April 2006
Volume 47, Issue 4
Free
Retinal Cell Biology  |   April 2006
A Frameshift Mutation in RPGR Exon ORF15 Causes Photoreceptor Degeneration and Inner Retina Remodeling in a Model of X-Linked Retinitis Pigmentosa
Author Affiliations
  • William A. Beltran
    From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York; and the
    School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
  • Pamela Hammond
    From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York; and the
  • Gregory M. Acland
    From the James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York; and the
  • Gustavo D. Aguirre
    School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Investigative Ophthalmology & Visual Science April 2006, Vol.47, 1669-1681. doi:10.1167/iovs.05-0845
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      William A. Beltran, Pamela Hammond, Gregory M. Acland, Gustavo D. Aguirre; A Frameshift Mutation in RPGR Exon ORF15 Causes Photoreceptor Degeneration and Inner Retina Remodeling in a Model of X-Linked Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2006;47(4):1669-1681. doi: 10.1167/iovs.05-0845.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To characterize the course of retinal disease in X-linked progressive retinal atrophy 2 (XLPRA2), a canine model of early onset X-linked retinitis pigmentosa (XLRP) caused by a two-nucleotide microdeletion in RPGR ORF15.

methods. The retinas of 25 XLPRA2-affected dogs (age range, 2–40.6 weeks) and age-matched control subjects were collected, fixed, and embedded in epoxy resin for morphologic evaluation or in optimal cutting temperature (OCT) medium for TUNEL assay and immunohistochemistry. Cell-specific antibodies were used to examine changes in rods and cones and to evaluate the effects of the primary photoreceptor degeneration on inner retinal cells.

results. Abnormal development of photoreceptors was recognizable as early as 3.9 weeks of age. Outer segment (OS) misalignment was followed by their disorganization and fragmentation. Reduction in length and broadening of rod and cone inner segments (IS) was next observed, followed by the focal loss of rod and cone IS at later time points. The proportion of dying photoreceptors peaked at approximately 6 to 7 weeks of age and was significantly reduced after 12 weeks. In addition to rod and cone opsin mislocalization, there was early rod neurite sprouting, retraction of rod bipolar cell dendrites, and increased Müller cell reactivity. Later in the course of the disease, changes were also noted in horizontal cells and amacrine cells.

conclusions. XLPRA2 is an early-onset model of XLRP that is morphologically characterized by abnormal photoreceptor maturation followed by progressive rod–cone degeneration and early inner retina remodeling. The results suggest that therapeutic strategies for this retinal degeneration should target not solely photoreceptor cells but also inner retinal neurons.

X-linked retinitis pigmentosa (XLRP) is a group of diseases that comprises some of the most severe and early-onset forms of inherited retinal degeneration in humans with partial or complete blindness in the third or fourth decade of life, or earlier. 1 2 3 Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for more than 70% of patients with XLRP, and most of these mutations are found in exon ORF15. 4 A spectrum of disease phenotypes is associated with RPGR mutations. The most common reflect the typical rod–cone degeneration encountered in most forms of RP and consist first of night blindness and loss of the midperipheral visual field followed by loss of day vision and central visual acuity. 1 2 Yet, some other mutations result in equal rod–cone abnormalities, 5 cone–rod dystrophies, 6 or macular degeneration. 7  
To understand the retinal function of the RPGR protein, as well as the pathogenic mechanisms that link mutations in RPGR with the death of photoreceptor cells, several animal models have been used. These comprise two transgenic murine models (RPGR knockout mouse, 8 and a dominant gain-of-function mutant 9 ), and two naturally occurring canine mutations in exon ORF15 10 that cause two forms of X-linked progressive retinal atrophy (XLPRA). 
