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Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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retinoids in the visual cycle role of the Retinal g protein coupled receptor
Journal of Lipid Research, 2020Co-Authors: Elliot H. Choi, Henri Leinonen, Anahita Daruwalla, Susie Suh, Krzysztof PalczewskiAbstract:Driven by the energy of a photon, the visual pigments in rod and cone photoreceptor cells isomerize 11-Cis-Retinal to the all-trans configuration. This photochemical reaction initiates the signal transduction pathway that eventually leads to the transmission of a visual signal to the brain and leaves the opsins insensitive to further light stimulation. For the eye to restore light sensitivity, opsins require recharging with 11-Cis-Retinal. This trans-Cis back conversion is achieved through a series of enzymatic reactions composing the retinoid (visual) cycle. Although it is evident that the classical retinoid cycle is critical for vision, the existence of an adjunct pathway for 11-Cis-Retinal regeneration has been debated for many years. Retinal pigment epithelium (RPE)-Retinal G protein-coupled receptor (RGR) has been identified previously as a mammalian Retinaldehyde photoisomerase homologous to retinochrome found in invertebrates. Using pharmacological, genetic, and biochemical approaches, researchers have now established the physiological relevance of the RGR in 11-Cis-Retinal regeneration. The photoisomerase activity of RGR in the RPE and Muller glia explains how the eye can remain responsive in daylight. In this review, we will focus on retinoid metabolism in the eye and visual chromophore regeneration mediated by RGR.
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Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked Retinal analogue.
Journal of Biological Chemistry, 2017Co-Authors: Nathan S. Alexander, Kota Katayama, Wenyu Sun, David Salom, Sahil Gulati, Jianye Zhang, Muneto Mogi, Krzysztof Palczewski, Beata JastrzebskaAbstract:Abstract ABSTRACT Phototransduction is initiated when the absorption of light converts the 11-Cis-Retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-Cis-Retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the visual or retinoid cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-Cis-Retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-Cis-6-membered ring (11-Cis-6mr)-Retinal, which has therapeutic effects against certain degenerative Retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked Retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-Cis-6mr-Retinal and demonstrated that this Retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro205 in green cone opsin could prevent entry and binding of 11-Cis-6mr-Retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-Cis-6mr-Retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-Cis-6mr-Retinal. These results and our structural modeling, indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-Cis-6mr-Retinal.
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cellular Retinaldehyde binding protein different binding modes and micro solvation patterns for high affinity 9 Cis and 11 Cis Retinal substrates
Journal of Physical Chemistry B, 2013Co-Authors: Rachel E. Helbling, Marcin Golczak, Krzysztof Palczewski, Christin S. Bolze, Achim Stocker, Michele CascellaAbstract:We use molecular dynamics (MD) simulations to determine the binding properties of different retinoid species to cellular Retinaldehyde binding protein (CRALBP). The complexes formed by 9-Cis-Retinal or 11-Cis-Retinal bound to both the native protein and the R234W mutant, associated to Bothnia-retina dystrophy, are investigated. The presented studies are also complemented by analysis of the binding structures of the CRALBP/9-Cis-retinol and CRALBP/9,13- diCis-Retinal complexes. We find that the poor X-ray scattering properties of the polyene tail of the ligand in all wild-type complexes can be attributed to a high mobility of this region, which does not localize in a single binding conformation even at very low temperatures. Our simulations report a clear difference in the residual solvation pattern in CRALBP complexes with either 9-Cis- or 9,13-diCis-Retinal. The reported structures indicate that the microsolvation properties of the ligand are the key structural element triggering the very recently discovered isomerase activity of this protein. The binding geometries obtained by MD simulations are validated by calculation of the respective optical spectra by the ZINDO/S semiempirical method, which can reproduce with good qualitative agreement the different red-shifts of the first absorption band of the different complexes.
