Distinct mouse models of Stargardt disease display differences in pharmacological targeting of ceramides and inflammatory responses.

Mutations in many visual cycle enzymes in photoreceptors and retinal pigment epithelium (RPE) cells can lead to the chronic accumulation of toxic retinoid byproducts, which poison photoreceptors and the underlying RPE if left unchecked. Without a functional ATP-binding cassette, sub-family A, member 4 (ABCA4), there is an elevation of all-trans-retinal and prolonged buildup of all-trans-retinal adducts, resulting in a retinal degenerative disease known as Stargardt-1 disease. Even in this monogenic disorder, there is significant heterogeneity in the time to onset of symptoms among patients. Using a combination of molecular techniques, we studied Abca4 knockout (simulating human noncoding disease variants) and Abca4 knock-in mice (simulating human misfolded, catalytically inactive protein variants), which serve as models for Stargardt-1 disease. We compared the two strains to ascertain whether they exhibit differential responses to agents that affect cytokine signaling and/or ceramide metabolism, as alterations in either of these pathways can exacerbate retinal degenerative phenotypes. We found different degrees of responsiveness to maraviroc, a known immunomodulatory CCR5 antagonist, and to the ceramide-lowering agent AdipoRon, an agonist of the ADIPOR1 and ADIPOR2 receptors. The two strains also display different degrees of transcriptional deviation from matched WT controls. Our phenotypic comparison of the two distinct Abca4 mutant-mouse models sheds light on potential therapeutic avenues previously unexplored in the treatment of Stargardt disease and provides a surrogate assay for assessing the effectiveness for genome editing.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Restoring retinal polyunsaturated fatty acid balance and retina function by targeting ceramide in AdipoR1-deficient mice.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration. This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 fatty acids, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium. Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in Bardet-Biedl syndrome-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including age-related macular degeneration.

Noninvasive two-photon optical biopsy of retinal fluorophores.

High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.

Epigenetic hallmarks of age-related macular degeneration are recapitulated in a photosensitive mouse model.

Age-related macular degeneration (AMD) is a chronic, multifactorial disorder and a leading cause of blindness in the elderly. Characterized by progressive photoreceptor degeneration in the central retina, disease progression involves epigenetic changes in chromatin accessibility resulting from environmental exposures and chronic stress. Here, we report that a photosensitive mouse model of acute stress-induced photoreceptor degeneration recapitulates the epigenetic hallmarks of human AMD. Global epigenomic profiling was accomplished by employing an Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq), which revealed an association between decreased chromatin accessibility and stress-induced photoreceptor cell death in our mouse model. The epigenomic changes induced by light damage include reduced euchromatin and increased heterochromatin abundance, resulting in transcriptional and translational dysregulation that ultimately drives photoreceptor apoptosis and an inflammatory reactive gliosis in the retina. Of particular interest, pharmacological inhibition of histone deacetylase 11 (HDAC11) and suppressor of variegation 3-9 homolog 2 (SUV39H2), key histone-modifying enzymes involved in promoting reduced chromatin accessibility, ameliorated light damage in our mouse model, supporting a causal link between decreased chromatin accessibility and photoreceptor degeneration, thereby elucidating a potential new therapeutic strategy to combat AMD.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration.

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4-/-Rdh8-/-), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.

Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Cone photoreceptors are essential for vision under moderate to high illuminance and allow color discrimination. Their fast dark adaptation rate and resistance to saturation are believed to depend in part on an intraretinal visual cycle that supplies 11- cis-retinaldehyde to cone opsins. Candidate enzymes of this pathway have been reported, but their physiologic contribution to cone photoresponses remains unknown. Here, we evaluate the role of a candidate retinol isomerase of this pathway, sphingolipid δ4 desaturase 1 (Des1). Single-cell RNA sequencing analysis revealed Des1 expression not only in Müller glia but also throughout the retina and in the retinal pigment epithelium. We assessed cone functional dependence on Müller cell-expressed Des1 through a conditional knockout approach. Floxed Des1 mice, on a guanine nucleotide-binding protein subunit α transducin 1 knockout ( Gnat1-/-) background to allow isolated recording of cone-driven photoresponses, were bred with platelet-derived growth factor receptor α (Pdgfrα)-Cre mice to delete Des1 in Müller cells. Conditional knockout of Des1 expression, as shown by tissue-selective Des1 gene recombination and reduced Des1 catalytic activity, caused no gross changes in the retinal structure and had no effect on cone sensitivity or dark adaptation but did slightly accelerate the rate of cone phototransduction termination. These results indicate that Des1 expression in Müller cells is not required for cone visual pigment regeneration in the mouse.-Kiser, P. D., Kolesnikov, A.V., Kiser, J. Z., Dong, Z., Chaurasia, B., Wang, L., Summers, S. A., Hoang, T., Blackshaw, S., Peachey, N. S., Kefalov, V. J., Palczewski, K. Conditional deletion of Des1 in the mouse retina does not impair the visual cycle in cones.

