6 research outputs found
Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function
Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms
Blue light regenerates functional visual pigments in mammals through a retinyl-phospholipid intermediate.
The light absorbing chromophore in opsin visual pigments is the protonated Schiff base of 11-cis-retinaldehyde (11cRAL). Absorption of a photon isomerizes 11cRAL to all-trans-retinaldehyde (atRAL), briefly activating the pigment before it dissociates. Light sensitivity is restored when apo-opsin combines with another 11cRAL to form a new visual pigment. Conversion of atRAL to 11cRAL is carried out by enzyme pathways in neighboring cells. Here we show that blue (450-nm) light converts atRAL specifically to 11cRAL through a retinyl-phospholipid intermediate in photoreceptor membranes. The quantum efficiency of this photoconversion is similar to rhodopsin. Photoreceptor membranes synthesize 11cRAL chromophore faster under blue light than in darkness. Live mice regenerate rhodopsin more rapidly in blue light. Finally, whole retinas and isolated cone cells show increased photosensitivity following exposure to blue light. These results indicate that light contributes to visual-pigment renewal in mammalian rods and cones through a non-enzymatic process involving retinyl-phospholipids.It is currently thought that visual pigments in vertebrate photoreceptors are regenerated exclusively through enzymatic cycles. Here the authors show that mammalian photoreceptors also regenerate opsin pigments in light through photoisomerization of N-ret-PE (N-retinylidene-phosphatidylethanolamine
Function of mammalian M-cones depends on the level of CRALBP in MĂŒller cells
Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the MĂŒller cell-driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in MĂŒller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by âŒ50% in both the RPE and retina of Rlbp1+/- mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/- mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/- mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the MĂŒller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1-/- mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation
Caractérisation structurale et de liaison membranaire de rétinol déshydrogénases
Les rĂ©tinol dĂ©shydrogĂ©nases ou RDHs sont des oxydorĂ©ductases inhĂ©rentes Ă lâaccomplissement de la fonction visuelle de la rĂ©tine. Elles sont en effet impliquĂ©es dans le cycle visuel rĂ©tinien. Suite Ă lâabsorption de la lumiĂšre par le pigment visuel des photorĂ©cepteurs, la rhodopsine, la RDH8 est la premiĂšre enzyme qui va intervenir dans le cycle visuel aprĂšs la libĂ©ration du chromophore de la rhodopsine, le tout-trans rĂ©tinal. Ainsi, la RDH8 dĂ©toxifie les photorĂ©cepteurs car le tout-trans rĂ©tinal est une espĂšce trĂšs rĂ©active qui peut induire des dommages Ă la rĂ©tine. La RDH11, quant Ă elle, agit de concert avec la RDH5 au niveau de la derniĂšre Ă©tape du cycle visuel dans lâĂ©pithĂ©lium pigmentaire rĂ©tinien en transformant le 11-cis rĂ©tinol en 11-cis rĂ©tinal, qui sera rĂ©acheminĂ© vers les photorĂ©cepteurs pour rĂ©gĂ©nĂ©rer le pigment visuel. Toutefois, la structure tertiaire des RDHs nâa encore jamais Ă©tĂ© rĂ©solue. Ces enzymes sont nĂ©anmoins reconnues pour ĂȘtre associĂ©es aux membranes cellulaires par leur segment N- et/ou C-terminal. Nous avons alors entrepris ce travail afin de caractĂ©riser la structure de ces enzymes et mieux comprendre leur interaction avec les membranes. Nous avons Ă©tudiĂ© dans un premier temps diffĂ©rentes portions du segment N- et C-terminal de la RDH11 et la RDH8 respectivement, par diffĂ©rentes mĂ©thodes spectroscopiques. Nous avons alors observĂ© que les segments de ces deux enzymes agissent par deux modes dâaction totalement diffĂ©rents. La RDH11 ferait appel Ă un segment N-terminal transmembranaire qui adopte une conformation hĂ©licale peu importe sa longueur, alors que la RDH8 utiliserait un segment C-terminal qui adopte une structure secondaire variable selon la longueur et dont la liaison est pĂ©riphĂ©rique Ă la membrane. En plus, la liaison de la RDH8 par son segment C-terminal serait potentiellement facilitĂ©e par une ou plusieurs acylations situĂ©es au niveau de certaines cystĂ©ines. Les mesures de pression dâinsertion maximale ont permis de comparer les