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probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to p23h opsin mutations
Journal of Biological Chemistry, 2011Co-Authors: Sanae Sakami, Artur V Cideciyan, Marcin Golczak, Tadao Maeda, Grzegorz Bereta, Kiichiro Okano, Alexander Sumaroka, Alejandro J Roman, Samuel G Jacobson, Krzysztof PalczewskiAbstract:Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-Cis-Retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its Retinal cytotoxicity is enhanced by lack of the 11-Cis-Retinal chromophore during rod outer segment development.
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the role of the 11 Cis Retinal ring methyl substituents in visual pigment formation
ChemInform, 2007Co-Authors: Marta Domínguez, Krzysztof Palczewski, Rosana Alvarez, Martin Perez, Angel R. De LeraAbstract:Artificial visual pigment formation from ring-demethylated Retinals was studied in an effort to understand the effect that methyl groups on the chromophore cyclohexenyl ring have on the visual cycle. The stereoselective synthesis of the 11-Cis-ring-demethylated analogues involves thallium-accelerated Suzuki cross-coupling reactions and highly stereocontrolled Wittig reactions to form key bonds. Only 11-Cis-1,1,5-trisdemethylRetinal (2) failed to form an artificial pigment, whilst variable pigment-formation yields were determined for the remaining analogues, increasing with the number (and location) of the chromophore hydrophobic ring methyl groups. Our results with the monodemethylated analogues 11-Cis-5-demethylRetinal (4) and 11-Cis-1-demethylRetinal (5) show that the C1–2-CH3 groups are more important for pigment formation than the C5-CH3 substituent. This is reflected in the absorption maxima of the artificial pigments, with values closer to that of native rhodopsin for 4. Docking studies based on a rhodopsin crystal structure, however, predict a lower pigment stability for 4 than for 5. Gas-phase DFT (B3LYP/6-31G*) computations of the free-ligand geometries, conformational searches about the C6� C7 bond, and docking studies revealed that, although the conformation of bound 5 is close to that of the native chromophore, the ligand needs to overcome the energy cost of shifting the unbound favored 6-s-trans conformation to the bound 6-s-Cis form. In addition, the presence of an extra methyl group at C18 (11-Cis-18-methylRetinal, 7) is tolerated well and adds further stability to the complex, most probably due to increased hydrophobic interactions.
Akiko Maeda - One of the best experts on this subject based on the ideXlab platform.
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Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice
Journal of Biological Chemistry, 2015Co-Authors: Bhubanananda Sahu, Wenyu Sun, Lindsay Perusek, Vipulkumar Parmar, Michael D. Griswold, Akiko MaedaAbstract:Regeneration of the visual chromophore, 11-Cis-Retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the Retinal pigmented epithelium (RPE). Oxidation of 11-Cis-retinol to 11-Cis-Retinal is accomplished by a family of enzymes termed 11-Cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-Cis-Retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-Cis-Retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-Cis-Retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-Cis-retinyl ester content also seen in Rdh5(-/-)Rdh11(-/-) mice as compared with Rdh5(-/-) mice. Normal Retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5(-/-) and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-Cis-Retinal chromophore needed for vision.
