Nancy J. Mangini

Effect of hydroxylamine on the subcellular distribution of arrestin (S-antigen) in rod photoreceptors

The immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3-amino-9-ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor outer segment (OS) layer (0.061 +/- 0.004; average +/- S.E.M.) was less than that of the inner segment (IS) layer (0.138 +/- 0.011). In light-adapted control retinas, OS labeling density (0.139 +/- 0.007) exceeded IS labeling density (0.095 +/- 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 +/- 0.006 (OS) and 0.183 +/- 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 +/- 0.004 (OS) and 0.182 +/- 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed to 3 min or 13 min of bright light and then immediately fixed.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of ubiquitin and related enzymes in human retina and retinal pigment epithelium

Abstract• Purpose: To examine the localization of ubiquitin (Ub) and related enzymes in human retina with emphasis on the retinal pigment epithelium (RPE)-Bruchs membrane complex. • Methods: Thirty human eyes enucleated for various disease processes were examined. Immunohistochemistry was performed on paraffin sections using antibodies against Ub, Ub-conjugating enzyme (E2), Ub carboxylterminal hydrolase (PGP 9.5), and, for comparison, arrestin (Arr). Immunoreactivity (IR) was tested using the avidin-biotin method.• Results: Ub was present throughout retina but was particularly prominent in ganglion cells and RPE. Most intriguing was the presence of Ub IR in age-related, sub-RPE deposits such as drusen and basal laminar deposits (BLD). RPE immunolabeling was more intense in older tissue, but otherwise no pattern of Ub IR could be linked to specific diseases. E2 IR colocalized with Ub, with one exception; E2 IR was not found in drusen or BLD. PGP 9.5 IR was intense in nerve fibers, ganglion cells, and the inner nuclear and plexiform layers. RPE staining was faint and patchy; sub-RPE deposits were not labeled. Arr IR was present in photoreceptors but not within or beneath RPE cells. • Conclusion: The ubiquitination process is important in human retina and particularly in ganglion cells. Ub-related processes are also active in RPE and may be involved in the degradation and disposal of proteins from these cells. The presence of Ub in sub-RPE deposits — without related Ub-processing enzymes — raises the possibility that certain proteins become ubiquitinated within RPE but that further degradation of the Ub-protein complexes does not occur.

A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, approximately 50% of photoreceptors remained and an approximately 45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium

Abstract · Background: Impaired calcium (Ca2+) metabolism has been implicated in the pathogenesis of various ocular diseases, suggesting that regulation of Ca2+ homeostasis in the retina is of major significance for normal function. There are known families of transport proteins that can catalyze net Ca2+ efflux across the plasma membrane, one of which is the Na+/Ca2+ exchanger. Using immunohistochemistry, we have investigated the human retina and retinal pigment epithelium (RPE) for the presence and distribution of the so-called cardiac-type Na+/Ca2+ exchanger. · Methods: Paraffin sections of ten eyes enucleated for various disease processes were incubated according to the ABC method with a polyclonal antibody (π) produced against the canine cardiac sarcolemmal Na+/Ca2+ exchanger. The reaction was visualized with aminoethylcarbazole. · Results: There was a positive reaction with anti-Na+/Ca2+ exchanger in the retina and RPE in all eyes, but the labeling varied among the different specimens. In neural retina, staining was most intense in Mueller cells, in cells of the inner nuclear layer, and in cone inner segments. Immunoreactivity was less pronounced in ganglion cells, nerve fibers, the outer nuclear layer and in rod inner segments. Outer segments appeared mostly negative. In the RPE, positive staining was present but the intensity of staining varied both within and among the specimens. Reactive RPE cells revealed the most intense labeling. · Conclusion: Na+/Ca2+ exchanger of the cardiac type is present in human retina and RPE. The variation in immunoreactivity among the different specimens may reflect the different diseases of these eyes and their different metabolic states. A specific relation between certain diseases and malfunction of the Na+/Ca2+ exchanger could have a major impact on therapeutic regimens.

Immunodetection of an arrestin-like protein in human retinal pigment epithelium

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.

Anti-arrestin immunoreactivity in the human retina: Difference between light- and dark-adaptation

Differences in arrestin (Arr) immunolocalization between light(LA)- and dark(DA)-adapted retinae have been described in various species. We have for the first time studied 5 LA and 5 DA human retinae from surgically enucleated eyes, each group comprising 1 exenteration specimen and globes with malignant melanoma or 2 degrees glaucoma. To examine the distribution of Arr, immunohistochemistry was performed on paraffin sections using three different antibodies to Arr: a48k, 3D1.2, and 5c6.47. Immunoreactivity (IR) was tested using the avidin-biotin method, and results were visualized with diaminobenzidine. With both a48k and 5c6.47, labeling was most intense in the photoreceptor layer. When comparing LA and DA retinae, IR of photoreceptor outer segments (OS) was clearly different with very little IR in OS of DA but distinct positivity in OS of LA retina. This was most obvious in melanoma eyes where retinal morphology was well-preserved while in glaucomatous eyes with retinal degeneration this pattern was less apparent. 3D1.2 did not react in any of the specimens. Although there was some variation within each group, we demonstrated a distinct difference in anti-Arr IR between LA and DA specimens. Thus Arr-IR might now be used as a promising tool to further study retinal diseases on human surgical specimens involving photoreceptor degeneration.

A Morphometric and Immunopathologic Study of Retinal Photic Injury in Primate

Clinical, histopathological, and ultrastructural studies examining the pathogenesis of retinal photic injury in animal models were performed by methods and yielded important qualitative information1,2. Morphometric studies of retinal photic injury in rodents had provided us with additional understanding of the more subtle pathophysiologic factors. For example, with morphometric techniques, it was determined that superior and temporal quadrants of the rodent retina arc more sensitive to retinal photic injury than the inferior and nasal quadrants3; aging rats are more susceptible to retinal photic injury4; light-adapted animals suffer less than the dark-adapted animals5; and different schedules of light exposure inflicted different severity of retinal lesions6. Hormonal influences on the severity of retinal lesions have also been defined7. Furthermore, with morpho-metric comparative methods, pharmacologic agents such as vitamin C8, dimethylthiourea9, flunarizine10, dexamethasone11, and methylprcdnisolone12 were demonstrated to rescue photoreceptor cells from light exposure.