In XLPRA1, a five-nucleotide deletion (del1028-1032) in exon ORF15 causes an immediate premature stop codon that results in a protein truncated of its 230 C-terminal amino acids. This mutation causes a loss of function of RPGR. Morphologic characterization showed that photoreceptor cells develop and function normally, but then undergo progressive rod–cone degeneration. The earliest histologic signs of rod degeneration are detected at 11 months of age, and are followed at later stages by cone death. 10 11  
In XLPRA2, preliminary results from our group have shown that the disease is a much more severe and earlier form of retinal degeneration than XLPRA1. 10 A two-nucleotide deletion (del 1084-1085) in exon ORF15 results in a frameshift that changes the deduced peptide sequence by the inclusion of 34 additional basic residues and increases the isoelectric point of the truncated protein. 10 In addition, the mutant ORF15 protein was also shown to accumulate in the endoplasmic reticulum of transfected COS7 cells. These results suggest that the mutation in XLPRA2 causes a toxic gain of function and is comparable to the severe human phenotype resulting from microdeletions that cause a frameshift. 12  
Although the precise subcellular location of the RPGR ORF15 protein in photoreceptor cells is still debated, 13 14 findings in the null mutant mouse suggest that it may play a role in maintaining a polarized distribution of proteins between inner (IS) and outer (OS) segments. 8 Yet, it is still unclear how the loss of function of RPGR or the expression of a toxic mutant RPGR protein in XLPRA2, may initiate a cascade of molecular events that ultimately lead to photoreceptor cell death. To begin to address this question, we examined the retinal structural alterations that occur in XLPRA2 and characterized the time course of photoreceptor disease, degeneration, and death and the subsequent alterations that occur in the inner retina. We found an early onset of photoreceptor disease leading to cell death, as well as early inner retina remodeling. Our results identify the critical stages in the pathogenesis of the disease and define the time windows for testing novel therapies. 
Materials and Methods
Animals
Thirty-five dogs were used in the study (Table 1) . This included 25 crossbred XLPRA2 affected dogs (age range, 2-40.6 weeks), and 10 nonaffected beagles that were used as normal control subjects (age range, 2–24 weeks). All affected dogs (11 hemizygous males, 14 homozygous females) were bred at the Retinal Disease Studies Facility (RDSF; University of Pennsylvania, New Bolton Center, Kennett Square, PA), and their genotype was determined either from the known status of their progenitors or from genetic testing for the disease-causing mutation. 10 All nonmutant beagles came from the Baker Institute colony of specific pathogen-free dogs. After an ocular examination to identify abnormalities not associated with the primary retinal disease, all animals were anesthetized by intravenous injection of pentobarbital sodium, the eyes enucleated, and the dogs euthanatized. All procedures involving animals were done in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Retinal Histology
The left eyes of 16 XLPRA2 dogs (age range, 2–40.6 weeks) were used for morphologic examination of disease expression using plastic embedding (Table 1 , morphology). The retinas of three normal beagles (ages: 2.3, 5.4, and 8.3 weeks) were used as control specimens. By 8 weeks of age the canine retina is structurally mature. 15 16 Immediately after enucleation, the posterior segments were isolated and fixed, using a triple fixative protocol (3% glutaraldehyde-2% formaldehyde; 2% glutaraldehyde-1% osmium tetroxide; and 2% osmium tetroxide), as previously reported. 15 The posterior segments were then trimmed into pieces that extended from the optic nerve to the ora serrata along the superior and inferior meridians, dehydrated, and embedded in epoxy resin (PolyBed 812; Polysciences, Warrington, PA). Tissues were sectioned with glass knives at 1 μm with a supercut microtome (Reichert Jung model 2065; Leica, Deerfield, IL), stained with azure II-methylene blue and a paraphenylenediamine counterstain. 
Sections from both the superior and inferior meridians were examined with a 40× objective on a light microscope (Axioplan; Carl Zeiss Meditec GmbH Oberkochen, Germany). The sections were examined in contiguous fields from the optic disc to the ora serrata. This included evaluation of the retinal pigment epithelium (RPE), the rod and cone OS and IS, and the thickness and density of the outer (ONL) and inner (INL) nuclear layers. For each dog, a single section from both quadrants was used for quantitative evaluation of the photoreceptor cells, and INL cells at three specific locations: S1, 2000 ± 500 μm from the optic nerve; S3, 2000 ± 500 μm from the ora serrata; and S2, midway (±500 μm) between these two points. At each of these sites, the number of rows of nuclei in the ONL and INL were counted in at least three areas of a 40× field and averaged. For the same areas, the thickness (in micrometers) of the ONL and INL were measured on digitally captured images (Spot 4.0 camera, Diagnostic Instruments, Inc., Sterling Heights, MI). 
The kinetics of photoreceptor cell loss were analyzed by fitting the ONL thickness data to solutions of the following differential equations reported by Clarke et al. 17 We are also reporting below the integral equations used for the statistical analysis, since we found typographical errors confirmed by the authors (Geoff Clarke, personal communication, November 29, 2005) in the integral equations provided in the supplementary information that accompanied their paper (http://www.nature.com/nature/journal/v406/n6792/suppinfo/406195a0.html). 