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evaluation of 9 Cis retinyl acetate therapy in rpe65 mice
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Tadao Maeda, Akiko Maeda, Gemma Casadesus, Philippe MargaronAbstract:Visual perception results from the biological conversion of light energy to electrical signaling by Retinal photoreceptors in the eye, a process called phototransduction. Visual pigments, consisting of the chromophore 11-Cis-Retinal bound to apoprotein G protein-coupled receptor opsins,1 initiate this process on the absorption of a photon that triggers photoisomerization of the chromophore into its trans form.1,2 The isomerized chromophore, all-trans-Retinal, then is reduced to all-trans-retinol, transported to the Retinal pigment epithelium (RPE), and converted to fatty acid all-trans-retinyl esters by lecithin/retinol acyltransferase (LRAT). Regeneration of 11-Cis-Retinal completes this retinoid (visual) cycle and is critical for maintaining vision.3 Defects in 11-Cis-Retinal production are associated with a number of inherited degenerative retinopathies.4 Two examples are Leber congenital amaurosis (LCA), a childhood-onset Retinal disease causing severe visual impairment, and retinitis pigmentosa (RP), another retinopathy with a more variable age of onset. At or soon after birth, LCA patients characteristically exhibit severe visual impairment exhibited by wandering nystagmus, amaurotic pupils, a pigmentary retinopathy with loss of cone and rod sensitivity, absent or greatly attenuated electroretinographic (ERG) responses, and an approximately 100-fold decrease in cone flicker amplitudes.5–7 RPE65, a 65-kDa protein specific to and abundant in the RPE8 that catalyzes the isomerization of fatty acid all-trans-retinyl esters to 11-Cis-retinol, has been recently described as the retinoid isomerase involved in the regeneration of 11-Cis-Retinal.9–11 Mutations in the RPE65 gene account for up to 16% of LCA cases and 2% of autosomal recessive RP cases.4,12–15 Spontaneous or engineered deletions of Rpe65 in mice and dogs result in 11-Cis-Retinal deficiency, an early-onset and slowly progressive Retinal degeneration with dramatically reduced ERG responses and typical LCA pathology16–19 accompanied by an accumulation of fatty acid all-trans-retinyl esters in the RPE.16,20 LCA is incurable, but several possible therapies are being investigated. RPE65 gene augmentation therapy and Retinal prostheses have shown preliminary encouraging signs of visual rescue in early-stage clinical evaluations.21–23 Recently, visual chromophore replacement therapy with 9-Cis-Retinal has been proposed as a novel pharmacologic approach to bypass the defective retinoid cycle.24–27 9-Cis-Retinal binds to opsin to form the rod cell pigment, iso-rhodopsin, which initiates phototransduction similar to that of rhodopsin.1 Oral administration of 9-Cis-Retinal or its precursors have regenerated opsin as iso-rhodopsin in the eyes, improved Retinal function as assessed by ERG responses, and ameliorated the pupillary light reflex in Rpe65 and Lrat knockout mice, two genetic models of LCA.24–27 These observations have led to the development of synthetic 9-Cis-retinoids as orally administered, prodrug forms of 9-Cis-Retinal for the treatment of various forms of inherited Retinal degeneration caused by defects in the retinoid cycle. Here we report pharmacokinetic and pharmacodynamic effects of the prodrug 9-Cis-R-Ac that is converted to another prodrug in the liver (i.e., mostly to 9-Cis-retinyl palmitate) in the Rpe65−/− mouse model. We describe the selection of soybean oil as an appropriate vehicle for administering the 9-Cis-R-Ac prodrug by gastric gavage, based on postabsorptive levels of its pharmacologically active metabolites in plasma. We assessed 9-Cis-R-Ac efficacy with the use of electroretinography and vision-dependent behavioral tests in single, intermittent, and daily dosing studies that also included biochemical quantification of retinoids in the eye. Dose-dependent improvement of the level and duration of Retinal function was observed in these knockout animals. Importantly, pharmacologic activity was sustained for sufficiently long periods after dosing to enable the formulation of a flexible, intermittent dosing schedule.
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role of photoreceptor specific retinol dehydrogenase in the retinoid cycle in vivo
Journal of Biological Chemistry, 2005Co-Authors: Akiko Maeda, Tadao Maeda, Wenyu Sun, Yoshikazu Imanishi, Houbin Zhang, Vladimir A Kuksa, Andrei Alekseev, Darin J Bronson, Li Zhu, David A SapersteinAbstract:Abstract The retinoid cycle is a recycling system that replenishes the 11-Cis-Retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-Retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH–/–) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH–/– mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-Cis-Retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-Retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-Retinal resulted in slight overproduction of A2E, a condensation product of all-trans-Retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-Retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.
Tadao Maeda - One of the best experts on this subject based on the ideXlab platform.
