Declines in Arrestin and Rhodopsin in the Macula with Progression of Age-Related Macular Degeneration

Abstract

purpose. Biochemical analysis of age-related macular degeneration (AMD) at distinct stages of the disease will help further understanding of the molecular events associated with disease progression. This study was conducted to determine the ability of a new grading system for eye bank eyes, the Minnesota Grading System (MGS), to discern distinct stages of AMD so that retinal region-specific changes in rod photoreceptor protein expression from donors could be determined.

methods. Donor eyes were assigned to a specific level of AMD by using the MGS. Expression of the rod photoreceptor proteins rhodopsin and arrestin was evaluated by Western immunoblot analysis in the macular and peripheral regions of the neurosensory retina from donors at different stages of AMD.

results. A significant linear decline in both arrestin and rhodopsin content correlated with progressive MGS levels in the macula. In contrast, the peripheral region showed no significant correlation between MGS level and the content of either protein.

conclusions. The statistically significant relationship between decreasing macular rod photoreceptor proteins and progressive MGS levels of AMD demonstrates the utility of the clinically based MGS to correspond with specific protein changes found at known, progressive stages of degeneration. Future biochemical analysis of clinically characterized donor eyes will further understanding of the pathobiochemistry of AMD.

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness in individuals over the age of 65 in the developed world. ^{1 2 3} This disease is characterized by the loss of central vision that arises from altered retinal function in the macula, the central portion of the retina. There are two types of AMD, atrophic (aAMD) and exudative (eAMD). Both degeneration types have drusen (clinically visible yellow deposits) and a loss of photoreceptors at end stages. ⁴ Larger areas of retinal pigment epithelium (RPE) degeneration in the center of the macula result in vision loss. In aAMD, the progressive loss of vision is relatively slow. In contrast, eAMD involves the development of choroidal neovascularization in the subretinal space, which may result in severe, acute vision loss. In general, eAMD or aAMD involving the macula are considered late or end-stage AMD. 

Progress toward understanding the molecular changes associated with the progression of AMD has been limited by insufficient clinical classification of donor eyes. Curcio et al. ⁵ have developed a classification of eyes (the Alabama Grading System) that is useful for histopathologic analysis of retinal changes in progressive levels of AMD. However, processing eyes with the tissue-fixation techniques necessary for histologic evaluation decreases the quality of subsequent biochemical studies, such as analysis of the proteome. We recently developed a grading system for eye bank eyes, referred to as the Minnesota Grading System (MGS), that optimizes tissue for biochemical analysis. This system takes into account the unique challenges of grading postmortem tissue. ⁶ The MGS is based directly on criteria from the Age-related Eye Disease Study (AREDS), the current standard in clinical studies of AMD, and the Wisconsin Age-Related Maculopathy Grading System (WARMGS), a basis for epidemiologic studies. ^{2 7 8 9} Four distinct levels of AMD are defined by the MGS and are based on specific clinical features, including drusen size and surface area, pigmentary changes, RPE atrophy, and the presence or absence of subretinal neovascularization. The MGS identifies early (MGS2), intermediate (MGS3), and late (MGS4) stages of AMD. However, it should be noted that the MGS has not yet been histologically verified. Biochemical analysis of donors at each distinct stage of AMD provides a unique opportunity for studying retinal changes during progression of the disease. 

Prior studies have shown that rod photoreceptors are lost at advanced stages of AMD. ^{4 10 11} In the present study, we monitored the expression of two rod-specific proteins, arrestin and rhodopsin, by Western immunoblot analysis, to determine whether these biochemical measures match the observed histologic and clinical features associated with advanced AMD. In addition, we investigated how content of these proteins changes at earlier stages of the disease, particularly at a time when obvious rod loss is not evident. Although the diagnosis of AMD is based on clinically defined changes in the macula, the same features of RPE cell loss and drusen have also been observed in the peripheral retina. Thus, one would expect that equivalent rod loss could occur in both regions. Conflicting data are present in the literature. In support, some early reports show rod loss in the periphery, ^{12 13} whereas later studies refute the idea of disease-related changes in peripheral rods. ^{4 14} We have included separate analysis of both macular and peripheral regions to determine whether there are region-specific differences in rod content that can be distinguished biochemically. 

