C20D3-Vitamin A Prevents Retinal Pigment Epithelium Atrophic Changes in a Mouse Model.

Purpose: This study aimed to evaluate the contribution of vitamin A dimerization to retinal pigment epithelium (RPE) atrophic changes. Leading causes of irreversible blindness, including Stargardt disease and age-related macular degeneration (AMD), occur as a result of atrophic changes in RPE. The cause of the RPE atrophic changes is not apparent. During the vitamin A cycle, vitamin A dimerizes, leading to vitamin A cycle byproducts, such as vitamin A dimers, in the RPE. Methods: To study the consequence of vitamin A dimerization to RPE atrophic changes, we used a rodent model with accelerated vitamin A dimerization, Abca4-/-/Rdh8-/- mice, and the vitamin A analog C20D3-vitamin A to selectively ameliorate the accelerated rate of vitamin A dimerization. Results: We show that ameliorating the rate of vitamin A dimerization with C20D3-vitamin A mitigates pathological changes observed in the prodromal phase of the most prevalent retinal degenerative diseases, including fundus autofluorescence changes, dark adaptation delays, and signature RPE atrophic changes. Conclusions: Data demonstrate that the dimerization of vitamin A during the vitamin A cycle is sufficient alone to cause the prerequisite RPE atrophic changes thought to be responsible for the leading causes of irreversible blindness and that correcting the dimerization rate with C20D3-vitamin A may be sufficient to prevent the RPE atrophic changes. Translational Relevance: Preventing the dimerization of vitamin A with the vitamin A analog C20D3-vitamin A may be sufficient to alter the clinical course of the most prevalent forms of blindness, including Stargardt disease and age-related macular degeneration (AMD).

Vitamin A cycle byproducts explain retinal damage and molecular changes thought to initiate retinal degeneration.

In the most prevalent retinal diseases, including Stargardt disease and age-related macular degeneration (AMD), byproducts of vitamin A form in the retina abnormally during the vitamin A cycle. Despite evidence of their toxicity, whether these vitamin A cycle byproducts contribute to retinal disease, are symptoms, beneficial, or benign has been debated. We delivered a representative vitamin A byproduct, A2E, to the rat's retina and monitored electrophysiological, histological, proteomic, and transcriptomic changes. We show that the vitamin A cycle byproduct is sufficient alone to damage the RPE, photoreceptor inner and outer segments, and the outer plexiform layer, cause the formation of sub-retinal debris, alter transcription and protein synthesis, and diminish retinal function. The presented data are consistent with the theory that the formation of vitamin A byproducts during the vitamin A cycle is neither benign nor beneficial but may be sufficient alone to cause the most prevalent forms of retinal disease. Retarding the formation of vitamin A byproducts could potentially address the root cause of several retinal diseases to eliminate the threat of irreversible blindness for millions of people.

Vitamin A cycle byproducts impede dark adaptation.

Impaired dark adaptation (DA), a defect in the ability to adjust to dimly lit settings, is a universal hallmark of aging. However, the mechanisms responsible for impaired DA are poorly understood. Vitamin A byproducts, such as vitamin A dimers, are small molecules that form in the retina during the vitamin A cycle. We show that later in life, in the human eye, these byproducts reach levels commensurate with those of vitamin A. In mice, selectively inhibiting the formation of these byproducts, with the investigational drug C20D3-vitamin A, results in faster DA. In contrast, acutely increasing these ocular byproducts through exogenous delivery leads to slower DA, with otherwise preserved retinal function and morphology. Our findings reveal that vitamin A cycle byproducts alone are sufficient to cause delays in DA and suggest that they may contribute to universal age-related DA impairment. Our data further indicate that the age-related decline in DA may be tractable to pharmacological intervention by C20D3-vitamin A.

Mechanisms of neurodegeneration in a preclinical autosomal dominant retinitis pigmentosa knock-in model with a RhoD190N mutation.

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.

Towards Treatment of Stargardt Disease: Workshop Organized and Sponsored by the Foundation Fighting Blindness.