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probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to p23h opsin mutations
Journal of Biological Chemistry, 2011Co-Authors: Sanae Sakami, Artur V Cideciyan, Marcin Golczak, Tadao Maeda, Grzegorz Bereta, Kiichiro Okano, Alexander Sumaroka, Alejandro J Roman, Samuel G Jacobson, Krzysztof PalczewskiAbstract:Rhodopsin, the visual pigment mediating vision under dim light, is composed of the apoprotein opsin and the chromophore ligand 11-Cis-Retinal. A P23H mutation in the opsin gene is one of the most prevalent causes of the human blinding disease, autosomal dominant retinitis pigmentosa. Although P23H cultured cell and transgenic animal models have been developed, there remains controversy over whether they fully mimic the human phenotype; and the exact mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H opsin knock-in mice, we found that the P23H protein was inadequately glycosylated with levels 1-10% that of wild type opsin. Moreover, the P23H protein failed to accumulate in rod photoreceptor cell endoplasmic reticulum but instead disrupted rod photoreceptor disks. Genetically engineered P23H mice lacking the chromophore showed accelerated photoreceptor cell degeneration. These results indicate that most synthesized P23H protein is degraded, and its Retinal cytotoxicity is enhanced by lack of the 11-Cis-Retinal chromophore during rod outer segment development.
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evaluation of 9 Cis retinyl acetate therapy in rpe65 mice
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Tadao Maeda, Akiko Maeda, Gemma Casadesus, Philippe MargaronAbstract:Visual perception results from the biological conversion of light energy to electrical signaling by Retinal photoreceptors in the eye, a process called phototransduction. Visual pigments, consisting of the chromophore 11-Cis-Retinal bound to apoprotein G protein-coupled receptor opsins,1 initiate this process on the absorption of a photon that triggers photoisomerization of the chromophore into its trans form.1,2 The isomerized chromophore, all-trans-Retinal, then is reduced to all-trans-retinol, transported to the Retinal pigment epithelium (RPE), and converted to fatty acid all-trans-retinyl esters by lecithin/retinol acyltransferase (LRAT). Regeneration of 11-Cis-Retinal completes this retinoid (visual) cycle and is critical for maintaining vision.3 Defects in 11-Cis-Retinal production are associated with a number of inherited degenerative retinopathies.4 Two examples are Leber congenital amaurosis (LCA), a childhood-onset Retinal disease causing severe visual impairment, and retinitis pigmentosa (RP), another retinopathy with a more variable age of onset. At or soon after birth, LCA patients characteristically exhibit severe visual impairment exhibited by wandering nystagmus, amaurotic pupils, a pigmentary retinopathy with loss of cone and rod sensitivity, absent or greatly attenuated electroretinographic (ERG) responses, and an approximately 100-fold decrease in cone flicker amplitudes.5–7 RPE65, a 65-kDa protein specific to and abundant in the RPE8 that catalyzes the isomerization of fatty acid all-trans-retinyl esters to 11-Cis-retinol, has been recently described as the retinoid isomerase involved in the regeneration of 11-Cis-Retinal.9–11 Mutations in the RPE65 gene account for up to 16% of LCA cases and 2% of autosomal recessive RP cases.4,12–15 Spontaneous or engineered deletions of Rpe65 in mice and dogs result in 11-Cis-Retinal deficiency, an early-onset and slowly progressive Retinal degeneration with dramatically reduced ERG responses and typical LCA pathology16–19 accompanied by an accumulation of fatty acid all-trans-retinyl esters in the RPE.16,20 LCA is incurable, but several possible therapies are being investigated. RPE65 gene augmentation therapy and Retinal prostheses have shown preliminary encouraging signs of visual rescue in early-stage clinical evaluations.21–23 Recently, visual chromophore replacement therapy with 9-Cis-Retinal has been proposed as a novel pharmacologic approach to bypass the defective retinoid cycle.24–27 9-Cis-Retinal binds to opsin to form the rod cell pigment, iso-rhodopsin, which initiates phototransduction similar to that of rhodopsin.1 Oral administration of 9-Cis-Retinal or its precursors have regenerated opsin as iso-rhodopsin in the eyes, improved Retinal function as assessed by ERG responses, and ameliorated the pupillary light reflex in Rpe65 and Lrat knockout mice, two genetic models of LCA.24–27 These observations have led to the development of synthetic 9-Cis-retinoids as orally administered, prodrug forms of 9-Cis-Retinal for the treatment of various forms of inherited Retinal degeneration caused by defects in the retinoid cycle. Here we report pharmacokinetic and pharmacodynamic effects of the prodrug 9-Cis-R-Ac that is converted to another prodrug in the liver (i.e., mostly to 9-Cis-retinyl palmitate) in the Rpe65−/− mouse model. We describe the selection of soybean oil as an appropriate vehicle for administering the 9-Cis-R-Ac prodrug by gastric gavage, based on postabsorptive levels of its pharmacologically active metabolites in plasma. We assessed 9-Cis-R-Ac efficacy with the use of electroretinography and vision-dependent behavioral tests in single, intermittent, and daily dosing studies that also included biochemical quantification of retinoids in the eye. Dose-dependent improvement of the level and duration of Retinal function was observed in these knockout animals. Importantly, pharmacologic activity was sustained for sufficiently long periods after dosing to enable the formulation of a flexible, intermittent dosing schedule.
