RESUMO
Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd-/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and lipid deposits in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Furthermore, the RPE of Prcd-/- mice exhibit a 1.7-fold increase in the expression of lipid transporter gene ATP-binding cassette transporter A1 (Abca1). Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd-/- mice exhibited age-related pathological features such as lipofuscin accumulation, Bruch's membrane (BrM) deposits and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd-/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd-/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.
Assuntos
Modelos Animais de Doenças , Epitélio Pigmentado da Retina , Tomografia de Coerência Óptica , Animais , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Camundongos , Metabolismo dos Lipídeos , Camundongos Knockout , Camundongos Endogâmicos C57BL , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Distrofias de Cones e Bastonetes/metabolismo , Distrofias de Cones e Bastonetes/genética , Eletrorretinografia , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Imuno-Histoquímica , Degeneração Macular/congênitoRESUMO
The outer segments of photoreceptors are specialized sensory cilia crucial for light detection. Any disruption that alters outer segment morphology can impair photoreceptor function and therefore vision. Progressive rod-cone degeneration (PRCD) is an integral membrane protein exclusively present in the photoreceptor OS with an unknown function. Multiple mutations in PRCD are linked with retinitis pigmentosa. The most common PRCD mutation observed in both human and multiple dog breeds, PRCD-C2Y, lacks the lipid modification "palmitoylation," which is crucial for protein stability and trafficking to the OS. Previous studies including ours show impaired disc morphogenesis and rhodopsin distributions in the absence of PRCD, but the precise role of PRCD in maintaining OS structure and function remains unclear. In this chapter, we discuss the potential role of PRCD in the maintenance of photoreceptor OS structural and functional integrity.
Assuntos
Cílios , Proteínas do Olho , Animais , Cães , Humanos , Proteínas do Olho/genética , Cílios/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Homeostase , LipídeosRESUMO
Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases.
Assuntos
Retinose Pigmentar , Rodopsina , Animais , Cisteína/genética , Cães , Proteínas do Olho/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Estabilidade Proteica , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Rodopsina/genética , Rodopsina/metabolismoRESUMO
The purpose of this study was to establish spectral domain optical coherence tomography (SD-OCT) assessment data in well-established canine models of inherited retinal dystrophies: PDE6B-rod-cone dysplasia 1 (RCD1: early onset retinitis pigmentosa), PRCD-progressive rod-cone degeneration (PRCD: late onset retinitis pigmentosa), CNGB3-achromatopsia, and RPE65-Leber congenital amaurosis (LCA). High resolution SD-OCT images of the retina were acquired from both eyes in 5 planes: temporal; superotemporal; superior; nasal; and inferior in adult dogs with: RCD1 (n = 4 dogs, median age: 1.5 yrs); PRCD (n = 2, 4.3 yrs); LCA (n = 3, 5.2 yrs); achromatopsia (n = 3, 4.2 yrs); and wild types (wt, n = 6, 5.5 yrs). Total, inner and outer retinal thicknesses and ellipsoid zone were analyzed. In selected animals, histomorphometric evaluations were performed. In dogs with RCD1, PRCD, and LCA, the thickness of the outer retina was, compared to wt, significantly decreased (p ≤ 0.02) in all OCT imaging planes, and in superotemporal and inferior imaging planes in dogs with achromatopsia. No significant thinning was observed in inner retina thickness in any disease model except in the inferior imaging plane in dogs with RCD1. Dogs with RCD1, PRCD, and LCA had significantly more areas with disrupted ellipsoid zone in the presumed area centralis than wt (p ≤ 0.001). OCT findings provide baseline information for research of retinal dystrophies using these canine models.
Assuntos
Defeitos da Visão Cromática , Distrofias Retinianas , Retinose Pigmentar , Animais , Defeitos da Visão Cromática/diagnóstico por imagem , Defeitos da Visão Cromática/genética , Cães , Retina/diagnóstico por imagem , Distrofias Retinianas/diagnóstico por imagem , Distrofias Retinianas/genética , Retinose Pigmentar/diagnóstico por imagem , Retinose Pigmentar/genética , Tomografia de Coerência ÓpticaRESUMO
PRCD (progressive rod-cone degeneration) is a small ~6 kDa protein with unknown function that specifically resides in photoreceptor discs and interacts with rhodopsin. PRCD's discovery resulted from decades-long study of a canine retinal disease called progressive rod-cone degeneration which is one of the most frequent causes of blindness in dogs characterized by the slow, progressive death of rod photoreceptors followed by cones. A series of genetic studies eventually mapped the disease to a single point mutation in a novel gene which was then named Prcd. Highlighting the importance of this gene, this and several other mutations have been identified in human patients suffering from retinitis pigmentosa. In this review, we highlight what is currently known about PRCD protein, including the etiology and pathology of the retinal disease caused by its mutation, the protein's trafficking, localization, and biochemical characterization.