Accumulation of fluorescent metabolic byproducts of the visual (retinoid) cycle is associated with photoreceptor and retinal pigment epithelial cell death in both Stargardt disease and atrophic (nonneovascular) age-related macular degeneration (AMD). As a consequence of this observation, small molecular inhibitors of enzymes in the visual cycle were recently tested in clinical trials as a strategy to protect the retina and retinal pigment epithelium in patients with atrophic AMD. To address the clinical translational needs for therapies aimed at both diseases, a workshop organized by the Foundation Fighting Blindness was hosted by the Department of Pharmacology at Case Western Reserve University on February 17, 2017, at the Tinkham Veale University Center, Cleveland, OH, USA. Invited speakers highlighted recent advances in the understanding of the pathophysiology of Stargardt disease, in terms of its clinical characterization and the development of endpoints for clinical trials, and discussed the comparability of therapeutic strategies between atrophic age-related macular degeneration (AMD) and Stargardt disease. Investigators speculated that reducing the concentrations of visual cycle precursor substances and/or their byproducts may provide valid therapeutic options for the treatment of Stargardt disease. Here we review the workshop's presentations in the context of published literature to help shape the aims of ongoing research endeavors and aid the development of therapies for Stargardt disease.

Identification of ginkgolide targets in brain by photoaffinity labeling.

Ginkgolides are terpene trilactones in Ginkgo biloba, a popular medicinal herb for memory disorders. Although ginkgolides are known for various neurobiological effects, their macromolecular target in brain is unknown. In this work, we employed benzophenone derivatives of ginkgolides to identify their binding target in brain. Photolabeling of bovine hippocampus homogenates identified a series of α-tubulin isotypes. Selective photolabeling of α-tubulin over ß-tubulin, which is equally abundant in brain, suggested that ginkgolides might modulate microtubule biology differently than typical microtubule-binding agents, such as taxol. In fact, ginkgolide A did not affect microtubule polymerization or cell proliferation; instead, it inhibited detyrosination of α-tubulin and reorientation of microtubule-organizing centers. Taken together, the current findings indicate that ginkgolides constitute a new class of microtubule-binding agents with distinct effects on α-tubulin biology.

Sequestration of ubiquitous dietary derived pigments enables mitochondrial light sensing.

Animals alter their physiological states in response to their environment. We show that the introduction of a chlorophyll metabolite, a light-absorbing pigment widely consumed in human diets, to Caenorhabditis elegans results in animals whose fat mass can be modulated by exposure to light, despite the worm consuming the same amount of food. In the presence of the chlorophyll metabolite, exposing the worms to light increased adenosine triphosphate, reduced oxidative damage, and increased median life spans, without an effect on animal reproduction. Mice fed a dietary metabolite of chlorophyll and exposed to light, over several months, showed reductions in systemic inflammation as measured by plasma α-macroglobulin. We propose that dietary chlorophyll metabolites can enable mitochondria to use light as an environmental cue, by absorbing light and transferring the energy to mitochondrial coenzyme Q.

The Rate of Vitamin A Dimerization in Lipofuscinogenesis, Fundus Autofluorescence, Retinal Senescence and Degeneration.

One of the earliest events preceding several forms of retinal degeneration is the formation and accumulation of vitamin A dimers in the retinal pigment epithelium (RPE) and underlying Bruch's membrane (BM). Such degenerations include Stargardt disease, Best disease, forms of retinitis pigmentosa, and age-related macular degeneration (AMD). Since their discovery in the 1990's, dimers of vitamin A, have been postulated as chemical triggers driving retinal senescence and degeneration. There is evidence to suggest that the rate at which vitamin A dimerizes and the eye's response to the dimerization products may dictate the retina's lifespan. Here, we present outstanding questions, finding the answers to which may help to elucidate the role of vitamin A dimerization in retinal degeneration.

Can Vitamin A be Improved to Prevent Blindness due to Age-Related Macular Degeneration, Stargardt Disease and Other Retinal Dystrophies?

We discuss how an imperfect visual cycle results in the formation of vitamin A dimers, thought to be involved in the pathogenesis of various retinal diseases, and summarize how slowing vitamin A dimerization has been a therapeutic target of interest to prevent blindness. To elucidate the molecular mechanism of vitamin A dimerization, an alternative form of vitamin A, one that forms dimers more slowly yet maneuvers effortlessly through the visual cycle, was developed. Such a vitamin A, reinforced with deuterium (C20-D3-vitamin A), can be used as a non-disruptive tool to understand the contribution of vitamin A dimers to vision loss. Eventually, C20-D3-vitamin A could become a disease-modifying therapy to slow or stop vision loss associated with dry age-related macular degeneration (AMD), Stargardt disease and retinal diseases marked by such vitamin A dimers. Human clinical trials of C20-D3-vitamin A (ALK-001) are underway.

Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization.

Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.

Morphological and physiological retinal degeneration induced by intravenous delivery of vitamin A dimers in rabbits.