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role of photoreceptor specific retinol dehydrogenase in the retinoid cycle in vivo
Journal of Biological Chemistry, 2005Co-Authors: Akiko Maeda, Tadao Maeda, Wenyu Sun, Yoshikazu Imanishi, Houbin Zhang, Vladimir A Kuksa, Andrei Alekseev, Darin J Bronson, Li Zhu, David A SapersteinAbstract:Abstract The retinoid cycle is a recycling system that replenishes the 11-Cis-Retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-Retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH–/–) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH–/– mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-Cis-Retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-Retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-Retinal resulted in slight overproduction of A2E, a condensation product of all-trans-Retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-Retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.
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recovery of visual functions in a mouse model of leber congenital amaurosis
Journal of Biological Chemistry, 2002Co-Authors: Preston J Van Hooser, Henry K.w. Fong, Tadao Maeda, Geeng Fu Jang, Vladimir A Kuksa, Yan Liang, Fred Rieke, Peter B Detwiler, Krzysztof PalczewskiAbstract:The visual process is initiated by the photoisomerization of 11-Cis-Retinal to all-trans- Retinal. For sustained vision the 11-Cis-chromophore must be regenerated from all-trans-Retinal. This requires RPE65, a dominant Retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the Retinal pigment epithelium, lack of 11-Cis-Retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623–8628) that in Rpe65−/− mice, oral administration of 9-Cis-Retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9 -Cis- Retinal administration significantly attenuated Retinal ester accumulation and supported rod Retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65−/− mice. This rod function is likely a consequence of low efficiency production of 11-Cis- Retinal by photo-conversion of all-trans- Retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65−/− mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and Retinal degenerations.
Rosalie K Crouch - One of the best experts on this subject based on the ideXlab platform.
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rpe65 and lrat mice comparable models of leber congenital amaurosis
Investigative Ophthalmology & Visual Science, 2008Co-Authors: Jie Fan, Jeanne M Frederick, Wolfgang Baehr, Baerbel Rohrer, Rosalie K CrouchAbstract:PURPOSE. The Rpe65 -/- mouse, used as a model for Leber congenital amaurosis, has slow rod degeneration and rapid cone loss, presumably because of the mistrafficking of cone opsins. This animal does not generate 11-Cis Retinal, and both cone loss and rod response are restored by 11-Cis Retinal administration. Similarly, the Lrat -/- mouse does not produce 11-Cis Retinal. The authors sought to determine whether the same effects on rod and cone opsins in the Rpe65 -/- mouse are also present in the Lrat -/- mouse, thereby establishing that these changes can be attributed to the lack of 11-Cis Retinal rather than to some unknown function of RPE65. METHODS. Rod and cone opsins were localized by immunohistochemical methods. Functional opsin levels were determined by regeneration with 11-Cis Retinal. Isorhodopsin levels were determined from pigment extraction. Opsin phosphorylation was determined by mass spectrometry. RESULTS. Rods in both models degenerated slowly. Regenerable rod opsin levels were similar over the 6-month time course investigated, rod opsin was phosphorylated at a low level (approximately 10%), and minimal 9-Cis Retinal was generated by a nonphotic process, giving a trace light response. In both models, S-opsin and M/L-opsin failed to traffic to the cone outer segments appropriately, and rapid cone degeneration occurred. Cone opsin mistrafficking in both models was arrested on 11-Cis Retinal administration. CONCLUSIONS. These data show that the Lrat -/- and Rpe65 -/- mice are comparable models for studies of Leber congenital amaurosis and that the destructive cone opsin mistrafficking is caused by the lack of 11-Cis Retinal.