interactions entre des segments de longueur variable en N-terminal de la RDH11 et en C-terminal de la RDH8 avec des monocouches de diffĂ©rents phospholipides. Ainsi, nous avons dĂ©terminĂ© les interactions les plus favorables pour chacun de ces segments. Nous nous sommes focalisĂ©s par la suite sur lâĂ©tude de lâenzyme RDH8 et la comparaison de ses propriĂ©tĂ©s structurales, de stabilitĂ© et de liaison membranaire avec celles de sa forme tronquĂ©e RDH8t, dĂ©pourvue de son segment en C-terminal. Notons que nous avons mis au point un protocole adaptĂ© pour surexprimer et purifier la RDH8 et sa forme tronquĂ©e. Ă notre connaissance, il sâagit ici des premiers travaux de recherche rapportant la surexpression et la purification dâune RDH8 (bovine) complĂšte dans un systĂšme procaryote (E. coli). Nous avons alors constatĂ© que les deux formes de la RDH8, complĂšte et tronquĂ©e, comprenaient majoritairement des hĂ©lices α en plus de la prĂ©sence de feuillets ÎČ, en accord avec le motif de Rossmann fold suggĂ©rĂ© dans la littĂ©rature pour cette famille dâenzymes. Il sâest avĂ©rĂ© Ă©galement que le segment C-terminal a un impact sur la stabilitĂ© de la RDH8 comme dĂ©montrĂ© par les mesures du contenu en structure secondaire de ces protĂ©ines en fonction des conditions de stockage et dans les expĂ©rimentations de dĂ©naturation thermique. Enfin, les mesures de pression dâinsertion maximale (PIM) et de synergie ont dĂ©montrĂ© que le segment C-terminal facilitait la liaison membranaire de la forme complĂšte par rapport Ă la forme tronquĂ©e, notamment dans le contexte de phospholipides portant une tĂȘte polaire chargĂ©e nĂ©gativement. Lâinteraction membranaire de la RDH8 pourrait donc impliquer des interactions Ă©lectrostatiques. Des expĂ©riences de spectroscopie de fluorescence ont permis de confirmer lâimplication du segment C-terminal dans la liaison de la RDH8 avec des bicouches lipidiques grĂące Ă la prĂ©sence de deux rĂ©sidus tryptophanes uniquement dans son segment C-terminal.In the retina, retinol dehydrogenases (RDHs) play a crucial role in the visual cycle allowing a good vision. The first step of the visual cycle is taking place in photoreceptors where RDH8 is located and then in the retinal pigmented epithelium (RPE) where RDH11 can be found. RDH11 is likely anchored to membranes by means of its N-terminal segment whereas RDH8 has been postulated to be membrane bound via its C-terminal segment. So, to better evaluate the role of the N-terminal segment of RDH11 and the C-terminal segment of RDH8 in the membrane binding of these proteins, different variants (Long and Short) of the aforementioned segments have been studied. In addition, mutations of the C-terminal segment of RDH8 have been introduced to monitor their interaction with lipid monolayers or bilayers. We have thus analyzed the secondary structure content of these segments by conventional spectroscopic methods such as circular dichroism (CD) and attenuated total reflectance (ATR) infrared spectroscopy whereas their interaction with phospholipids have been mainly monitored by surface pressure measurements when using monolayers and fluorescence spectroscopy for bilayers. Overall, we found that the N-terminal segment of RDH11 adopts an α-helix conformation acting as a transmembrane domain. Values of maximum insertion pressure (MIP) and synergy suggested a preferential binding of the RDH11 Long-peptide to phosphoethanolamine, which are abundant in the RPE. In the case of RDH8, our findings suggest an important role of the long C-terminal segment in membrane binding, which is supported by its helical content and the larger values of MIP and synergy. We also compared the behavior of RDH8 and its truncated form (RDH8t, without its C-terminal segment) to better understand the involvement of this segment in membrane binding. Thus, both enzymes have been expressed in E. coli, purified by affinity chromatography and studied by the spectroscopic methods mentioned above and by using MIP and synergy measurements. RDH8 and RDH8t display a secondary structure content in agreement with their predicted Rossmann fold. Interestingly, the removal of the C-terminal segment decreased the temporal and thermal stability of these enzymes. In addition, this segment contributes to protein-lipid interaction especially in presence of negatively charged phospholipids likely through electrostatic interactions. The involvement of the C-terminal segment of the RDH8 in its membrane anchoring has been further confirmed by fluorescence measurements of its two Trp residues located in this segment. The present characterization of RDH8 is a first step paving the way for the elucidation of its structural and functional features to gain a better understanding of its role within the visual cycle and investigating mechanisms of retinal pathogenesis