Dephosphorylation by protein phosphatase 2A regulates visual pigment regeneration and the dark adaptation of mammalian photoreceptors.

Resetting of G-protein-coupled receptors (GPCRs) from their active state back to their biologically inert ground state is an integral part of GPCR signaling. This "on-off" GPCR cycle is regulated by reversible phosphorylation. Retinal rod and cone photoreceptors arguably represent the best-understood example of such GPCR signaling. Their visual pigments (opsins) are activated by light, transduce the signal, and are then inactivated by a GPCR kinase and arrestin. Although pigment inactivation by phosphorylation is well understood, the enzyme(s) responsible for pigment dephosphorylation and the functional significance of this reaction remain unknown. Here, we show that protein phosphatase 2A (PP2A) acts as opsin phosphatase in both rods and cones. Elimination of PP2A substantially slows pigment dephosphorylation, visual chromophore recycling, and ultimately photoreceptor dark adaptation. These findings demonstrate that visual pigment dephosphorylation regulates the dark adaptation of photoreceptors and provide insights into the role of this reaction in GPCR signaling.

Protective Effect of a Locked Retinal Chromophore Analog against Light-Induced Retinal Degeneration.

Continuous regeneration of the 11-cis-retinal visual chromophore from all-trans-retinal is critical for vision. Insufficiency of 11-cis-retinal arising from the dysfunction of key proteins involved in its regeneration can impair retinal health, ultimately leading to loss of human sight. Delayed recovery of visual sensitivity and night blindness caused by inadequate regeneration of the visual pigment rhodopsin are typical early signs of this condition. Excessive concentrations of unliganded, constitutively active opsin and increased levels of all-trans-retinal and its byproducts in photoreceptors also accelerate retinal degeneration after light exposure. Exogenous 9-cis-retinal iso-chromophore can reduce the toxicity of ligand-free opsin but fails to prevent the buildup of retinoid photoproducts when their clearance is defective in human retinopathies, such as Stargardt disease or age-related macular degeneration. Here we evaluated the effect of a locked chromophore analog, 11-cis-6-membered ring-retinal against bright light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Using in vivo imaging techniques, optical coherence tomography, scanning laser ophthalmoscopy, and two-photon microscopy, along with in vitro histologic analysis of retinal morphology, we found that treatment with 11-cis-6-membered ring-retinal before light stimulation prevented rod and cone photoreceptor degradation and preserved functional acuity in these mice. Moreover, additive accumulation of 11-cis-6-membered ring-retinal measured in the eyes of these mice by quantitative liquid chromatography-mass spectrometry indicated stable binding of this retinoid to opsin. Together, these results suggest that eliminating excess of unliganded opsin can prevent light-induced retinal degeneration in Abca4-/-Rdh8-/- mice.

MicroRNA-processing Enzymes Are Essential for Survival and Function of Mature Retinal Pigmented Epithelial Cells in Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss. The neovascular or "wet" form of AMD can be treated to varying degrees with anti-angiogenic drugs, but geographic atrophy (GA) is an advanced stage of the more prevalent "dry" form of AMD for which there is no effective treatment. Development of GA has been linked to loss of the microRNA (miRNA)-processing enzyme DICER1 in the mature retinal pigmented epithelium (RPE). This loss results in the accumulation of toxic transcripts of Alu transposable elements, which activate the NLRP3 inflammasome and additional downstream pathways that compromise the integrity and function of the RPE. However, it remains unclear whether the loss of miRNA processing and subsequent gene regulation in the RPE due to DICER1 deficiency also contributes to RPE cell death. To clarify the role of miRNAs in RPE cells, we used two different mature RPE cell-specific Cre recombinase drivers to inactivate either Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), thus removing RPE miRNA regulatory activity in mice by disrupting two independent and essential steps of miRNA biogenesis. In contrast with prior studies, we found that the loss of each factor independently led to strikingly similar defects in the survival and function of the RPE and retina. These results suggest that the loss of miRNAs also contributes to RPE cell death and loss of visual function and could affect the pathology of dry AMD.

A Combination of G Protein-Coupled Receptor Modulators Protects Photoreceptors from Degeneration.