Constant risk:  
\[\frac{dONL(t)}{dt}\ {=}\ {-}{\mu}_{0}\ {\times}\ ONL(t)\ \begin{array}{l}\mathrm{integral}\\{\rightarrow}\end{array}\ ONL(t)\ {=}\ ONL(0)e^{{-}{\mu}_{0}t}\]
Exponentially decreasing risk:  
\[\frac{dONL(t)}{dt}\ {=}\ {-}{\mu}_{0}e^{{-}At}\ {\times}\ ONL(t)\ \begin{array}{l}\mathrm{integral}\\{\rightarrow}\end{array}\ ONL(t)\ {=}\ ONL(0)e^{{[}(e^{{-}At}{-}1){\mu}_{0}/A{]}}\]
Exponentially increasing risk:  
\[\frac{dONL(t)}{dt}\ {=}\ {-}{\mu}_{0}e^{At}\ {\times}\ ONL(t)\ \begin{array}{l}\mathrm{integral}\\{\rightarrow}\end{array}\ ONL(t)\ {=}\ ONL(0)e^{{[}{-}(e^{At}{-}1){\mu}_{0}/A{]}}\]
Data-fitting was performed with nonlinear regression analysis (with PROC NLIN in SAS 9.1 software; SAS Institute Inc., Cary, NC). This is a least-squares procedure for estimating parameters in nonlinear models. The parameter estimates (using the Gaussian method) and 95% confidence intervals, along with probabilities, (based on the Wald test) were computed. The R 2 was used to assess the overall goodness of fit of the model. Please note that it was not possible to fit our data to mathematical models that account for the time period between birth and onset of cell death (called the “delay” parameter by Clarke et al. 17 ), because there were insufficient data at early time points, which prevented us from getting an estimate for the delay parameter. 
Phagocytic cells present in the photoreceptor layer were counted throughout the entire length of both the superior and inferior retinal meridians and expressed as the number of phagocytic cells per unit length of retina. The unit length was set as 10,000 μm. Pyknotic photoreceptor nuclei were counted in the ONL in both the superior and inferior meridians and expressed as the number of pyknotic nuclei per unit area of ONL. The unit area was set as 1 million μm2 (1 M μm2) of ONL. A similar count was used to quantify TUNEL-positive photoreceptor cells. 
TUNEL Assay
In 18 XLPRA2-affected dogs, one eye was processed immediately after enucleation for TUNEL assays and/or immunohistochemistry (Table 1 : TUNEL, IHC). After enucleation, a slit was made through the globe at the level of the ora serrata, and the entire globe was fixed for 3 hours in 4% paraformaldehyde in 0.1 M phosphate-buffered saline at 4°C. The posterior segment then was isolated, the vitreous gently removed, and the eye cup fixed for an additional 24 hours at 4°C in 2% paraformaldehyde in 0.1 M phosphate-buffered saline. The tissue was then trimmed, cryoprotected sequentially for 24 hours in a solution of 15% and 30% sucrose in 0.1 M sodium phosphate and 0.15 M sodium chloride (pH 7.2; BupH, phosphate-buffered saline; Pierce, Rockford, IL; referred in the text as PBS) at 4°C, and embedded in OCT medium. 
Cryosections (7 μm thick) along the superior meridian of 17 XLPRA2 dogs (age range, 3.9–40.6 weeks) were used for TUNEL assay, according to the manufacturer’s protocol (In situ cell death detection kit; Roche) and stained with 4′,6′-diamino-2-phenylindole (DAPI). Sections along the superior meridian of three normal beagles (ages, 4, 5, and 6 weeks) were also used. Positive control specimens included sections pretreated with DNase I (3 U/mL in 50 mM Tris-HCl [pH 7.5] and 1 mg/mL BSA for 10 minutes at room temperature). For negative control subjects, the terminal transferase enzyme was omitted from the TUNEL reaction mixture. Sections were examined from the optic disc to the ora serrata by epifluorescence microscopy with the 40× objective. TUNEL-labeled cells in the ONL were counted throughout the entire length of the section (i.e., from disc to ora serrata). In determining the proportion of photoreceptor cells that undergo cell death as a function of time, we express our results as the number of TUNEL-labeled photoreceptor cells per 1 M μm2 of ONL. The area of the ONL of each section was obtained by measuring the entire length of the ONL from optic disc to ora serrata, and multiplying it by the average thickness of the ONL throughout the section (mean value of the thickness measured in the three locations S1, S2, and S3). This method may slightly underestimate, in areas of decreased photoreceptor density, the proportion of cells that are TUNEL positive. Yet, it was selected because individual cell count could not be determined on 7-μm-thick DAPI-stained cryosections. For each dog, this procedure was performed in triplicate with sequential sections from the superior meridian. The values were averaged and reported as the mean ± SD. 
