RESUMO
Leber congenital amaurosis (LCA) is the most serious form of the autosomal recessive childhood-onset retinal dystrophies. Mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, account for 10-15% of LCA cases. Whereas previous studies of RPE65 deficiency in both animal models and patients attributed remaining visual function to cones, we show here that light-evoked retinal responses in fact originate from rods. For this purpose, we selectively impaired either rod or cone function in Rpe65-/- mice by generating double- mutant mice with models of pure cone function (rhodopsin-deficient mice; Rho-/-) and pure rod function (cyclic nucleotide-gated channel alpha3-deficient mice; Cnga3-/-). The electroretinograms (ERGs) of Rpe65-/- and Rpe65-/-Cnga3-/- mice were almost identical, whereas there was no assessable response in Rpe65-/-Rho-/- mice. Thus, we conclude that the rod system is the source of vision in RPE65 deficiency. Furthermore, we found that lack of RPE65 enables rods to mimic cone function by responding under normally cone-isolating lighting conditions. We propose as a mechanism decreased rod sensitivity due to a reduction in rhodopsin content to less than 1%. In general, the dissection of pathophysiological processes in animal models through the introduction of additional, selective mutations is a promising concept in functional genetics.
Assuntos
Atrofias Ópticas Hereditárias/genética , Epitélio Pigmentado Ocular/fisiologia , Proteínas/genética , Células Fotorreceptoras Retinianas Bastonetes/fisiopatologia , Visão Ocular/fisiologia , Animais , Proteínas de Transporte , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho , Camundongos , Camundongos Mutantes , Atrofias Ópticas Hereditárias/fisiopatologia , cis-trans-IsomerasesRESUMO
PURPOSE: Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. METHODS: An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. RESULTS: Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal-corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin). CONCLUSIONS: The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.
Assuntos
Movimento Celular/genética , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Perfilação da Expressão Gênica , Limbo da Córnea/metabolismo , Limbo da Córnea/patologia , Pterígio/genética , Biomarcadores/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Clusterina/genética , Clusterina/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Imunofluorescência , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Queratinas/genética , Queratinas/metabolismo , Limbo da Córnea/efeitos dos fármacos , Poliaminas/farmacologia , Pterígio/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The term, 'retinitis pigmentosa', refers to a heterogeneous group of inherited diseases that cause degeneration of rod and cone photoreceptors in the human retina. Loss of photoreceptor cells is usually followed by alterations in the retinal pigment epithelium and retinal glia. Ultimately, degenerative changes occur in the inner retinal neurons, blood vessels, and optic nerve head. This chapter provides background information on the genetics of retinitis pigmentosa and a summary of the histopathologic alterations in human retinas caused by this disease. Detailed information is provided on the effects of the primary disease process on the rod photoreceptors and changes in the other retinal components, all of which are important considerations for understanding and developing therapies for retinitis pigmentosa.
Assuntos
Retina/patologia , Retinose Pigmentar/patologia , Astrócitos/patologia , Axônios/patologia , Humanos , Neuroglia/patologia , Disco Óptico/patologia , Células Fotorreceptoras/patologia , Células Ganglionares da Retina/patologia , Vasos Retinianos/patologia , Retinose Pigmentar/genéticaRESUMO
Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.