Methods

Grading of the Level of AMD

Eyes were obtained from the Minnesota (MN) Lions Eye Bank and maintained at 4°C in a moist chamber until dissection and photographing. All tissue was acquired with consent of the donor or donor’s family for use in medical research. The consent is in accordance with the principals outlined in the Declaration of Helsinki. In one eye of each pair, dissection of the sensory retina was performed to expose the underlying choroid and RPE in the macular region. Criteria established by the MGS were used to determine the stage of AMD. ⁶ The partner eye was snap frozen in liquid nitrogen and stored at −80°C for biochemical analysis. The stages of AMD will be referred to as MGS1 (minimal or no AMD) through MGS4 (severe AMD). 

Preparation of Retinal Homogenates

In the undissected partner eye, the vitreous humor was removed while the eyes were still frozen to minimize vitreous contamination. The eyecup was stabilized in an upright position by using an embedding medium for frozen tissue. A trephine punch of 8-mm diameter was centered over the macular area to separate the macula from the periphery. The major retinal capillaries were then used to separate the peripheral retina into nasal, superior, inferior, and temporal regions. The neurosensory retina was carefully peeled away from the RPE and rinsed with PBS to remove potential contaminating RPE cells. The superior and nasal sections (representing the peripheral sections used in the study) were combined and gently homogenized (∼15 passes in a glass homogenizer with a Teflon pestle) in 250 μL of medium containing 20% sucrose, 20 mM Tris-acetate (pH 7.2), 2 mM MgCl₂, 10 mM glucose, and 2% CHAPS (3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate). The retinal homogenates were then centrifuged for 15 minutes at 100g, the supernatant retained, and the pellet rehomogenized in 200 μL additional buffer. The supernatants from each step were combined and centrifuged at 600g for 15 minutes. The supernatant was then stored at −80°C. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay (Pierce). Bovine serum albumin was used as a standard. 

Rod Outer Segment Preparation

Characterization of the loss of rod outer segments (ROS) in retinal preparations was performed according to a slightly modified version of an established protocol for purification of ROS. ¹⁵ Retinal dissections were performed as just described. The neurosensory retina from the periphery and the macular region was collected separately in homogenization buffer (20% wt/vol sucrose, 2 MgCl₂, 20 mM Tris-acetate [pH 7.2]). ROS were sheared by flicking the tubes (Eppendorf, Fremont, CA). The RPE underlying the neurosensory retina were collected in phosphate-buffered saline. Samples of either RPE or neurosensory retina were layered over 25% to 60% continuous sucrose gradients before centrifugation at 75,000g for 75 minutes at 4°C. The ROS band was collected off the gradient and brought up in ∼10 to 15 mL of 10 mM Tris and centrifuged at 17,369g for 30 minutes. The ROS pellet was collected in 10 mM Tris and protein concentration was determined as just described. 

One-Dimensional Gel Electrophoresis

Before Coomassie blue staining and Western immunoblot analysis, retinal proteins were electrophoretically separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Mighty Small SE 250; Hoeffer Scientific Instruments, San Francisco, CA) system and a 10% resolving gel with a 3% stacking gel. ¹⁶ Protein loads for preparations from human retina were 5 μg per lane. Homogenates were not boiled before loading to prevent aggregation of rhodopsin. 