Immunohistochemistry
Sections along the superior retinal meridian of nine XLPRA2 dogs (age range, 2–40.6 weeks) and six normal dogs (age range, 2–24 weeks) that were processed as described earlier were used for fluorescent immunohistochemistry. We used a battery of cell-specific primary antibodies, 9 18 19 20 21 22 23 24 25 26 27 28 29 30 of which more than half worked on canine retina (see details in Table 2 ). Because of the lack of specific antibodies directed against all subpopulations of ganglion cells in the dog, our study did not include assessment of their density or morphology. Because previous testing of RPGR and RPGRIP antibodies conducted in our laboratory failed to show any cross-reactivity or specificity on canine retina, we did not include them in this study. 10 Cryosections (7–10-μm thick) were incubated overnight with the primary antibodies after a blocking step with 10% normal serum from the appropriate species. The antigen–antibody complexes were visualized with fluorochrome-labeled secondary antibodies (Alexa Fluor, 1:200; Invitrogen, Carlsbad, CA). DAPI stain was used to detect cell nuclei. Slides were mounted with a medium composed of polyvinyl alcohol and DABCO (1,4 diazobizyklo-[2.2.2]oktan) (Gelvatol; Sigma-Aldrich, St. Louis, MO), and examined with an epifluorescence microscope (Axioplan; Carl Zeiss Meditec). Images were digitally captured (Spot 4.0 camera; Diagnostic Instruments, Inc.) and imported into a graphics program (Photoshop; Adobe, Mountain View, CA) for display. 
Results
Photoreceptor Disease
Gross examination of the eyes at the time of enucleation did not reveal significant differences in size between affected and normal dogs. Measurements of retinal length between the optic disc and the ora serrata along the superior meridian confirmed that in XLPRA2 the growth of the eye was not affected by the disease (data not shown). 
Normal retinal development in the dog is complete at approximately 7 to 8 weeks of age. 15 16 At birth, photoreceptor cells have not completely differentiated and are located in the sclerad portion of the outer neuroblastic layer, which then give rise to the ONL. Maturation of photoreceptors occurs in waves from the central to peripheral retina. 31 IS are first seen as short bulges of cytoplasm protruding from the external limiting membrane between 1 day and 1 week after birth. 32 At approximately 2 weeks of age, IS are visible throughout the entire length of the retina, and OS formation is underway centrally (Fig. 1A) . By 5.4 weeks of age, OS are formed and begin to elongate (Fig. 1B) . Full maturation of photoreceptors is reached at approximately 8 weeks of age, at which time the retina resembles that of the adult (Fig. 1C) . Pyknotic figures in the ONL were extremely rare (0 to 1 per 1 M μm2 of ONL) at all ages examined. Concurrent with the maturation of the photoreceptors, there are changes in the inner retinal layers which are most prominent in the INL. These primarily consist of a decrease in the number of nuclei, presumably because of an increase in eye size (see Figs. 1A 1B 1C ). 
Disease stages of XLPRA2 were defined at early time points on the basis of the structural changes observed in the photoreceptor layer (IS, OS). At later ages, reduction in ONL thickness was also taken into account. Because no major differences in the course of the disease were observed between the superior and inferior retina, the description is provided for the superior meridian (Fig. 1) . Table 3provides more specific details (phagocyte count, ONL thickness, pyknosis in ONL, and INL thickness) for both the superior and inferior meridians. 
The earliest time point examined in the affected dogs was 2 weeks of age (Fig. 1D) , and no evidence of abnormal development or arrested differentiation was observed (stage 0; normal). The typical layering of the neuroretina was preserved from the optic disc to the ora serrata along both superior (tapetal) and inferior (nontapetal) meridians. Nuclei in the ONL were elongated, which is a normal characteristic of immature photoreceptor cells. Nuclear pyknosis was negligible in the ONL (Table 3) . Cone and rod IS were present throughout the photoreceptor layer. In the central retina, elongating IS, with budding OS, were observed, whereas in the periphery, short IS were in close contact with the apical RPE. 