Assuntos
Gatos/fisiologia , Proteínas de Membrana/fisiologia , Degeneração Neural/fisiopatologia , Segmento Externo da Célula Bastonete/fisiologia , Animais , Transporte Biológico , Imuno-Histoquímica , Valores de ReferênciaRESUMO
Morphogenesis of photoreceptor outer segment disks appears to occur by an evagination of the ciliary plasma membrane (Steinberg et al., J Comp Neurol 190:501-519, '80). We tested if polymerized actin (F-actin) was necessary for the regulation of this postulated process by incubating Xenopus eyecups with 5 or 25 microM cytochalasin D for 6-28 hours. During the second hour, the incubation medium contained 3H-leucine. Both concentrations of cytochalasin resulted in: 1) dissolution of the rhodamine-phalloidin labeling pattern of photoreceptors, and 2) collapse of the calycal processes (which are normally filled with actin filaments) and disappearance of the inner segment microfilaments. In addition, the few most basal rod and cone outer segment disks appeared several times their normal diameter. These oversized disks had incorporated 3H-leucine and extended along the margin of the outer or inner segment. The nature of the overgrown disks is consistent only with a morphogenetic process involving evaginations of the ciliary plasma membrane. Deregulation by cytochalasin D was manifest by excessive growth of a few nascent disks rather than normal growth of many. Therefore, the normal network of actin filaments is apparently not necessary for continued evagination of the membrane, but it does seem to be an essential part of the mechanism that initiates the evagination of the ciliary plasma membrane and/or the mechanism that controls how far nascent disks grow.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Citocalasinas/farmacologia , Citoesqueleto/ultraestrutura , Células Fotorreceptoras/ultraestrutura , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Citocalasina D , Substâncias Macromoleculares , Microscopia Eletrônica , Modelos Anatômicos , Morfogênese/efeitos dos fármacos , Células Fotorreceptoras/efeitos dos fármacos , Xenopus laevisRESUMO
In cephalopods, the complex rhodopsin-retinochrome system serves to regenerate metarhodopsin and metaretinochrome after illumination. In the dark, a soluble protein, retinal-binding protein (RALBP), shuttles 11-cis retinal released from metaretinochrome located in the photoreceptor inner segments to metarhodopsin present in the rhabdoms. While in the rhabdoms, RALBP delivers 11-cis retinal to regenerate rhodopsin and in turn binds the all-trans isomer released by metarhodopsin. RALBP then returns all-trans retinal to the inner segments to restore retinochrome. The conventional interpretation of retinoid cycling is contradicted by immunocytochemical studies showing that, in addition to rhodopsin, retinochrome is present in the rhabdomal compartment, making possible the direct exchange of chromophores between the metapigments with the potential exclusion of RALBP. By using immunofluorescence and laser scanning confocal microscopy, we have precisely located opsin, aporetinochrome, and RALBP in light-/dark-adapted octopus retinas. We found differences in the distribution of all three proteins throughout the retina. Most significantly, comparison of cross sections though light- and dark-adapted rhabdoms showed a dramatic shift in position of the proteins. In the dark, opsin and retinochrome colocalized at the base of the rhabdomal microvilli. In the light, opsin redistributed along the length of the microvillar membranes, and retinochrome retreated to a location that is perhaps extracellular. RALBP was present in the core cytoplasm of the photoreceptor outer segments in the dark, and RALBP moved to the periphery in the light. Because of the colocalization of opsin and retinochrome in the dark, we believe that the two metapigments participate directly in chromophore exchange. RALBP may serve to transport additional chromophore from the inner segments to the rhabdoms and may not be immediately involved in the exchange process.
Assuntos
Adaptação Ocular/fisiologia , Adaptação à Escuridão/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Octopodiformes/metabolismo , Retina/metabolismo , Retinoides/metabolismo , Animais , Imunofluorescência , Histocitoquímica , Microscopia Confocal , Octopodiformes/anatomia & histologia , Células Fotorreceptoras/metabolismo , Retina/ultraestrutura , Pigmentos da Retina/metabolismo , Rodopsina/metabolismo , Opsinas de Bastonetes/metabolismoRESUMO
The macula of the neural retina from 12 adult rhesus monkeys (Macaca mulatta) was detached from the overlying retinal pigment epithelium (RPE) by subretinal injection of a balanced salt solution. Seven days later, the two layers were reapposed by draining fluid from the vitreous cavity and replacing it with a 3:1 mixture of sulphur hexafluride gas and air. Animals were sacrificed at 1 hr, 2 days and 7 days after detachment, and at periods ranging from 3 to 14 days after reattachment. At 2-7 days prior to sacrifice, some eyes received an intravitreal injection of 3H-L-fucose. The eyes were then fixed for light and electron microscopy (EM), and tissue sections were processed for autoradiography (ARG) or immunocytochemistry. During the 7-day detachment interval, rod outer segments (ROSs) and cone outer segments (COSs) degenerated, but inner segments remained intact and the rest of the retina appeared normal. The apical RPE surface dedifferentiated during the detachment interval. At 3 days after reattachment, a regrowth of rudimentary ROSs and COSs had occurred, but the disc stacking was clearly abnormal. ROSs and COSs both showed an increase in length and a tendency to return to their normal configurations with increasing time after reattachment. ROSs and COSs regained approximately 40% of their normal lengths after a 2-week reattachment period; however, persistent outer segment abnormalities were frequently found in otherwise well regenerated areas. Autoradiographic results confirmed that new disc members were synthesized subsequent to reattachment. Newly synthesized rod disc membranes were uniformly labeled using antibodies to bovine opsin. Regenerating outer segments interdigitated with newly formed apical RPE processes, and radiolabeled phagosomes were identified within the RPE cytoplasm by 1 week after reattachment. Proliferation of the RPE cell layer was identified at some locations in all animals, and was strongly correlated with a lack of underlying outer segment regeneration. Because of the short detachment interval, and the absence of underlying pathology or trauma, the recovery process described here probably represents an example of optimum recovery after retinal reattachment.