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chromophore switch from 11 Cis dehydroRetinal a2 to 11 Cis Retinal a1 decreases dark noise in salamander red rods
The Journal of Physiology, 2007Co-Authors: Petri Alalaurila, Rosalie K Crouch, Kristian Donner, Carter M CornwallAbstract:Dark noise, light-induced noise and responses to brief flashes of light were recorded in the membrane current of isolated rods from larval tiger salamander retina before and after bleaching most of the native visual pigment, which mainly has the 11-Cis-3,4-dehydroRetinal (A2) chromophore, and regenerating with the 11-Cis-Retinal (A1) chromophore in the same isolated rods. The purpose was to test the hypothesis that blue-shifting the pigment by switching from A2 to A1 will decrease the rate of spontaneous thermal activations and thus intrinsic light-like noise in the rod. Complete recordings were obtained in five cells (21°C). Based on the wavelength of maximum absorbance, λmax,A1 = 502 nm and λmax,A2 = 528 nm, the average A2 : A1 ratio determined from rod spectral sensitivities and absorbances was ∼0.74 : 0.26 in the native state and ∼0.09 : 0.91 in the final state. In the native (A2) state, the single-quantum response (SQR) had an amplitude of 0.41 ± 0.03 pA and an integration time of 3.16 ± 0.15 s (mean ± s.e.m.). The low-frequency branch of the dark noise power spectrum was consistent with discrete SQR-like events occurring at a rate of 0.238 ± 0.026 rod−1 s−1. The corresponding values in the final state were 0.57 ± 0.07 pA (SQR amplitude), 3.47 ± 0.26 s (SQR integration time), and 0.030 ± 0.006 rod−1 s−1 (rate of dark events). Thus the rate of dark events per rod and the fraction of A2 pigment both changed by ca 8-fold between the native and final states, indicating that the dark events originated mainly in A2 molecules even in the final state. By extrapolating the linear relation between event rates and A2 fraction to 0% A2 (100% A1) and 100% A2 (0% A1), we estimated that the A1 pigment is at least 36 times more stable than the A2 pigment. The noise component attributed to discrete dark events accounted for 73% of the total dark current variance in the native (A2) state and 46% in the final state. The power spectrum of the remaining ‘continuous’ noise component did not differ between the two states. The smaller and faster SQR in the native (A2) state is consistent with the idea that the rod behaves as if light-adapted by dark events that occur at a rate of nearly one per integration time. Both the decreased level of dark noise and the increased SQR amplitude must significantly improve the reliability of photon detection in dim light in the presence of the A1 chromophore compared to the native (A2) state in salamander rods.