The eye uses vitamin A as a cofactor to sense light and, during this process, some vitamin A molecules dimerize, forming vitamin A dimers. A striking chemical signature of retinas undergoing degeneration in major eye diseases such as age-related macular degeneration (AMD) and Stargardt disease is the accumulation of these dimers in the retinal pigment epithelium (RPE) and Bruch's membrane (BM). However, it is not known whether dimers of vitamin A are secondary symptoms or primary insults that drive degeneration. Here, we present a chromatography-free method to prepare gram quantities of the vitamin A dimer, A2E, and show that intravenous administration of A2E to the rabbit results in retinal degeneration. A2E-damaged photoreceptors and RPE cells triggered inflammation, induced remolding of the choroidal vasculature and triggered a decline in the retina's response to light. Data suggest that vitamin A dimers are not bystanders, but can be primary drivers of retinal degeneration. Thus, preventing dimer formation could be a preemptive strategy to address serious forms of blindness.

The photosynthetic eukaryote Nannochloris eukaryotum as an intracellular machine to control and expand functionality of human cells.

To construct an intracellular machine, we sought a symbiotic relationship between a photosynthetic green alga and human cells. Human cells selectively take up the minimal eukaryote Nannochloris eukaryotum and the resulting symbionts are able to survive and proliferate. Host cells can utilize N. eukaryotum's photosynthetic apparatus for survival, and expression of cellular vascular endothelial growth factor can be controlled with input of photonic energy. This seemingly rare spontaneous association provides an opportunity to fabricate light-controlled, intracellular machines.

Light-harvesting chlorophyll pigments enable mammalian mitochondria to capture photonic energy and produce ATP.

Sunlight is the most abundant energy source on this planet. However, the ability to convert sunlight into biological energy in the form of adenosine-5'-triphosphate (ATP) is thought to be limited to chlorophyll-containing chloroplasts in photosynthetic organisms. Here we show that mammalian mitochondria can also capture light and synthesize ATP when mixed with a light-capturing metabolite of chlorophyll. The same metabolite fed to the worm Caenorhabditis elegans leads to increase in ATP synthesis upon light exposure, along with an increase in life span. We further demonstrate the same potential to convert light into energy exists in mammals, as chlorophyll metabolites accumulate in mice, rats and swine when fed a chlorophyll-rich diet. Results suggest chlorophyll type molecules modulate mitochondrial ATP by catalyzing the reduction of coenzyme Q, a slow step in mitochondrial ATP synthesis. We propose that through consumption of plant chlorophyll pigments, animals, too, are able to derive energy directly from sunlight.

The retina rapidly incorporates ingested C20-D3-vitamin A in a swine model.

PURPOSE: To determine how the retina uses vitamin A for vision, we studied the flux of oral vitamin A into and out of the swine retina. METHODS: We administered labeled vitamin A to swine daily for 30 days and measured the percent of the labeled vitamin A to native unlabeled vitamin A in the retinal epithelium, neuroretina, plasma, liver, lung, and kidney. RESULTS: We show that during normal vitamin A homeostasis, the retina rapidly assimilates newly ingested dietary vitamin A, which replaces native vitamin A. Retinal vitamin A is turned over faster than previously thought. Provitamin A carotenoids do not significantly contribute to retinal vitamin A pools when consuming diets adequate in vitamin A. CONCLUSIONS: Fast vitamin A turnover in the retina has direct implications for emerging therapies to prevent major forms of blindness based on controlling the concentrations of retinal vitamin A.

Dietary chlorophyll metabolites catalyze the photoreduction of plasma ubiquinone.

Ubiquinol is a plasma antioxidant. The mechanisms responsible for maintenance of plasma ubiquinol are poorly understood. Here, we show that metabolites of chlorophyll can be found in blood plasma of animals that are given a chlorophyll-rich diet. We also show that these metabolites catalyze the reduction of plasma ubiquinone to ubiquinol in the presence of ambient light, in vitro. We propose that dietary chlorophyll or its metabolites, together with light exposure, regulate plasma redox status through maintaining the ubiquinol pool.

Retinal coenzyme Q in the bovine eye.

Coenzyme Q plays an integral role in oxygen metabolism and management, and there is a positive correlation between low tissue coenzyme Q concentrations and the progression of many degenerative diseases. Retinal oxidative damage plays a role in the pathogenesis of many degenerative eye diseases; nevertheless, despite the retina's high rate of oxygen metabolism, there is little data relating to retinal coenzyme Q concentrations. In this study, we quantified coenzyme Q in the model bovine eye and determined whether it could function as a retinal lipid antioxidant. We found that the neural retina's ubiquinone concentration exceeded those of the vitreous humor, lens, choroid, and extraocular muscle, but it was lower than those measured in heart, kidney, liver, and brain tissues. Ubiquinol was found to be as effective as vitamin E as a retinal lipid antioxidant. The overall relatively low levels of ubiquinone found in the retina, coupled with the retina's need for lipid antioxidants and oxidative metabolism, suggests that retinal function might be sensitive to changes in ubiquinone concentrations.