Western Immunoblot Analysis of 1-D Gels

After resolution by 10% SDS-PAGE, retinal proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Mini Trans-Blot Cell; Bio-Rad, Hercules CA) at 110 V for 3 hours. PVDF membranes were probed with one of the following primary monoclonal antibodies: rhodopsin (1:1000; Biodesign, Saco, ME) or A9C6 arrestin 1:40. The rhodopsin antibody recognizes the first 10 amino acids of the N terminus (GTEGPNFYV). The A9C6 arrestin antibody specifically detects residues 376-386 (VFEEFARHNLK) at the carboxyl terminus of S-antigen. Goat anti-mouse alkaline phosphatase–conjugated secondary antibody (1:3000) was used in conjunction with the substrate 5-bromo-4-chlor-3′-iodolyl phosphate p-toluidine/nitro blue tetrazolium chloride (BCIP-NBT) to visualize the immunoreaction. Images were then captured (Fluor-S MultiImaging System; Bio-Rad). 

Densitometry of Immune Reaction

To determine the relative content of arrestin or rhodopsin in human retinal homogenates, densitometric analysis was performed on the immunoreaction of individual protein bands (SigmaScan; SPSS Science, Chicago, IL). Preliminary experiments showed that retinal protein loads between 1 and 10 μg produced a linear signal for both the arrestin and rhodopsin antibody. Therefore, 5 μg was chosen as the amount of total protein to load for the semiquantitative Western blot analysis. All band densities were normalized to a reference sample from a peripheral retinal homogenate that was included in each Western blot. This allowed for blot-to-blot comparisons. 

Statistical Analysis

Measures of immune reaction were considered outliers and were eliminated from statistical analysis when they were outside the 95% confidence interval and skewed the normal distribution. Linear regression analysis was used to determine whether there was a significant linear relationship between protein content and the level of MGS. Between-group differences were tested with a one-way analysis of variance (ANOVA), with the level of significance set at P ≤ 0.05. When appropriate, post hoc analysis was performed using the Tukey-Kramer test. 

Results

Clinical Information of Donors

Demographic and clinical information for donors obtained from written records provided by the MN Lions Eye Bank is summarized in Table 1 . Because of the difference in AMD prevalence noted between individuals with lighter and darker pigmentation, ^{17 18} only eyes from white donors were used in the study. We excluded donors when ocular histories indicated glaucoma. All MGS4 donor eyes exhibited eAMD. The average amount of time from death to tissue freezing of donor eyes used in the study was 16.7 ± 4.7 hours (mean ± SD, n = 46). There was a bias toward male donors, which is representative of the donor eyes we received from the MN Lions Eye Bank. Although we have attempted to match ages between groups, the ages of the donors with MGS1 and MGS2 eyes were considerably younger than donors with later stages of AMD. Each level of MGS contained donors with multiple causes of death, including cancers and pulmonary and cardiac etiologies. These variations in sample populations from different stages of AMD still allowed for detection of retinal differences between groups. 

Loss of Rod-Specific Proteins with AMD Progression

In studies in which immunohistochemistry and topographical counts of photoreceptors were used, investigators have reported that rod photoreceptors in the macular region are selectively lost at advanced stages of AMD. ^{4 10} In the present study, the relative content of rod photoreceptors was monitored in the macula and periphery during the progression of AMD by measuring the expression of two rod-specific proteins, arrestin and rhodopsin. Before processing retinas for biochemical analysis, we determined the stage of AMD in donor eyes according to the MGS. The protein profile of Coomassie blue–stained retinal homogenate proteins was reproducible from all donors, regardless of region or assigned MGS level (Fig. 1) . 

We evaluated the relative content of rod arrestin (S-antigen), a 48-kDa soluble protein, by Western immunoblot analysis in retinal homogenates. A representative immunoreaction from the macula and periphery of each level of MGS is shown (Fig. 2A) . Based on densitometric analysis of arrestin immune reaction in macular sections for each MGS level (Fig. 2B) , a significant negative linear relationship was observed between the arrestin content and progressive levels of MGS (P = 0.0005). Comparison of the mean density from each group showed the relative content of arrestin in MGS4 eyes was significantly less than in the control MGS1 eyes, providing corroborative evidence of the linear association. This decline in arrestin content with advancing AMD, as defined by MGS level in the macula is in sharp contrast to the peripheral region (Fig. 2B) , which showed no statistically significant change between AMD groups (P = 0.65). 