The development of a clinical trial protocol and functional biomarkers for age-related macular degeneration
Age-related macular degeneration (AMD) is the leading cause of blindness amongst older adults in the developed world. With the predicted rise in the ageing population over the next decades, the prevalence of this debilitating disease will simply continue to increase. The only treatments currently available are for advanced neovascular AMD. The retina is already severely compromised by this stage in disease development. Therefore, there is a pressing need to evaluate potential novel interventions that aim to prevent the development of advanced disease in people with early AMD, to prevent sight loss from occurring. Furthermore, it is necessary to develop tests that are sensitive to subtle changes in visual function in order to evaluate the efficacy of these emerging treatments.
There is a growing body of evidence to suggest that hypoxia contributes to the development of AMD. Hypoxia is most acute at night when the retinal photoreceptors are most metabolically active, due to the demands of the rod dark current. Increasing the light levels at night will cause the oxygen demand, and hence the hypoxia, to be substantially diminished. This leads to the hypothesis that providing low level night time light therapy to people with early AMD may slow disease progression by reducing hypoxia.
In order to evaluate the potential effectiveness of such an intervention, it is necessary to select appropriate outcome measures. The inherent variability of the standard test of visual function, visual acuity, renders it inappropriate for use as a primary outcome measure in proof of concept clinical trials. Therefore, the first aim of this thesis was to evaluate the diagnostic validity and repeatability of alternative functional tests that may be used as biomarkers for early macular disease.
Dark adaptation was evaluated using three stimuli, a spot of 2o radius and annuli of 7o and 12o radii, in 21 healthy adults (on two occasions) and in 11 participants with early AMD. All stimuli were found to be highly diagnostic for early AMD. The spot of 2o radius provided the best separation between groups with respect to the time constant of cone recovery (area under the ROC curve 0.91). The repeatability of chromatic and flicker thresholds were also assessed in 30 healthy adults. The coefficient of repeatability, expressed as a percentage of the mean threshold, was 17.1% for red-green chromatic thresholds, 31.1% for blue-yellow, 53.4% for 14Hz flicker thresholds, and ranged between 36.4%-53.3% for parameters of dark adaptation. A small learning effect was found for both chromatic thresholds and the 14-Hz flicker test, indicating that a control group is needed in studies of new therapeutic interventions.
The second aim of this thesis was to develop a protocol for a clinical trial that seeks to determine if low level night time light therapy can prevent the progression of early AMD. The level of retinal illuminance required to suppress the rod dark current, the maximum retinal illuminance which prevents substantial suppression of melatonin secretion, and the most appropriate means of delivering the dose of retinal illumination were evaluated. The final protocol employed an organic LED illuminated light mask, worn during hours of sleep, as the mode of intervention.
In conclusion, this thesis has confirmed that cone dark adaptation is a sensitive functional biomarker for AMD, and that all three functional tests have a good inter-session repeatability. These biomarkers will be validated in the prospective clinical trial of low-level light therapy to confirm their prognostic and predictive capabilities. The proposed trial will also evaluate the effectiveness of the low level night time light therapy, delivered by means of an illuminated light mask, at slowing the progression of early AMD