Assuntos
Células Fotorreceptoras/patologia , Regeneração , Descolamento Retiniano/terapia , Animais , Proteínas do Olho/metabolismo , Fixadores , Fucose/metabolismo , Macaca mulatta , Retina/fisiologia , Retina/ultraestrutura , Descolamento Retiniano/patologia , Opsinas de Bastonetes , Fatores de Tempo , CicatrizaçãoRESUMO
OBJECTIVES: To immunocytochemically characterize the neural and glial elements of idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy, and to correlate them with the outcome of surgery. METHODS: Opercula were collected from eyes undergoing vitrectomy for stage 3 FTMH and processed for transmission electron microscopy, light epifluorescence, and laser scanning confocal microscopy. Glia were identified using anti-glial fibrillary acid protein (GFAP), antivimentin, and anti-cellular retinaldehyde binding protein antibodies. Anti-phosphodiesterase gamma and antirhodopsin were used for cone and rod photoreceptors, and anticytokeratin was used for retinal pigment epithelium. The findings were correlated with the clinical data before and after surgery. For statistical analysis, data were combined with those of a previous study by the authors of 18 opercula. RESULTS: Opercula from 12 consecutive eyes of 12 patients were studied. In all opercula, GFAP, vimentin, and cellular retinaldehyde binding protein-positive glia were present. Six (50%) of 12 opercula contained more than 5 photoreceptors with somata and internal photoreceptor fibres, but lacking outer segments, demonstrating strong immunoreactivity to anti-phosphodiesterase gamma without antirhodopsin reactivity consistent with cones. Further, 2 (17%) of 12 opercula showed few cones (1-5 cones), and 4 (33%) of 12 contained only glia. Clinicopathologic correlation of the 30 opercula from the 2 studies showed that eyes with opercula containing more than 5 photoreceptors were associated with a worse anatomical closure rate after initial surgery, compared with those with fewer than 5 photoreceptors (P =.004). Once closure had been achieved with reoperation, median postoperative vision was similar in both groups (20/40 and 20/60, respectively). CONCLUSIONS: A spectrum of opercula occur in FTMH ranging from those containing only glia to those containing numerous cones. The extent of foveal neuroretinal tissue loss may affect the outcome of surgery.