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cone opsin mislocalization in rpe65 mice a defect that can be corrected by 11 Cis Retinal
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Baerbel Rohrer, Heather R. Lohr, Peter Humphries, Mathias W. Seeliger, Michael T Redmond, Rosalie K CrouchAbstract:PURPOSE. In Retinal degenerative diseases, rod photoreceptors typically deteriorate more rapidly than cone photoreceptors. In the Rpe65 / mouse, a model for Leber’s congenital amaurosis, cones degenerate much more rapidly than rods. In this model, the retinoid processing pathway in the Retinal pigment epithelium is disrupted, and 11-Cis Retinal is not generated. This study was designed to investigate the feasibility of restoring functional cones with exogenous 11-Cis Retinal. METHODS. Rpe65 / ::Rho / mice were used to remove any interference of rods and compared with wild-type (wt) mice. Pups were injected intraperitoneally with 11-Cis Retinal, starting at postnatal day (P)10, and were maintained in complete darkness. At P25, cone function was assessed with photopic single-flash and flicker ERGs. Cone survival was determined immunohistochemically with cone-specific antibodies, and cone opsin levels were obtained by quantitative RT-PCR. RESULTS. At P25, cone density and transcript levels of cone opsins were drastically reduced, but a minute cone electroretinogram was detected, indicating that the cones were functional. Confocal microscopy revealed that the cone opsins were mislocalized, suggesting that their transport to the outer segments was impaired. Intraperitoneal administrations of 11Cis Retinal before P25 led to increased transport of cone opsins to the outer segments and preserved cones anatomically and functionally. CONCLUSIONS. The results suggest that the ligand is required during cone opsin synthesis for successful opsin trafficking and that without 11-Cis Retinal, cones may degenerate because of opsin mislocalization. These results may have important consequences for the treatment of cone dystrophies. (Invest Ophthalmol Vis Sci. 2005;46:3876 –3882) DOI:10.1167/iovs.050533
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breaking the covalent bond a pigment property that contributes to desensitization in cones
Neuron, 2005Co-Authors: Vladimir J Kefalov, Maureen E Estevez, Massahiro Kono, Patrice W Goletz, Rosalie K Crouch, Carter M Cornwall, Kingwai YauAbstract:Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-Cis Retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-Cis Retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-Cis Retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.
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11-Cis-Retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors.
Journal of Biological Chemistry, 2002Co-Authors: Zsolt Ablonczy, Patrice W Goletz, Rosalie K Crouch, T. Michael Redmond, Jian Xing, Daniel R. Knapp, Baerbel RohrerAbstract:Abstract Rpe65 −/− mice produce minimal amounts of 11-Cis-Retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65 −/− mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65 −/− mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-Cis-Retinal to Rpe65 −/−mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-Cis-Retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-Cis-Retinal, does not generate the activity generally associated with the bleached apoprotein.
Yiannis Koutalos - One of the best experts on this subject based on the ideXlab platform.
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Relative Contributions of All-Trans and 11-Cis Retinal to Formation of Lipofuscin and A2E Accumulating in Mouse Retinal Pigment Epithelium.
Investigative ophthalmology & visual science, 2021Co-Authors: Nicholas P. Boyer, Debra A. Thompson, Yiannis KoutalosAbstract:Purpose Bis-retinoids are a major component of lipofuscin that accumulates in the Retinal pigment epithelium (RPE) in aging and age-related macular degeneration (AMD). Although bis-retinoids are known to originate from Retinaldehydes required for the light response of photoreceptor cells, the relative contributions of the chromophore, 11-Cis Retinal, and photoisomerization product, all-trans Retinal, are unknown. In photoreceptor outer segments, all-trans Retinal, but not 11-Cis Retinal, is reduced by retinol dehydrogenase 8 (RDH8). Using Rdh8-/- mice, we evaluated the contribution of increased all-trans Retinal to the formation and stability of RPE lipofuscin. Methods Rdh8-/- mice were reared in cyclic-light or darkness for up to 6 months, with selected light-reared cohorts switched to dark-rearing for the final 1 to 8 weeks. The bis-retinoid A2E was measured from chloroform-methanol extracts of RPE-choroid using HPLC-UV/VIS spectroscopy. Lipofuscin fluorescence was measured from whole flattened eyecups (excitation, 488 nm; emission, 565-725 nm). Results Cyclic-light-reared Rdh8-/- mice accumulated A2E and RPE lipofuscin approximately 1.5 times and approximately 2 times faster, respectively, than dark-reared mice. Moving Rdh8-/- mice from cyclic-light to darkness resulted in A2E levels less than expected to have accumulated before the move. Conclusions Our findings establish that elevated levels of all-trans Retinal present in cyclic-light-reared Rdh8-/- mice, which remain low in wild-type mice, contribute only modestly to RPE lipofuscin formation and accumulation. Furthermore, decreases in A2E levels occurring after moving cyclic-light-reared Rdh8-/- mice to darkness are consistent with processing of A2E within the RPE and the existence of a mechanism that could be a therapeutic target for controlling A2E cytotoxicity.