Assuntos
Proteínas de Transporte/genética , Proteínas do Olho/genética , Retinose Pigmentar/genética , Animais , Cães , Humanos , Mutação , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/patologia , Rodopsina/metabolismoRESUMO
Progressive retinal atrophy (PRA) due to the c.5G>A mutation in the progressive rod-cone degeneration (PRCD) gene is an important genetic disease in English cocker spaniel (ECS) dogs. Because the prevalence of this disease has not been verified in Brazil, this study aimed to evaluate the allele frequency of the c.5G>A mutation in the PRCD gene. Purified DNA from 220 ECS dogs was used for genotyping, of which 131 were registered from 18 different kennels and 89 were unregistered. A clinical eye examination was performed in 28 of the genotyped animals; 10 were homozygous mutants. DNA fragments containing the mutation region were amplified by PCR and subjected to direct genomic sequencing. The prcd-PRA allele frequency was 25.5%. Among the registered dogs, the allele frequency was 14.9%; among the dogs with no history of registration, the allele frequency was 41%. Visual impairment was observed in 80% (8/10) of the homozygous mutant animals that underwent clinical eye examination. The high mutation frequency found in this study emphasizes the importance of genotyping ECSs as an early diagnostic test, especially as part of an informed breeding program, to avoid clinical cases of PRA.
RESUMO
Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.
Assuntos
Proteínas de Membrana/deficiência , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Distrofias de Cones e Bastonetes/veterinária , Modelos Animais de Doenças , Cães , Espaço Extracelular/metabolismo , Proteínas do Olho/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Retinose Pigmentar/genética , Retinose Pigmentar/patologiaRESUMO
Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein with unknown function that is associated with retinitis pigmentosa (RP). The most common mutation in PRCD linked with severe RP phenotype is substitution of the only cysteine to tyrosine (C2Y). In this study, we find that PRCD is post-translationally modified by a palmitoyl lipid group at the cysteine residue linked with RP. Disrupting PRCD palmitoylation either chemically or by genetically eliminating the modified cysteine dramatically affects the stability of PRCD. Furthermore, in vivo electroporation of PRCD C2Y mutant in the mouse retina demonstrates that the palmitoylation of PRCD is important for its proper localization in the photoreceptor OS. Mutant PRCD C2Y was found in the inner segment in contrast to normal localization of WT PRCD in the OS. Our results also suggest that zDHHC3, a palmitoyl acyltransferase (PAT), catalyzes the palmitoylation of PRCD in the Golgi compartment. In conclusion, we find that the palmitoylation of PRCD is crucial for its trafficking to the photoreceptor OS and mislocalization of this protein likely leads to RP-related phenotypes.
Assuntos
Proteínas do Olho/química , Proteínas do Olho/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Retinose Pigmentar/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas do Olho/genética , Humanos , Lipoilação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade Proteica , Transporte Proteico , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genéticaRESUMO
The objective of the study was to examine whether a nutritional antioxidant supplementation could improve visual function in healthy dogs as measured by electroretinography (ERG) and autorefraction. A total of twelve Beagles, 6 to 8 years of age, with normal eyes upon indirect ophthalmoscopy and slit lamp biomicroscopy, were age and sex matched and randomly assigned to receive a feeding regimen for 6 months with or without a daily antioxidant supplementation. Portable, mini-Ganzfeld ERG and a Welch Allyn hand-held autorefractor were used to test retinal response and refractive error in the dogs at baseline and at the end of the supplementation period. All ERG a-wave amplitudes obtained were increased in the treatment group compared with those of dogs in the control group, with significant improvements in the scotopic high and photopic single flash cone ERG responses (P < 0·05 for both). For the b-wave amplitudes, all responses were similarly increased, with significant improvements in responses for the scotopic high light intensity stimulation (P < 0·05), and for photopic single flash cone and 30 Hz flicker (P < 0·01 for both) recordings. Change in refractive error was significantly less in the treatment group compared with that of the control group during the 6-month study (P < 0·05). Compared with the control group, the antioxidant-supplemented group showed improvement to varying degrees for retinal function and significantly less decline in refractive error. Dogs, like humans, experience retinal and lens functional decline with age. Antioxidant supplementation as demonstrated may be beneficial and effective in the long-term preservation and improvement of various functions of the canine eye.
RESUMO
Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration. Mutations of the PRCD gene are associated with RP in both dogs and humans. To date, four distinct PRCD mutations have been reported worldwide. Here we report the clinical phenotype of another patient with PRCD mutations, carrying the known p.R18X mutation and a novel missense mutation, p.P25T. This mutation affects a highly conserved amino acid, is predicted to be damaging by several prediction tools, and was not found in the public databases or in 115 ethnically-matched control individuals. The phenotype of this patient resembles that of previously reported patients with PRCD mutations, including bull's eye maculopathy, which appears to be a hallmark of the PRCD-induced phenotype. PRCD encodes for a 54 amino acids long protein with unknown function. The first 20 amino acids appear to encode for a signal peptide (SP), suggesting that PRCD is a secreted protein. To study PRCD secretion, C-terminally myc-tagged PRCD was expressed in cultured cells. Cells and conditioned media were analyzed by Western blot. PRCD was found in both cell extracts and media. However, a truncated PRCD protein lacking the first 20 amino acids was present only in cell extracts and not in media, confirming that PRCD extracellular secretion is mediated by its N-terminal SP. To characterize the secretory pathway of PRCD, various pharmacological agents which interfere with transport of proteins through the ER and Golgi to the plasma membrane were used. PRCD secretion was significantly inhibited by all tested pharmacological agents, confirming that it is secreted through the classic ER/Golgi-dependent secretory pathway. We tested the effect of two mutations on the PRCD protein, and found that p.C2Y, but not p.P25T, affects protein stability, and that neither mutation affects secretion. Our data suggest that PRCD functions as a secreted protein. These findings shed a new light on PRCD function and the etiology of RP.