increased the activities of glutathione peroxidase (GPx) and superoxide dismutase and decreased the levels of malondialdehyde and lipofuscin. Furthermore, using a mouse model of lung tumors, we found that SZMs significantly decreased the number of tumor nodes with an increase in the activity of GPx. SZMs had a greater effect on the increase in both antioxidant and antitumor activities than did sodium selenite and zinc sulfate. These findings suggest that SZMs may be effective for improving antioxidant capacity and preventing tumors.

Molecular medicine in ophthalmic care.

BACKGROUND: Lipofuscin is the most consistent and phylogenically constant morphologic marker of cellular aging. Autofluorescence of the A2E fluorophore within retinal pigment epithelial (RPE) lipofuscin affords the opportunity for noninvasive evaluation of age- and disease-related pathophysiological changes in the human retina. It is being used in National Eye Institute/Age-Related Eye Disease Study II to evaluate age-related macular degeneration (AMD) geographic atrophy expansion. Experiments show lipofuscin can be reversed in cell culture and animal models in heart, brain, spinal cord, and retinal tissues, using an array of antioxidants and iron chelators. METHODS: An 80-year-old man with a gastric resection presented with complaints of unremitting night driving difficulty despite treatment with lutein and omega III fatty acids. Notable parafoveal deposition of retinal lipofuscin by 50 degrees fundus auto-fluorescence (580 nm excitation/660 barrier filters) and concurrent abnormalities in non-Snellen measures of visual function-Contrast Sensitivity Function, 6.5 degrees large field tritan threshold, 10 degrees threshold visual fields, and deficits in the National Institutes of Health/National Eye Institute Visual Function Questionnaire (VFQ) 25 subjective night driving/mental health subscale questionnaire were obtained. The patient was placed on an over-the-counter daily oral polyphenolic mixture containing resveratrol and re-evaluated 5 months later. RESULTS: The data reveal improvements in all measures of visual function, subjective improvement in vision and mental functioning on the VFQ 25, and visible clearing of RPE lipofuscin. CONCLUSION: To our knowledge, we believe this to be the first reported human clinical case of lipofuscin reversal in the human eye correlated with measured clinical and subjective improvement in visual and mental function after nutraceutical intervention.

The effect of Polbax extract on lipofuscin accumulation in cultured neonatal rat cardiac myocytes.

Polbax, a water-soluble extract of fresh pollen grains and pistils, was tested for its ability to influence the accumulation of lipofuscin (age pigment) in cultured neonatal rat cardiac myocytes. Exposure for 3 weeks to Polbax at concentrations of 0.1, 1.0 or 10 mg/L decreased lipofuscin accumulation morphometrically assayed using laser scanning microscopy images (green excitation light) of formaldehyde-fixed cells, by 24%, 41% or 43%, respectively. Based on the knowledge that oxidative stress and iron-catalysed peroxidation play an important role in lipofuscinogenesis, we suggest that Polbax may slow lipofuscin formation due to antioxidant activities, perhaps involving intralysosomal dismutation of superoxide produced by autophagocytosed mitochondria and/or iron-chelation.

Carnitine as a free radical scavenger in aging.

Carnitine (4-N-trimethylammonium-3-hydroxybutyric acid) plays an important role in the translocation of acetyl moieties from the mitochondria into the cytoplasm for acetylcholine synthesis in the brain. Previous studies in our laboratory have shown that L-carnitine suppresses oxidative damage during aging. This study was carried out to see the effect of L-carnitine on the status of non-enzymatic antioxidants and lipofuscin accumulation in various regions of the aged rat brain. We observed a decrease in the status of ascorbic acid, glutathione and vitamin E in aged rats. Histological work showed that the accumulation of lipofuscin increased as a function of age. The extent of damage varied between the regions we have investigated. Supplementation of L-carnitine to aged rat improved the antioxidant status in a duration dependent manner. The accumulation of lipofuscin was also found to be decreased after L-carnitine administration. The data suggests that decrement of lipofuscin accumulation by L-carnitine may be partially due to its antioxidant promoting action.

Ascorbic acid 2-O-alpha-glucoside-induced redox modulation in human keratinocyte cell line, SCC: mechanisms of photoprotective effect against ultraviolet light B.

We previously reported that the topical application of ascorbic acid 2-O-alpha-glucoside (AA-2G) suppressed the cutaneous inflammation by ultraviolet irradiation in human and guinea pigs (Miyai et al., Nishinihon J. Dermatol., 58, 439-443 (1996)). In this paper, the effect of AA-2G on the lethal damage induced by ultraviolet B (UVB) was studied using a human keratinocyte cell line, SCC, established from squamous cell carcinoma. The photoprotective effect of AA-2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125-1 mM) and more effective than that of ascorbic acid (AsA) at 1 mM. This protection was completely abolished in the presence of an alpha-glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA-2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. AA-2G exhibited a preventive effect against the cytotoxicity produced by tert-butylhydroperoxide, an inducer of lipid peroxidation, in the presence of alpha-tocopherol, but not in the absence of alpha-tocopherol. Cytotoxicity of UVB was also effectively reduced by the combination of AA-2G and alpha-tocopherol. In addition, AA-2G reduced UVB-promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide. These data suggest that AA-2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and potentiating the antioxidative activity of alpha-tocopherol.