To confirm these findings with arrestin, we also measured the relative content of rhodopsin in the same donor retinas used for analysis of arrestin. Rhodopsin content was evaluated with an antibody that specifically recognizes 39-kDa rod opsin in human tissue. ¹⁹ In contrast to the sharp immune reaction of a single protein band for arrestin, the immune reaction with the rhodopsin antibody was more diffuse, due to glycosylation and phosphorylation of the protein, which alters the migration (Fig. 3A) . 

Results of the densitometric analysis for macular rhodopsin showed a nonsignificant linear relationship (P = 0.19). This lack of linear response was due to the control values (MGS1) being lower than expected when compared with values for MGS2. If we consider densities only from among disease retinas (i.e., MGS2 to -4), there is a significant negative linear response between rhodopsin content and the progression of the disease (P = 0.002). Comparison of mean density for each group showed a significant decline in rhodopsin content in MGS4 compared with MGS2 retinas. When we compared rhodopsin content in the periphery (Fig. 3B) , no linear relationship was found throughout the MGS levels (P = 0.71). These results are consistent with peripheral arrestin data. 

ROS Loss to the RPE Cell Layer

To explore the potential basis for the lower than expected rhodopsin content in the macula of MGS1 eyes, we considered the possibility of incomplete recovery of ROS during dissection of the neurosensory retina. An anatomic feature of rod photoreceptors is the narrow cilium that physically separates the rod inner segments (RIS) from the ROS and are packed full of membrane disks containing most of the rhodopsin in the cell. Because the tips of the ROS interdigitate with the RPE cell layer, ROS may remain lodged in the RPE after mechanical separation of the neurosensory retina from the RPE. Thus, incomplete recovery of ROS may explain the lower than expected values for rhodopsin content in the control (MGS1) group. 

The potential differential loss of ROS from the macular neurosensory retina between MGS1 and -2 retinas was explored by preparing ROS via sucrose gradient separation from both the neurosensory retina and the underlying RPE cell layer. We found that 19% ± 5.5% (mean ± SEM, n = 3/MGS level) of the total ROS protein was associated with the RPE cell layer in MGS1 donors. In contrast, 9.1% ± 1.9% (mean ± SEM, n = 3/MGS level) was recovered from the RPE cells in MGS2 donors. These results show that more ROS are lost to the RPE cell layer in MGS1 eyes, thus providing an explanation for the lower macular rhodopsin content. However, these results do not explain why arrestin content does not follow the same trend. 

Analysis of the relative content of rhodopsin and arrestin was determined by Western blot analysis in homogenates (containing both RIS and ROS) and sucrose gradient purified ROS from the neurosensory retina of the same donor eye. Protein loads were equivalent for each sample. However, since the homogenates contain a mixture of cell types, the rhodopsin immune reaction was used to normalize the content of photoreceptor cells. When comparing the ratio of rhodopsin to arrestin immune reactions for six donors varying in the amount of postmortem time until freezing (9.8–16.4 hours; Fig. 4A ), we found that there was 3.1 ± 0.4-fold (mean ± SEM) more arrestin present in the homogenate than in the ROS. Because the homogenate contains both RIS and ROS, this difference represents the relative content of arrestin present in RIS. In addition, there was no significant time-dependent difference in the rhodopsin-arrestin ratio among the six samples studied (Fig. 4B) . These results suggest that arrestin translocation to the RIS was stabilized in donor retinas by 9.8 hours. These results also indicate that arrestin content is less affected by the loss of ROS to the RPE cell layer during dissection. 