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Photooxidation mediated by 11-Cis and all-trans Retinal in single isolated mouse rod photoreceptors
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2020Co-Authors: Chunhe Chen, Masahiro Kono, Yiannis KoutalosAbstract:Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis Retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans Retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-Cis Retinal continually supplied to photoreceptors. The released all-trans Retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-Cis and all-trans Retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-Cis or all-trans Retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-Cis Retinal. The results indicate that both 11-Cis and all-trans Retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans Retinal generated by light through its reduction to retinol minimizes all-trans Retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-Cis Retinal, 11-Cis-Retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.
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The 11-Cis Retinal Origins of Lipofuscin in the Retina.
Progress in Molecular Biology and Translational Science, 2015Co-Authors: Leopold Adler, Chunhe Chen, Nicholas P. Boyer, Zsolt Ablonczy, Yiannis KoutalosAbstract:Lipofuscin is a fluorescent mixture of partially digested proteins and lipids that accumulates with age in the lysosomal compartment of the Retinal pigment epithelium (RPE) of the eye. Because it has been found to have significant cytotoxic potential, lipofuscin is thought to play a role in Retinal degeneration diseases including age-related macular degeneration and Stargardt disease, a form of juvenile macular degeneration. The only known components of lipofuscin are bis-retinoids, the condensation products of two molecules of Retinal. The bulk of lipofuscin is thought to originate in the rod photoreceptor outer segments as a by-product of reactions involving the Retinal chromophore of rhodopsin. 11-Cis Retinal flows from the RPE into the rod outer segments, where it combines with opsin to form rhodopsin; all-trans Retinal is released into the rod outer segments by photoactivated rhodopsin following its excitation by light. Both 11-Cis and all-trans Retinal can generate lipofuscin-like fluorophores and bis-retinoids when added to rod outer segment membranes. The levels of lipofuscin precursor fluorophores present in the outer segments of dark-adapted rods are similar in cyclic-light- and dark-reared mice, as are the levels of accumulated lipofuscin in the RPE. Because the retinol dehydrogenase enzyme present in rod outer segments can reduce all-trans but not 11-Cis Retinal, lipofuscin precursors are more likely to form from 11-Cis than all-trans Retinal, even under cyclic light conditions. Thus, 11-Cis Retinal may be the primary source of lipofuscin in the retina.
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low aqueous solubility of 11 Cis Retinal limits the rate of pigment formation and dark adaptation in salamander rods
The Journal of General Physiology, 2012Co-Authors: Rikard Frederiksen, Yiannis Koutalos, Nicholas P. Boyer, Benjamin Nickle, Kalyan S Chakrabarti, Daniel D Oprian, Carter M CornwallAbstract:We report experiments designed to test the hypothesis that the aqueous solubility of 11-Cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-Cis-Retinal and an analogue retinoid, 11-Cis 4-OH Retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-Cis 4-OH Retinal is more soluble in aqueous buffer than 11-Cis-Retinal. (b) Both 11-Cis-Retinal and 11-Cis 4-OH Retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-Cis-Retinal is roughly 50-fold greater than that of 11-Cis 4-OH Retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-Cis-Retinal or 11-Cis 4-OH Retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-Cis 4-OH Retinal regenerated on average fivefold faster than rods treated with 11-Cis-Retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-Cis 4-OH Retinal than with 11-Cis-Retinal. Based on these observations, we propose a model in which aqueous solubility of Cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.