C20-D3-vitamin A slows lipofuscin accumulation and electrophysiological retinal degeneration in a mouse model of Stargardt disease.

Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D(3)-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D(3)-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects.

Deuterium enrichment of vitamin A at the C20 position slows the formation of detrimental vitamin A dimers in wild-type rodents.

Degenerative eye diseases are the most common causes of untreatable blindness. Accumulation of lipofuscin (granular deposits) in the retinal pigment epithelium (RPE) is a hallmark of major degenerative eye diseases such as Stargardt disease, Best disease, and age-related macular degeneration. The intrinsic reactivity of vitamin A leads to its dimerization and to the formation of pigments such as A2E, and is believed to play a key role in the formation of ocular lipofuscin. We sought a clinically pragmatic method to slow vitamin A dimerization as a means to elucidate the pathogenesis of macular degenerations and to develop a therapeutic intervention. We prepared vitamin A enriched with the stable isotope deuterium at carbon twenty (C20-D(3)-vitamin A). Results showed that dimerization of deuterium-enriched vitamin A was considerably slower than that of vitamin A at natural abundance as measured in vitro. Administration of C20-D(3)-vitamin A to wild-type rodents with no obvious genetic defects in vitamin A processing, slowed A2E biosynthesis. This study elucidates the mechanism of A2E biosynthesis and suggests that administration of C20-D(3)-vitamin A may be a viable, long-term approach to retard vitamin A dimerization and by extension, may slow lipofuscin deposition and the progression of common degenerative eye diseases.

Determination of coenzyme Q10 level in human retina by the high-performance liquid chromatography / 眼科研究

Background Researches have demonstrated that age-related macular degeneration (AMD) is associated with the oxidative stress injury of retina.Coenzyme Q10 (CoQ10) is an important antioxidant agent.CoQ10 level in blood plasma is a primary index of reflecting the oxidative stress ability of human.However,the study on CoQ10 content in retina has not been seen yet.ObjectiveThe aim of this study is to establish a method of detecting CoQ10 content in retina by the high-performance liquid chromatography (HPLC). Methods The retinas were isolated from 10 healthy eyes of donors aged 20-28 years.The donor eyes were obtained from National Development and Research Institute,Inc.USA.Isolated retina tissue was prepared into homogenate then lyophilized and deproteinized with methanol.Samples were extracted with heptane prior to the HPLC analysis with the chromatographic conditions as follows:RP-18 column,a mobile phase consisted of methanol-hexane-acetic acid-isopropanol (V/V=55:9:1:1) and 0.42% sodium acetate,ultraviolet rays (UV) detector at 275 nm.Results CoQ10 was effectively isolated from human retina.The limit of detection of CoQ10 was 0.14mg/L.The peak area and concentration of CoQ10 showed a good linear correlation within the concentration range of 0.2-395.00mg/L (R~2＝0.9943).Repeatability study showed that the relative standard deviations for CoQ10 at the concentration of 0.86mg/L,2.59mg/L and 3.45mg/L were 2.7%,0.1% and 3.3%,respectively.The within- and inter-day standard deviations for the analysis of CoQ10 were 1.6% and 3.7%,respectively.The recovery was 101%-113% for the human retina samples.The concentration of CoQ10 in 10 retinas from human donors was 0.51±0.20μg/eye in average.Conclusion A HPLC method for the quantified analysis of CoQ10 in human retina is developed.

Coenzyme Q10 in the human retina.

PURPOSE: To determine the concentration of coenzyme Q10 (CoQ10) in the human retina. METHODS: Eye tissues were lyophilized and exhaustively extracted with heptane. The extracts were analyzed for CoQ10 by high-performance liquid chromatography (HPLC). RESULTS: The average concentration of CoQ10 in the retina was 42+/-11 nanomoles/g dry retina for donors younger than 30 years of age and 24+/-13 nanomoles/g dry retina for donors older than 80 years of age. The average concentrations of CoQ10 in the choroid was 27+/-16 nanomoles/g dry choroid for donors younger than 30 age and 18+/-11 nanomoles/g dry choroid for donors older than 80. CONCLUSIONS: CoQ10 levels in the retina can decline by approximately 40% with age. This decline may have two consequences: a decrease in antioxidant ability and a decrease in the rate of ATP synthesis in the retina and, as such, this decline may be linked to the progression of macular degeneration.