Discussion

The purpose of this study was to document the correspondence of the MGS to specific biochemical changes previously documented to occur in progressive stages of AMD. We used expression of rod-specific proteins as a biochemical measure to detect changes in photoreceptor rods that were previously demonstrated using histologic analysis and functional measures of AMD. ^{4 10 14 20 21} This is the first study in which parallel measures of rhodopsin and arrestin have been examined for changes in protein content within progressive levels of AMD defined in the context of the MGS. We found that macular content of rod-specific proteins decreased with MGS, but peripheral content did not change. These region-specific changes in rod protein content at advanced stages of MGS were consistent with previous reports of rod loss restricted to the macula. ^{4 10 11}  

In the present study, we used well-characterized antibodies that recognized rhodopsin and S-arrestin in retinal homogenates to obtain a relative measure of protein content. This approach circumvents many of the technical challenges and complications of indirect measurements, such as fundus reflectometry, anatomic studies, and spectroscopy, which have produced inconclusive quantitative results. ^{22 23 24 25} Studies using the kinetic activity of rhodopsin to determine the quantity have been complicated by both postmortem bleaching that occurs during eye bank tissue processing and attempts at rhodopsin regeneration. ^{22 24} Finally, some studies have used multistep purification procedures specifically for rhodopsin that may result in variable extractions. ^{22 24}  

In our measures of rhodopsin content, we observed a lower than expected level in MGS1 eyes. The results of quantitation of ROS proteins that remained associated with RPE cells after dissection of the overlying neurosensory retina show a twofold greater loss of ROS in MGS1 maculae. These results provide a potential explanation for the discrepancy in the rhodopsin measurements. We propose that as the MGS grade increases, drusen formation and RPE degeneration increase the likelihood of dissociation between the neurosensory retina and the RPE cell layer, resulting in better recovery of ROS within progressing stages. 

Arrestin is also found in the ROS, but unlike the transmembrane protein rhodopsin, arrestin has demonstrated reversible migration between RIS to ROS in response to light conditions. ^{26 27 28} In the dark, arrestin migrates from ROS to RIS in an energy-dependent manner. Postmortem human donor photoreceptors have been analyzed for their metabolic capacity by examining donor eyes that had been in the dark 1 to 5 hours after death. ^{29 30} The studies show that rhodopsin phosphorylation occurs up to 4.5 hours after death, indicating that the retina retains metabolic activity for ∼4.5 hours after which levels of guanosine triphosphate (GTP) and adenosine triphosphate (ATP) decrease. ³⁰  

Average time to refrigeration and enucleation are typically 4 and 6 hours after death, respectively, in eyes from the MN Lions Eye Bank obtained for this study. ³¹ Because the eyes have been maintained in the dark during the critical time before energy levels decline, arrestin concentration is higher in the RIS and therefore less subject to problems associated with incomplete removal of the ROS from the RPE during retinal dissection. Our results show that postmortem translocation of arrestin does occur in our donor eyes, as evidenced by a threefold increase in content in the RIS. 

A potential ambiguity with the A9C6 monoclonal antibody is that it recognizes arrestin in human blue cones in addition to rods. ^{32 33} This may complicate interpretation of our data since cone photoreceptors are preferentially spared in the macula until later stages of the disease. ^{4 10 11 34 35} However, blue cones constitute approximately 7% of cones within the macula, ³⁶ which accounts for <1% of the total photoreceptors in the macula. Within the periphery, there are even fewer blue cones that are well dispersed. ^{34 37} Because blue cones are such a minor component, they should not significantly influence our results. 

As our donor information in Table 1illustrates, there is an increase in mean age of approximately 25 years when comparing MGS1 to -4. This age difference highlights the difficulty in finding older donors having no clinical signs of AMD, which is an intrinsic problem of studying a prevalent age-related disease. In addition, we acknowledge the potential for selection bias in the type of donor contributing eyes for research. To determine whether the difference in age between different levels of MGS would influence our results, we consulted previous studies examining age-dependent changes in rhodopsin and arrestin content. In studies using fundus reflectometry, spectroscopy, and radioimmunoassay to compare rhodopsin and arrestin content in younger and older individuals, no substantial age-dependent change was noted. ^{22 23 24 25 38} However, a weakness in these studies was that the macula and the periphery were not separated. Using the entire retina could mask the changes that are occurring specifically within the macula. 

