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.
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.
The primary event in mammalian vision is a light-initiated cisto-trans isomerization of the retinal chromophore bound, via a protonated Schiff base, to a lysine residue in the opsin apoprotein ( Figure 1A). 1 This isomerization activates the protein which triggers a series of events resulting in a signal to the brain. This has been a basic tenet of our understanding of vision. Rod-rhodopsin, the opsin-retinal complex responsible for night vision, is a G-proteincoupled receptor, activated by light with an absorbance maximum at 500 nm. As the absorption from rhodopsin is minute above 600 nm, the pigment is not believed to be involved in vision at longer wavelengths.It has been proposed that the visual signal transduction pathway in a species of deep-sea fish involves the use of photosensitizers. 2 Molecules derived from chlorophyll 650 are thought to absorb longer-wavelength light and transfer the gained energy to shorterwavelength visual pigments ( Figure 1B), thus adding an extra step to their transduction pathway. This is based on the observations that: (i) the fish only possess visual pigments with λ max e 545 nm, (ii) bleaching of its 545 nm pigment with 671 nm light is faster than bleaching with 654 nm light, and (iii) chlorophyll derivatives which have strong absorbances centered at 665 nm have been isolated along with the 545 nm pigment. A triplet-triplet energytransfer mechanism from the chlorophyll 650 derivatives to the 545 nm pigment has been speculated. 2 Enhanced visual sensitivity is reported as a common side effect in patients exposed to porphyrins during photodynamic therapy. 3 In related photosynthetic systems found in plants, carotenoids are believed to act as quenchers of chlorophylls and singlet oxygen, in addition to their primary roles as light-harvesting complexes. 4 The quenching involves triplet-state energy transfer from chlorophyll to carotenoid. 4b To expand rhodopsin sensitivity into the near-IR we have investigated the bleaching of bovine rhodopsin upon exposure to λ max ) 675 nm light in the presence of various chromophores which are potential photosensitizers with strong absorptions around 665 nm. We report rate enhancements on the order of up to 3 times compared to that for the bleaching of rhodopsin alone with λ max ) 675 nm light.In all experiments the bleaching rates of bovine rhodopsin were measured using UV-vis spectroscopy by monitoring the absorbance at 500 nm corresponding to that for the Schiff base. A 0.009 mmol solution of bovine rhodopsin (90% in ROS suspension) solublized in 5% dodecyl--D-maltoside 5 in phosphate-buffered saline was used for all bleaching experiments. The curve marked rhodopsin in Figure 2 depicts a bleaching rate set to 1.0, for the initial 30 min of bleaching of bovine rhodopsin, with λ max ) 675 nm light at 25°C.
Oxazolidinone-functionalized enecarbamates react stereoselectively with singlet oxygen to give methyldesoxybenzoin (MDB) in moderate to high enantiomeric excess. The stereochemical outcome depends on the E/Z substrate geometry, temperature, and solvent variables. The analysis of the differential activation parameters suggests a large contribution from the entropy term in determining the enantioselectivity. We demonstrate the utility of the temperature and solvent variables in determining the degree of the photochemical kinetic resolution of the enecarbamates; for example, in the photooxygenation at −70 °C in methanol, MDB may be obtained in methanol.
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-D 3 -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-D 3 -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-D 3 -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.S targardt disease, first described in 1909, is an autosomal recessive macular dystrophy affecting ∼1 in 10,000 people. The majority of people affected by the disease present with uncorrectable, decreased visual acuity during their teenage years, which most often progresses to legal blindness. To date, there is no approved intervention. Stargardt disease is marked by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE), degeneration of the neuroretina, and subsequent loss of vision. The condition results from mutations in the ATPbinding cassette, subfamily A, member 4 (ABCA4) gene (1), which encodes a transmembrane flippase localized in photoreceptor outer segments. The flippase transports the phosphatidyl-ethanolamineretinaldehyde Schiff base between the cytosol and the cytoplasmic disk surfaces (2). Mutations in ABCA4 also result in retinitis pigmentosa and cone-rod dystrophy and have been linked to age-related macular degeneration (AMD) (3, 4).The accumulation of lipofuscin in the RPE is a common denominator in retinopathies associated with mutations in the ABCA4 gene. Also known as "wear and tear pigment," lipofuscin accumulates as a byproduct of cumulative damage during aging. The brown-yellow, autofluorescent, electron-dense material is also found in cells of the liver, kidney, heart muscle, adrenals, nerve, and ganglion and is considered one of the most consistent morphologic features of aging with a rate of accumulation inversely related to longevity (5, 6). In Stargardt disease, changes in RPE lipof...
Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy resulting from mutations in the ABCA4 (ABCR) gene. The age of onset of Stargardt disease is typically 10 -20 years of age and leads in almost all cases to blindness by age 50 (1, 2). A hallmark of the disease is premature lipofuscin accumulation in the retinal pigment epithelium (RPE) 2 of the eye. The RPE is critical for the neurosensory retina homeostasis; it acts as a transport exchange system with blood capillaries and is critical for regeneration and phagocytosis of photoreceptor outer segments. It is hypothesized that when RPE lipofuscin reaches a critical level, it contributes to a decline in cell function (3-8) resulting in the degeneration of the macular region of the neurosensory retina leading to loss of central vision (9 -11).The defective gene in Stargardt, ABCA4, encodes for an outer segment rim protein (RmP). The function of RmP is to transport the all-trans-retinaldehyde-phosphatidylethanolamine (retinaldehyde-PE) Schiff base from the luminal side of the disk membrane to the cytosolic face, where retinaldehyde can then be converted back to retinol (3). In the absence of its proper transporter, the retinaldehyde-PE conjugates may react to form vitamin A dimers (A2E and ATR-dimer among others), which are then deposited in the RPE after phagocytosis of the photoreceptors outer segments. Numerous studies have demonstrated the toxicity of vitamin A dimers to cultured RPE cells and they are hypothesized to play a key role in lipofuscin formation and subsequent retinal degeneration (12, 13). Nevertheless, the exact mechanisms that lead to lipofuscin accumulation or to vision loss as a result of the impaired transport of the retinaldehyde-PE conjugates remain unclear.In the accompanying article, we have shown that the ratedetermining step in vitamin A dimerization is the cleavage of a C20 carbon-hydrogen bond of the retinaldehyde-PE Schiff base (14). Replacing the C20 hydrogen atoms of vitamin A with deuterium atoms (i.e. C20-D 3 -vitamin A) makes this bond harder to cleave and impedes vitamin A dimerization (14). In this study we sought to determine whether retarding the intrinsic reactivity of vitamin A to dimerize could slow lipofuscin formation in the RPE and delay changes associated with human Stargardt disease. To accomplish this we raised ABCA4 Ϫ/Ϫ mutant albino mice (the mouse model of human Stargardt's disease) on diets containing either C20-D 3 -vitamin A (the treated group) or vitamin A at its natural isotopic abundance (the control group) and measured the concentration of vitamin A dimers, lipofuscin and other biological markers indicative of ocular health in both groups. Treated mice exhibited an 80% reduction in A2E, a 95% reduction in ATR dimer and a 70% decrease in fundus autofluorescence at three months of age. After six months, the treated group showed fewer lipofuscin granules as visualized qualitatively by electron microscopy, and at 12 months they showed improved eye function as measured by electroretinogram (ERG). ...
Effective therapies for most solid cancers, especially those that have progressed to metastasis, remain elusive because of inherent and acquired resistance of tumor cells to conventional treatments. Additionally, the effective therapeutic window for many protocols can be very narrow, frequently resulting in toxicity. The present study explores an anticancer strategy that effectively eliminates resistant cancer cells without exerting deleterious effects on normal cells. This approach employs melanoma differentiationinduced gene-7/interleukin-24 (mda-7/IL-24), a cancer-specific, apoptosis-inducing cytokine, in combination with nontoxic doses of a chemical compound from the endoperoxide class that decomposes in water generating singlet oxygen. This combinatorial regimen specifically induced in vitro apoptosis in prostate carcinoma cells, with innate resistance to chemotherapy or engineered resistance to mda-7/IL-24, as well as pancreatic carcinoma cells inherently resistant to any treatment modality, including mda-7/ IL-24. Apoptosis induction correlated with increased cellular reactive oxygen species production and was prevented by general antioxidants, such as N-acetyl-L-cysteine or Tiron. Induction of apoptosis in combination-treated cancer cells correlated with a reduction in the antiapoptotic protein BCL-x L. In contrast, both normal prostate and pancreatic epithelial cells were unaffected by the single or combination treatment. These provocative findings suggest that this combinatorial strategy might provide a platform for developing effective treatments for therapy-resistant cancers.
The primary event in vision is light-initiated activation of visual pigments. All visual pigments consist of the protein opsin bound to 11-cis-retinal and are responsible for initiating the transformation of light into an electrical signal. In a mouse model, we show that derivatives of chlorophyll can act as visual pigments initiating the transformation of light into an electrical signal and thus change the primary event in vision to initial activation of a chlorophyll derivative. Electroretinographic b-wave amplitudes recorded in response to red and blue light were two-fold greater in mice administered chlorin e(6), which accumulated in photoreceptor outer segments.
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