Assuntos
Neuroglia/patologia , Células Fotorreceptoras de Vertebrados/patologia , Perfurações Retinianas/patologia , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Idoso , Proteínas de Transporte/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Queratinas/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neuroglia/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Perfurações Retinianas/metabolismo , Perfurações Retinianas/cirurgia , Rodopsina/metabolismo , Vimentina/metabolismo , VitrectomiaRESUMO
PURPOSE: To evaluate changes in the rods and amacrine cells and horizontal cells in human retinas with retinitis pigmentosa. METHODS: Seven retinas from patient donors with retinitis pigmentosa and 14 age- and postmortem-matched normal human retinas were processed for immunocytochemistry and confocal microscopy. The following cell-specific antibodies were used: anti-rhodopsin (rods), anti-gamma-aminobutyric acid (amacrine cells), anticalbindin (cones and horizontal cells), anti-glial fibrillary acidic protein (astrocytes and reactive Müller cells), and anti-synaptophysin and anti-SV2 (synaptic vesicles). RESULTS: In retinal regions with significant photoreceptor loss, the rods, gamma-aminobutyric acid-positive amacrine cells, and calbindin-positive horizontal cells had undergone neurite sprouting. The rod, amacrine and horizontal cell neurites were associated with the surfaces of glial fibrillary acidic protein-immunoreactive Müller cells. Most rod neurites that projected into the inner retina contacted the somata of gamma-aminobutyric acid-positive amacrine cells. CONCLUSIONS: Rods, amacrine and horizontal cells undergo neurite sprouting in human retinas with retinitis pigmentosa. These changes in the retinal neurons may contribute to the electroretinographic abnormalities and progressive decline in vision noted by patients with retinitis pigmentosa. These alterations may also complicate strategies for treatment of retinitis pigmentosa.
Assuntos
Neuritos/patologia , Neurônios Aferentes/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Calbindinas , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Neuritos/metabolismo , Neurônios Aferentes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/metabolismo , Rodopsina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
BACKGROUND/AIMS: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is normally synthesised by the retinal pigment epithelium (RPE) and deposited in Bruch's membrane. Mutations in the TIMP3 gene cause Sorsby's fundus dystrophy (SFD), which is characterised by thickening of Bruch's membrane, choroidal neovascularisation, and photoreceptor degeneration. To elucidate the role of TIMP-3 in human retinal degenerative diseases, we immunolocalised TIMP-3 in eyes with SFD caused by the Ser-181-Cys TIMP3 gene mutation or retinitis pigmentosa (RP; not caused by TIMP3 mutations). METHODS: Standard light microscopic immunocytochemistry, including antigen retrieval, was used to localise TIMP-3 in paraffin sections of human eyes: two with SFD, three with different genetic forms of RP, and two normal. RESULTS: In the SFD eyes, the thickened Bruch's membrane was strongly TIMP-3 positive except where RPE cells had degenerated. Similarly, in the RP eyes, Bruch's membrane was TIMP-3 positive except where RPE cells were lost, consistent with ongoing RPE mediated turnover of TIMP-3 in this region. In areas of total photoreceptor loss, migrated RPE cells formed cuffs around blood vessels in the RP retinas. Thick, TIMP-3 positive extracellular matrix (ECM) deposits associated with the migrated RPE cells occluded some vascular lumina, correlating with the observed loss of inner retinal neurons in RP. CONCLUSIONS: TIMP-3 is a component of the increased ECM sequestered in Bruch's membrane in SFD. Further information is needed on normal TIMP-3/ECM interactions in Bruch's membrane and the effect of mutant TIMP-3 on this process. The finding of TIMP-3 accumulations in retinas with RP not caused by TIMP-3 mutations emphasises the importance of ECM remodelling in normal and diseased human eyes.
