RESUMEN
Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.
Asunto(s)
Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Retinitis Pigmentosa , Animales , Ratones , Perros , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Mutación , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ratones Noqueados , Amaurosis Congénita de Leber/tratamiento farmacológico , Amaurosis Congénita de Leber/genética , Bromocriptina/farmacología , Bromocriptina/uso terapéutico , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismo , Humanos , Quimioterapia Combinada , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Femenino , AMP Cíclico/metabolismo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/genética , Masculino , Calcio/metabolismoRESUMEN
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.
Asunto(s)
Ceramidas , Receptores de Adiponectina , Retina , Animales , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/genética , Ratones , Ceramidas/metabolismo , Retina/metabolismo , Retina/patología , Ratones Noqueados , Ácidos Grasos Insaturados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genéticaRESUMEN
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.
Asunto(s)
Degeneración Macular , Degeneración Retiniana , Humanos , Ratones , Animales , Enfermedad de Stargardt/metabolismo , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/genética , Degeneración Macular/metabolismo , Retinaldehído/metabolismo , Retina/metabolismo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Modelos Animales de Enfermedad , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
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.
Asunto(s)
Epitelio Pigmentado de la Retina , Retinaldehído , Animales , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/metabolismo , Pigmentos Retinianos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neuroglía/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismoRESUMEN
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.
Asunto(s)
Retinopatía Diabética , Degeneración Macular , Degeneración Retiniana , Retinitis Pigmentosa , Humanos , Retina/metabolismo , Degeneración Retiniana/metabolismo , Retinitis Pigmentosa/metabolismo , Degeneración Macular/patología , Retinopatía Diabética/metabolismoRESUMEN
Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.
Asunto(s)
Amaurosis Congénita de Leber , Degeneración Retiniana , cis-trans-Isomerasas , Animales , Proteínas del Ojo/genética , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Ratones , Ratones Noqueados , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/complicaciones , Degeneración Retiniana/genética , Degeneración Retiniana/terapia , cis-trans-Isomerasas/genéticaRESUMEN
Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.
Asunto(s)
Ceramidasas/antagonistas & inhibidores , ADN/genética , Mutación , Receptores de Adiponectina/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Enfermedades de la Retina/genética , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores de Adiponectina/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patologíaRESUMEN
Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Ojo/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Bovinos , Membrana Celular/ultraestructura , Proteínas del Ojo/inmunología , Lipidómica , Proteínas de la Membrana/inmunología , Ratones Endogámicos BALB C , Ratones Noqueados , Microscopía Electrónica de Transmisión , Nanotecnología , Periferinas/metabolismo , Células Fotorreceptoras Retinianas Bastones/ultraestructura , Rodopsina/metabolismo , Anticuerpos de Dominio Único/inmunología , Tetraspaninas/metabolismoRESUMEN
The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.
Asunto(s)
Modelos Animales de Enfermedad , Ratones , Modelos Animales , Receptores de Estrógenos/genética , Enfermedades de la Retina/genética , Epitelio Pigmentado de la Retina/metabolismo , cis-trans-Isomerasas/genética , Animales , Técnicas de Sustitución del Gen , Integrasas/genética , Ratones Transgénicos , Reproducibilidad de los Resultados , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/fisiopatología , Epitelio Pigmentado de la Retina/fisiopatología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , cis-trans-Isomerasas/metabolismoRESUMEN
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.
Asunto(s)
Diterpenos/farmacología , Péptidos/farmacología , Degeneración Retiniana/prevención & control , Visión Ocular/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Oxidorreductasas de Alcohol/genética , Animales , Diterpenos/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Retiniana/fisiopatologíaRESUMEN
Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.
Asunto(s)
Adenina/metabolismo , Edición Génica/métodos , Enfermedades de la Retina/metabolismo , Visión Ocular/fisiología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Codón sin Sentido/genética , Vectores Genéticos/fisiología , Lentivirus/fisiología , Ratones Endogámicos C57BLRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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.
Asunto(s)
Biopsia/métodos , Imagen Óptica/métodos , Retina/diagnóstico por imagen , Animales , Modelos Animales de Enfermedad , Colorantes Fluorescentes , Ratones , Ratones Endogámicos C57BL , Retina/química , Enfermedades de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/química , Epitelio Pigmentado de la Retina/diagnóstico por imagenRESUMEN
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.
Asunto(s)
Epigénesis Genética/genética , Histona Desacetilasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Degeneración Macular/genética , Degeneración Retiniana/genética , Anciano , Animales , Cromatina/genética , Modelos Animales de Enfermedad , Histona Desacetilasas/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/patología , Ratones , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Trastornos por Fotosensibilidad/genética , Retina/metabolismo , Retina/patología , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
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.
Asunto(s)
Células Fotorreceptoras de Vertebrados/citología , Sustancias Protectoras/farmacología , Clorhidrato de Raloxifeno/farmacología , Degeneración Retiniana/prevención & control , Epitelio Pigmentado de la Retina/citología , Retinaldehído/toxicidad , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transportadoras de Casetes de Unión a ATP/fisiología , Oxidorreductasas de Alcohol/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/metabolismo , Oxidorreductasas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Visión Ocular/fisiología , Animales , Células Ependimogliales/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinaldehído/metabolismo , Transducina/metabolismoRESUMEN
Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.
Asunto(s)
Rayos Láser , Oftalmoscopía/métodos , Fotones , Retina/diagnóstico por imagen , Enfermedades de la Retina/diagnóstico por imagen , Transportadoras de Casetes de Unión a ATP/genética , Oxidorreductasas de Alcohol/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Rayos Infrarrojos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Imagen Óptica/métodos , Retina/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , cis-trans-Isomerasas/genéticaRESUMEN
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.
Asunto(s)
Sustancias Protectoras/farmacología , Retina/efectos de los fármacos , Degeneración Retiniana/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Animales , Diterpenos , Luz , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Opsinas/metabolismo , Retina/metabolismo , Retinaldehído/metabolismo , Retinoides/metabolismoRESUMEN
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.
Asunto(s)
Genes Dominantes , Retina/metabolismo , Retinitis Pigmentosa/genética , cis-trans-Isomerasas/genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Técnicas de Sustitución del Gen , Humanos , Ratones , Mutación , Agregación Patológica de Proteínas/genética , Pliegue de Proteína , Retina/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/patología , cis-trans-Isomerasas/químicaRESUMEN
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.