Lipofuscin accumulation in ageing myocardium & its removal by meclophenoxate.

A study was undertaken on the age-associated histochemical changes in the ventricular myocardium and the influence of meclophenoxate hydrochloride (MPH) on the age pigment lipofuscin. Sixty Wistar albino rats in three age-groups (3, 15 and 30 months old) were treated with meclophenoxate hydrochloride (100 mg/kg body wt/day, ip) for a period of 2-8 wk. Five animals each from the three age-groups served as controls. Various histochemical and micromorphometric studies were carried out on the myocardial tissue. A linear increase in the myocardial volume occupied by the pigment was observed with advancing age. As a result of meclophenoxate treatment, a gradual decrease in the myocardial volume occupied by the pigment was noted. After 4-6 wk treatment, the pigment bodies were found lodged into the capillary endothelium and the lumen, facilitating the removal of the pigment via blood stream. Histochemical and micromorphometric analyses of ventricular myocardium of albino rats have shown thus that deposition of the age-pigment, lipofuscin, can be accepted as an index of cellular ageing.

Prevention of lipofuscin development in neurons by anti-oxidants.

It was documented that ageing is associated with a progressive and highly significant proliferation of the total number of light microscopically visible lipofuscin granules in the grey substance of sections of the cervical spinal cord of Balb/c mice. The mean total numbers (+/- standard errors) of lipofuscin granules in standard sections of the glutaraldehyde-osmium fixed, epon embedded spinal cords that were examined with a phase contrast light microscope in 1 week, 1 month, 8 months and 18 months old mice were 0, 269 +/- 56, 1101 +/- 82 and 2464 +/- 318, respectively. The population densities of multiglobular lipofuscin units as seen with the electron microscope in random spinal cord neurons of the same 4 age groups corresponded well with the above quantitative, light microscopic data. Continuous treatment for 8 months with either the natural anti-oxidant Vitamin E (alpha-tocopherol) at 40 mg/mouse/week or the synthetic anti-oxidant butylated hydroxytoluene at about 100 mg/mouse/week diminished significantly the proliferation of lipofuscin granules in spinal cord neurons that developed during that period of ageing. No toxicity of any sort was caused by these two treatments. These results provide support for the peroxide theory of lipofuscin biogenesis and encourage further exploration of the possibilities of obtaining greater anti-lipofuscin effects with less molecular bulk of antioxidants.

Lipofuscin accumulation and its prevention by vitamin E in nervous tissue: quantitative analysis using snail buccal ganglia as a simple model system.

The total numbers and sizes of the lipofuscin granules in buccal ganglia of the pond snail Planorbis corneus were measured by light microscope morphometry of serial sections in different groups of animals. One group, weight range 0.5-5.0 g (age 1-4 years), was maintained on a lettuce diet (low Vitamin E content). Lipofuscin content increased with age by over a 100 times in this group. The lipofuscin deposits accumulate in the glial cells at the periphery of the ganglia. The other group, weight range 0.5-5.0 g was fed a supplemented Vitamin E diet for 4 months before quantitisation of the lipofuscin. This diet prevented lipofuscin appearance in the young animals, and reduced the lipofuscin content by 50% in the old animals. The findings provide direct evidence that Vitamin E reduces lipofuscin accumulation in glial cells in intact nervous tissue. The buccal ganglia of Planorbis provide a useful model system for studying age and dietary related alterations of lipofuscin in nervous tissue.

Lipofuscin in neuronal aging and diseases.

The increasing intraneuronal accumulation of lipofuscin has been linked to the aging process by a striking linear correlation between the degree of accumulation and chronological age. It has been established that age pigments are soluble in polar and nonpolar solvents; the pigment is autofluorescent and stains with PAS, Sudan black B, Nile blue osmic acid and ferric ferricyanide techniques. Whether all pigments exhibiting these properties are identical, or at least closely related, regardless of the surrounding tissue, animal species and age of the individual, is debatable. Pigment formation has been demonstrated in young and aged animals as well as in individual subjected to experimental stress and to dietetic and environmental interference. Electron microscopic studies in animals have shown a considerable variability in the fine structure of individual lipofuscin granules but the presence of "lucent vacuoles" surrounded by a unit membrane is one of the characteristic features of neuronal lipofuscin in the aged. Recently, electron microscopy, utilizing the freeze-etching technique, has provided convincing evidence which disproves the earlier view that lucent vacuoles are the remnant of lipid material dissolved and removed during the preparation of the tissues for microscopic examination. These vacuoles have also been demonstrated in freshly frozen material not previously fixed or immersed. Vacuolated pigment granules occur earlier in the area postrema than in other regions of the rat brain (Hasan and Heyder 1974). Regional differences in the time sequence of pigment deposition are present.