Curcio et al. ¹¹ focused specifically on the macula, and found that an ∼30% decline in the total number of macular rod photoreceptors was observed between the third and ninth decades. Specifically, a 9% decrease in rods was reported in donors ages 60 to 75 years, followed by a further ∼28% decline in rods in individuals aged 80 to 90 when compared with a group of young donors aged 27 to 37 years. ¹¹ We attempted to account for some of the potential variability simply due to aging by including donors from a broad range of ages in each MGS level. In our population sample, 100% of the donors were aged 75 years or less in MGS1 and -2. The percentage declined to 75% and 38% ≤75 years of age for MGS3 and -4, respectively. To estimate the contribution of the disease after accounting for age, we compared the changes in macular arrestin content in MGS1 and -4 retinas. Using values from donors of all ages, there was an ∼60% decrease in arrestin content in MGS4. Because a portion of this decrease could be due to age, we re-examined macular arrestin from only donors aged 70 to 75 years in MGS1 and -4 to estimate the percentage of change due specifically to pathologic degeneration. We found an ∼53% decrease in arrestin content in MGS4 eyes compared with age-matched MGS1 eyes, suggesting most of the changes we are seeing are disease-related and not due to normal aging. Thus, we acknowledge that the age difference between our control and groups at later stages of MGS could account for some of the decrease in expression of macular rhodopsin and arrestin. However, an age effect is likely a minor contribution. 

The significant linear relationship between the content of macular rod proteins with progression of MGS suggests that rod loss begins early in the disease process. However, it is not until later stages of AMD that the decline in protein content is significantly less than controls or the earlier stage (MGS2). There are two potential confounding variables that could influence the small but nonsignificant decline at the earlier stage. First, inclusion of all tissue in the 8-mm macular punch could be masking some of the subtle changes in rod density that are restricted exclusively to a subregion of the macula. Previous reports show that the greatest rod loss is 0.5 to 3.0 mm from the center of the macula. ⁴ This region accounts for only 14% of the total area included in the 8-mm punch. Sites progressively farther from the macular center showed less rod loss until at ∼8 mm disease-related differences were undetectable. ^{4 10} A second variable that could influence the rod content is the compensatory changes in rod photoreceptor size in the early stages of AMD. It has been noted that although the number of rod photoreceptors decreases, the diameter of the surviving rod’s inner segments increases spatially. ¹¹ Furthermore, it was suggested that the protein content per rod also increases, although the amount of rhodopsin and arrestin was not measured. ¹¹ Our biochemical measure of total protein content cannot distinguish whether this compensatory mechanism has occurred or whether changes of ROS length may contribute to the observed changes in ROS protein content. 

Although it is difficult to compare experimental results acquired by using different techniques, our results are in agreement with those in other studies. The 30% to 60% decrease we observed in macular rod protein content at later stages of AMD is consistent with reports of a 30% to 40% rod loss that occurs in advanced aAMD and eAMD, respectively. ⁴ The unchanging peripheral protein content we observed through progressive stages of AMD are in agreement with results from histologic, topographical, and functional studies that showed no rod loss in the periphery. ^{11 14 21}  

Using the MGS, macular protein expression changes corresponded to each stage of AMD, including the earliest clinical stages. Because the MGS is based on clinical definitions and standards, identification of changes in protein or gene expression at specific stages of degeneration could identify pathologic mechanisms involved with disease progression. This is the first step in developing strategies for therapy targeted at specific molecular defects. A universal system of grading eye bank eyes would also permit more direct comparison of the molecular details of degeneration with other clinical and epidemiologic information to help identify mechanisms of disease progression. 

This study detected specific biochemical changes that correspond to progression of AMD, as documented by the MGS. Furthermore, these experiments confirmed region-specific, biochemical changes in protein expression that occur between the periphery and macula at each stage of AMD. This information provides the framework for design of future in-depth protein analysis. 