Assuntos
Olho/metabolismo , Retinose Pigmentar/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Idoso , Idoso de 80 Anos ou mais , Lâmina Basilar da Corioide/metabolismo , Corioide/metabolismo , Células Epiteliais , Feminino , Humanos , Mutação , Reação do Ácido Periódico de Schiff , Retina/metabolismo , Vasos Retinianos , Retinose Pigmentar/genética , Inibidor Tecidual de Metaloproteinase-3/genéticaAssuntos
Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Luz , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Transdução de Sinais , Animais , Toxinas Bacterianas/genética , Bovinos , Imunofluorescência , Proteínas Hemolisinas/genética , Mutagênese Sítio-Dirigida , Fosforilação , Células Fotorreceptoras de Vertebrados/metabolismo , Coelhos , Rodopsina/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were used to compare domains within the interphotoreceptor matrices (IPM) of the cat and monkey, two species where the morphological relationship between the retinal pigment epithelium (RPE) and photoreceptors is distinctly different. In the monkey, PNA labeling was heaviest over the cone outer segments and a discrete region of the interphotoreceptor matrix bordering the cone inner and outer segments--a region which has been termed the cone matrix sheath. Near the apical border of the RPE, the outer margin of the PNA-labeled matrix is surrounded by a circular array of apical microvilli. In the cat retina, PNA labeling was highest over a region of the IPM lying between the outer margin of the cone sheath processes and surrounding rod matrix. In contrast, intracellular labeling of cone inner and outer segments was sparse. The RPE apical processes forming the cone sheath were not labeled. In the monkey retina, WGA preferentially labeled rod outer segments and the region of the IPM around rod inner and outer segments. The cone matrix sheath was not preferentially labeled using this lectin. Rod inner segments and cone inner and outer segments were labeled moderately. In the cat retina, WGA labeling was dense over both rod outer segments and cone outer segments as well as the cone sheath. Rod and cone inner segments, as well as the IPM around both rods and cones, were moderately labeled. These observations suggest that the specialized processes arising from the apical surface of retinal pigment epithelial cells, together with photoreceptor-specific extracellular matrix domains, contribute to the formation of specific microenvironments around rod and cone photoreceptor cells.
Assuntos
Gatos/anatomia & histologia , Matriz Extracelular/ultraestrutura , Macaca mulatta/anatomia & histologia , Células Fotorreceptoras/ultraestrutura , Animais , Arachis , Lectinas , Microscopia Eletrônica , Aglutinina de Amendoim , Lectinas de Plantas , Aglutininas do Germe de TrigoRESUMO
Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.
Assuntos
Cálcio/metabolismo , Retina/fisiologia , Rodopsina/fisiologia , Fosfolipases Tipo C/farmacologia , Animais , Bovinos , Fosforilação , Fosfolipases Tipo C/metabolismoRESUMO
Tunicamycin (TM), an inhibitor of dolichylphosphate-mediated protein glycosylation, was injected intravitreally into the eyes of diurnal rodents with cone-dominated retinas. Injection of 1 microgram of the B2 isomer led to a progressive degeneration of the photoreceptor outer segments and disruption of the RPE-photoreceptor interface that took place over a 10-day period. Cone outer segments were shortened by postinjection day 6 and virtually absent by day 9. The microvilli that normally protrude from the apical surface of the retinal pigment epithelium were replaced by a fringe of shortened processes. The other retinal layers showed no morphological evidence of disruption. Retinal sensitivity, as measured by electroretinographic b-wave threshold, showed a significant and progressive decline over the 10-day course of the experiment that paralleled the disruption of retinal morphology. These results suggest that TM leads to similar morphological and electrophysiological effects on rod and cone photoreceptors.
Assuntos
Células Fotorreceptoras/efeitos dos fármacos , Degeneração Retiniana/induzido quimicamente , Tunicamicina/toxicidade , Animais , Eletrorretinografia/efeitos dos fármacos , Células Fotorreceptoras/ultraestrutura , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/ultraestrutura , Degeneração Retiniana/patologia , Sciuridae , Corpo Vítreo/efeitos dos fármacosRESUMO
The temporal pattern of cone outer-segment disc shedding was examined in the retina of the California ground squirrel, Spermophilus beecheyi, under two different lighting conditions. Squirrels were entrained to either 10-180 lx (room lighting) or 1,400 lx. Cone shedding during the dark period was biphasic in both conditions, with increases occurring at 2-3 hr and 5-6 hr after light offset. Entrainment to 1400 lx resulted in an increase in shedding at 2 hr after light offset and a slight advance in the timing of both peaks. Dense granules were often found in photoreceptors, retinal neurons, Müller cells, microglia and vascular cells. These granules, which were found primarily during the dark period and early light period, were lipofuscin-like in their lipophilia, size and autofluorescence. Many of the granules were probably autophagic in origin, but some within Müller cells may have originated via endocytosis of extracellular material which was exocytosed by photoreceptors.