Degeneration of retinal photoreceptor cells can arise from environmental and/or genetic causes. Since photoreceptor cells, the retinal pigment epithelium (RPE), neurons, and glial cells of the retina are intimately associated, all cell types eventually are affected by retinal degenerative diseases. Such diseases often originate either in rod and/or cone photoreceptor cells or the RPE. Of these, cone cells located in the central retina are especially important for daily human activity. Here we describe the protection of cone cells by a combination therapy consisting of the G protein-coupled receptor modulators metoprolol, tamsulosin, and bromocriptine. These drugs were tested in Abca4-/-Rdh8-/- mice, a preclinical model for retinal degeneration. The specificity of these drugs was determined with an essentially complete panel of human G protein-coupled receptors. Significantly, the combination of metoprolol, tamsulosin, and bromocriptine had no deleterious effects on electroretinographic responses of wild-type mice. Moreover, putative G protein-coupled receptor targets of these drugs were shown to be expressed in human and mouse eyes by RNA sequencing and quantitative polymerase chain reaction. Liquid chromatography together with mass spectrometry using validated internal standards confirmed that metoprolol, tamsulosin, and bromocriptine individually or together penetrate the eye after either intraperitoneal delivery or oral gavage. Collectively, these findings support human trials with combined therapy composed of lower doses of metoprolol, tamsulosin, and bromocriptine designed to safely impede retinal degeneration associated with certain genetic diseases (e.g., Stargardt disease). The same low-dose combination also could protect the retina against diseases with complex or unknown etiologies such as age-related macular degeneration.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium.

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk-/- mice also accumulated fluorescent products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk-/- mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice.

Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4(-/-)Rdh8(-/-) mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4(-/-)Rdh8(-/-) mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE.

Serum levels of lipid metabolites in age-related macular degeneration.

Age-related macular degeneration (AMD) is a neurodegenerative disease that causes adult-onset blindness. There are 2 forms of this progressive disease: wet and dry. Currently there is no cure for AMD, but several treatment options have started to emerge making early detection critical for therapeutic success. Analysis of the eyes of Abca4(-/-)Rdh8(-/-) mice that display light-induced retinal degeneration indicates that 11-cis-retinal and docosahexaenoic acid (DHA) levels were significantly decreased as compared with the eyes of control dark-adapted C57BL/6J mice. In addition, exposure to intense light correlated with higher levels of prostaglandin G2 in the eyes of Abca4(-/-)Rdh8(-/-) mice. Intense light exposure also lowered DHA levels in the eyes of wild-type C57BL/6J mice without discernible retinal degeneration. Analysis of human serum from patients with AMD recapitulated these dysregulated DHA levels and revealed dysregulation of arachidonic acid (AA) levels as well (∼32% increase in patients with AMD compared with average levels in healthy individuals). From these observations, we then built a statistical model that included levels of DHA and AA from human serum. This model had a 74% probability of correctly identifying patients with AMD from controls. Addition of a genetic analysis for one of the most prevalent amino acid substitutions in the age-related maculopathy susceptibility 2 gene linked to AMD, Ala(69)→Ser, did not improve the statistical model. Thus, we have characterized a reliable method with the potential to detect AMD without a genetic component, paving the way for a larger-scale clinical evaluation. Our studies on mouse models along with the analysis of human serum suggest that our small molecule-based model may serve as an effective tool to estimate the risk of developing AMD.

Expansion of first-in-class drug candidates that sequester toxic all-trans-retinal and prevent light-induced retinal degeneration.

All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells.

G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.

Effect of caloric intake on Western-style diet-induced intestinal tumors in a mouse model for hereditary colon cancer.

Increased caloric intake has been associated with increased risk for cancer of the large intestine. We studied caloric intake effect on tumor formation in Apc1638( N/+ ) mice, a preclinical model for human familial adenomatous polyposis. Mice were fed a controlled AIN-76A diet or a new Western-style diet (NWD). Intestinal tumor development was evaluated after 6 mo of feeding 1) AIN-76A diet (fed ad libitum) vs. AIN-76A (caloric intake reduced 30%); 2) NWD (fed ad libitum) vs. NWD (caloric intake reduced 30%); and 3) AIN-76A (fed ad libitum) vs. NWD (paired-fed with NWD providing equal caloric intakes to AIN-76A). Intestinal tumor incidences were 78-100% with intergroup variation P > 0.05; however, tumor multiplicity responded differently to dietary treatment: 1) Tumor multiplicity was unchanged after AIN-76A (caloric intake reduced 30% vs. mice fed AIN-76A ad libitum); 2) tumor multiplicity was unchanged after NWD (caloric intake reduced 30% vs. NWD ad libitum); and 3) tumor multiplicity increased 130% after NWD was paired-fed with the same caloric intake as mice fed AIN-76A ad libitum (P < 0.05). Body weights showed no association with tumor development. Findings indicated modified nutrients in NWD were mainly responsible for increased tumors in mice fed NWD vs. AIN-76A in this preclinical mouse model for human FAP.