 Supported by the MN Lions Macular Degeneration Center, the University of Minnesota Vision Foundation, National Eye Institute Grants EY014176 (DAF) and AG025392 (TWO), the American Health Assistance Foundation, the Foundation Fighting Blindness, the American Federation for Aging Research (DAF), and an unrestricted grant to the Department of Ophthalmology from the Research to Prevent Blindness Foundation. CME was supported by National Eye Institute Grant T32 EY07133-12 and a Glenn/AFAR Scholarship from the American Federation for Aging Research.

 Submitted for publication July 9, 2004; revised October 25, 2004; accepted November 23, 2004.

 Disclosure: C.M. Ethen, None; X. Feng, None; T.W. Olsen, None; D.A. Ferrington, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Donor Demographic and Clinical Information

MGS Level	Number Analyzed					Age , † (y)	Range (y)	Cause of Death*
		Total	M	F
1	10	6	4	56 ± 13	37–72	Heart failure (3), cancer (2), respiratory failure (2), head trauma (1), pneumonia (1), aneurysm (1)
2	16	11	5	64 ± 11	38–75	Cancer (10), heart failure (2), respiratory failure (2), stroke (1), sepsis (1)
3	12	8	4	75 ± 9	63–91	Heart failure (3), cancer (3), renal failure (2), age (1), embolism (1), respiratory failure (1), brain injury (1)
4	8	6	2	83 ± 9	70–94	Heart failure (3), age (2), cancer (1), respiratory failure (1), aneurysm (1)

*  Number of donors in a category are within parentheses.

†  Mean age ± SD.

Figure 1.

 

Retinal protein profile in donors at different MGS levels in the macula and periphery. Representative Coomassie blue–stained peripheral (A) and macular (B) proteins resolved by 10% SDS-PAGE. Protein loads were 5 μg per lane.

Figure 2.

 

Arrestin content in the neurosensory retina. (A) Representative immunoreaction from macular (M) and peripheral (P) regions at MGS1 to 4. Migration of the molecular mass marker is indicated. Protein loads were 5 μg per lane. (B) Relative content determined from densitometric analysis of the immune reaction in retinal homogenates from macular (▪) and peripheral (|pR) regions. Normalized density is the sample immune reaction normalized to a standard reaction on all blots. Data are expressed as the mean ± SEM. Dashed and solid lines: slope of line fitted to peripheral (0.01) and macular (−0.14) data, respectively. * Significant difference between MGS4 and MSG1, 2, and 3 eyes. Results are from 6 to 11 donors for the macula and 8 to 14 donors for the periphery.

Figure 3.

 

Rhodopsin content in the neurosensory retina. (A) Representative immunoreaction from macular (M) and peripheral (P) regions at MGS1 to 4. Migration of molecular mass markers are indicated. Protein loads were 5 μg per lane. (B) Relative content determined from densitometric analysis of the immune reaction in retinal homogenates from macular (▪) and peripheral (|pR) regions. Normalized density is the sample immune reaction normalized to a standard reaction on all blots. Data are expressed as the mean ± SEM. Dashed and solid lines: slope of fitted line to peripheral (0.02) and macular (−0.07) data, respectively. * Significant difference between MGS4 and 2 eyes. Results are from 6 to 12 donors for the macula and 7 to 14 donors for the periphery.

Figure 4.

 

Arrestin translocates to the RIS of the retina postmortem. (A) Immune blots detecting arrestin (arr) and rhodopsin (rho) in both retinal homogenates (H) and purified ROS (R) from the same donor eye. Three representative samples and their time postmortem to freezing are shown. Protein loads were 5 μg per lane. Molecular mass markers are indicated. (B) Ratio of arrestin to rhodopsin content in retinal homogenate (▪) and purified ROS ( ) from the same donor eye. Six donor eyes at various times postmortem were analyzed.

The authors thank the personnel at the MN Lions Eye Bank for their assistance in acquiring eyes for the study, Kristin Pilon and Kristin Berg for retinal dissections, Dale S. Gregerson for his kind gift of arrestin antibody, and Cavan Reilly for discussions in statistical analysis.