Assuntos
Autofagia , Fagocitose , Células Fotorreceptoras/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Sciuridae/fisiologia , Animais , Contagem de Células , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Luz , Masculino , Microscopia Eletrônica , Fagossomos/ultraestrutura , Fotometria , Epitélio Pigmentado Ocular/ultraestrutura , Segmento Externo da Célula Bastonete/ultraestrutura , Fatores de TempoRESUMO
RGS9 is a member of the RGS family of GTPase accelerating proteins (GAPs) for heterotrimeric G proteins. We have explored its contribution to GTPase acceleration in mammalian rod and cone photoreceptors. When RGS9 was specifically removed from detergent extracts of rod outer segments by immunodepletion, the extracts lost nearly all of their GAP activity stimulatable by the inhibitory subunit of cGMP phosphodiesterase. Immunolocalization using monoclonal antibodies and confocal microscopy revealed that RGS9 is present in cones at significantly higher levels than in rods. Thus, RGS9 is the predominant source of GAP activity in rod outer segments, and RGS9 concentration emerges as a potentially important determinant of the faster response kinetics and lower sensitivity of mammalian cones, as compared with rods.
Assuntos
Proteínas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Western Blotting , Bovinos , Técnica Indireta de Fluorescência para Anticorpo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase , Proteínas de Membrana/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Visão OcularRESUMO
Mutations in tissue inhibitor of metalloproteinases (TIMP)-3 are found in some patients with Sorsby's fundus dystrophy, a retinal degeneration characterized by abnormal deposits in Bruch's membrane and choroidal neovascularization. The purpose of this study was to localize TIMP-3 in the retina/choroid of normal human and animal eyes. Immunolabeling was performed on unfixed and fixed sections of human eyes aged 24 to 85 years and unfixed sections of baboon, chicken, cow, pig, and rat eyes using a monoclonal antibody against a human TIMP-3 synthetic peptide. The antibody produced strong immunolabeling of Bruch's membrane and drusen and weak labeling of retina blood vessels in unfixed human and baboon eyes. Unfixed chicken, cow, pig, and rat tissues showed no reactivity. After antigen retrieval, all fixed human eyes showed specific labeling of Bruch's membrane and drusen, which was strongest in eyes from elderly donors. The results indicate that TIMP-3 is an extracellular matrix component of Bruch's membrane. Thus, abnormal local function of TIMP-3 may lead to the characteristic Bruch's membrane deposits and choroidal neovascularization found in Sorsby's fundus dystrophy. Specific labeling of drusen raises the possibility that altered TIMP-3-mediated matrix remodeling may contribute to age-related degenerative changes in Bruch's membrane.
Assuntos
Lâmina Basilar da Corioide/enzimologia , Inibidores de Proteases/análise , Proteínas/análise , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Western Blotting , Lâmina Basilar da Corioide/imunologia , Matriz Extracelular/enzimologia , Humanos , Dados de Sequência Molecular , Inibidores de Proteases/imunologia , Proteínas/imunologia , Inibidor Tecidual de Metaloproteinase-3RESUMO
Several retinal photoreceptor proteins involved in phototransduction have also been found in the mammalian pineal. This study demonstrates that rat and human pineals express protein kinases that are identical to the corresponding rod photoreceptor rhodopsin kinases. The deduced amino acid sequence of rat and human rhodopsin kinases have 84% sequence similarity to the earlier reported sequence of the bovine retinal enzyme, with complete conservation of the topological regions containing the position of the catalytic domain and sites of posttranslational modifications. Rat pineal also expresses rod opsin and putative blue cone opsin. Using immunocytochemistry, rod opsin and rhodopsin kinase were found to be co-localized in pinealocytes in the human tissue. These data demonstrate that the mammalian pineal contains light-sensitive opsins and a kinase involved in their inactivation. These findings correlate with an earlier report that neonatal rats show extraretinal light sensitivity, and suggest that a functional photoreceptive system may be present in the adult mammalian pineal.
Assuntos
Proteínas do Olho/metabolismo , Glândula Pineal/enzimologia , Proteínas Quinases/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Primers do DNA , Sondas de DNA/química , DNA Complementar/análise , Proteínas do Olho/genética , Receptor Quinase 1 Acoplada a Proteína G , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Glândula Pineal/citologia , Reação em Cadeia da Polimerase , Proteínas Quinases/genética , RNA Mensageiro/análise , Ratos , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismoRESUMO
OBJECTIVE: Little is known of the cytopathology of photoreceptors in human inherited retinal dystrophies that initially affect the central retina, including the macula. The current study sought to determine the cytologic features of dysfunctional cone and rod photoreceptors, as well as the pattern of degeneration of the cells in representative cases of central retinal dystrophy. STUDY DESIGN: Comparative human tissue study. MATERIALS: Four human donor eyes with the following forms of central retinal dystrophy: cone-rod dystrophy (CRD), central areolar choroidal dystrophy, Bardet-Biedl syndrome, and cone dystrophy-cerebellar ataxia. The cytologic features of retinal photoreceptors in these eyes were compared with those in an eye with retinitis pigmentosa and six normal human eyes. METHODS AND OUTCOME MEASURES: Immunocytochemistry and electron microscopy were used to evaluate the retinal histopathology in the donor eyes. RESULTS: Cone numbers were decreased in the case of CRD, particularly in the central and far peripheral retina, and both cone and rod outer segments were slightly shortened. Occasional degenerate cones had dense cytoplasm and pyknotic nuclei dislocated sclerad to the external-limiting membrane. The most prominent alteration in this retina was marked enlargement and distortion of the cone photoreceptor pedicles, which contained reduced numbers of synaptic vesicles. The retina with central areolar choroidal dystrophy contained a few cones with similarly abnormal synapses. However, comparable cone synapse abnormalities were not observed in the cases of Bardet-Biedl syndrome, cone dystrophy-cerebellar ataxia, retinitis pigmentosa, or in the normal retinas. CONCLUSIONS: The functional consequences of the cone synapse abnormalities in CRD are not known but may correlate with the electroretinographic abnormalities documented in some cases of CRD. To our knowledge, comparable synapse changes have not been noted in either rods or cones in other forms of retinal dystrophy, including retinitis pigmentosa, suggesting that different cytopathologic mechanisms may be involved.
Assuntos
Células Fotorreceptoras de Vertebrados/ultraestrutura , Degeneração Retiniana/patologia , Sinapses/ultraestrutura , Adulto , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Ataxia Cerebelar/patologia , Doenças da Coroide/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Síndrome de Laurence-Moon/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
A class 1 aldehyde dehydrogenase (ALDH) catalyzes oxidation of retinaldehyde to retinoic acid in bovine retina. We used immunocytochemistry and in situ hybridization to localize this enzyme in adult and fetal bovine retinas. Specific ALDH immunoreactivity was present in the cytoplasm of wide-field amacrine cells restricted in distribution to the dorsal part of the adult retina. The somata diameters ranged from approximately 8 microns to approximately 15 microns, and the cells increased in density from approximately 125 cells/mm2 near the horizontal meridian to approximately 425 cells/mm2 in the superior far periphery. The ALDH-positive cells had somata on both sides of the inner plexiform layer (IPL) and processes in two IPL strata. The majority of ALDH-positive cells were unreactive with antibodies against known amacrine cell enzymes and neurotransmitters, including GABA and glycine. The ALDH-positive amacrine cells also did not react with anti-cellular retinoic acid-binding protein, which was present in a subset of GABA-positive amacrine cells. In flat-mounted retinas processed by in situ hybridization, the larger ALDH-positive amacrine cells tended to be more heavily labeled. In addition to amacrine cells, Müller cell processes in the inner retina were weakly immunoreactive for ALDH; however, these glial cells did not contain ALDH mRNA. The pattern of ALDH expression in fetal bovine retinas was documented by immunocytochemistry. No ALDH reactivity was found before 5.5 months; for the remainder of the fetal period, ALDH immunoreactivity was present in amacrine cells similar to those in adult retina. The ALDH-positive amacrine cells in bovine retina are novel, being limited in distribution to the dorsal retina and unlabeled with other amacrine cell-specific markers. Identification of ALDH in amacrine cells provides additional evidence that cells of the inner retina are involved in retinoid metabolism.