At 3.9 weeks of age, there was a moderate increase in the number of pyknotic cells in the ONL (Table 3)and very subtle changes in the morphology of the OS. This early stage of OS disruption and misalignment was better seen when dogs were slightly older (≈5 weeks of age; stage 1; Fig. 1E ). At that age, nuclear pyknosis had increased in the ONL (Table 3)
By 7.9 weeks of age (stage 2; Fig. 1F ), there was severe OS disintegration, with abundant disorganized and disoriented membranous material persisting in the photoreceptor layer. ONL thickness was moderately reduced, and the number of pyknotic nuclei in the ONL had decreased (Table 3) . A similar decline in INL thickness, as seen in the normal retina, was observed between 2 and 7.9 weeks of age (see Figs. 1A 1B 1C 1D 1E 1F ) and reached approximately three to four rows of nuclei (see Table 3 ). 
By 11.9 and 16 weeks, there was narrowing of the subretinal space with shortening or loss of rod IS, and these abnormalities were comparable at both time points (stage 3; Fig. 1G ). Distorted OS persisted in the photoreceptor layer. There was marked rod loss, with the ONL reduced to approximately 60% of its original thickness. Pyknosis in the ONL was further reduced. A few phagocytic cells located in the subretinal space in close apposition to the RPE were first seen in the 11.9-week-old retina. Their number increased at 16 weeks (Table 3) . Disease-associated thinning of the INL was observed at both ages and was most pronounced in the peripheral retina (Table 3)
There was a clear decrease in the density of both rod and cone photoreceptors by 26 weeks of age (stage 4; Fig 1H ). This decrease was seen at the level of the photoreceptor layer, where there were areas devoid of any rod and cone IS and also in the ONL where internuclear spacing was increased. ONL thickness was less than 50% of its original thickness. In the photoreceptor layer, the remaining rod and cone IS appeared broader and, although the subretinal space was severely narrowed, shortened, and distorted, OS were still present. Cone nuclei displaced into the IS and extruding into the subretinal space were first seen at this age. Phagocytic cells persisted in the subretinal space. 
The latest stage of disease examined was at 40.6 weeks (stage 5; Fig. 1I ). At that time point, there were approximately two to three rows of nuclei left in the ONL, and the remaining cone and rod IS were short and broad. Numerous photoreceptor cells maintained a shortened and misaligned OS, and RPE cytoplasmic processes extended toward them. Phagocytes remained in the subretinal space, but were not migrating in the ONL or inner retina. Nuclear pyknosis persisted in the ONL but was not observed in the INL, despite a decrease in its thickness (Table 3)
To illustrate the rate of photoreceptor cell loss that occurs during the course of the disease along both the superior and inferior retinal meridians, we plotted the average thickness of the ONL (expressed as either the number of nuclei per column, or in micrometers) as a function of age. These graphs (Fig. 2)show a major and rapid early cell loss occurring from 4 to 12 weeks of age. Subsequently, the number of remaining photoreceptors continued to decrease but at a slower rate. The kinetics of photoreceptor cell loss was best described by a model of constant risk of cell death when the ONL thickness was measured as the number of nuclei. When the ONL thickness was measured in micrometers, the data were best fit by both a model of constant risk and of decreasing risk of cell death. 
To determine whether the disease and degeneration of photoreceptor cells was uniformly distributed throughout the retina, or, if there was a topographic distribution of the disease, we examined all sections from optic disc to the ora serrata. We observed that a comparable disease process occurred throughout the entire length of the retina along both the superior and inferior meridians. In the normal retina, there is a normal gradient in ONL thickness from central (10–14 nuclei) to peripheral (3–6 nuclei) retina. 33 A similar trend in ONL thickness occurred in young mutant retinas and therefore should not be considered as several different stages of the disease coexisting along the same retinal meridian. 
Cell Death
Because of the uniform distribution and rate of disease along the superior and inferior meridians, we determined cell death by using TUNEL assays in sections from the superior meridian. The earliest age when cell death was examined by TUNEL assay was 3.9 weeks, because this was the age when the first morphologic signs of disease were detected. Approximately 31 to 48 TUNEL-labeled cells per 1 M μm2 of ONL were counted. The proportion of photoreceptors undergoing cell death was higher at 5 and 6 weeks, and reached a peak of more than 300 TUNEL-positive cells per 1 M μm2 of ONL at 6.7 weeks of age. In normal retinas of 4, 5, and 6 week-old beagles, the number of TUNEL-positive cells per unit area of ONL was significantly lower and did not exceed six per 1 M μm2 of ONL. At 8 weeks of age, the proportion of photoreceptors undergoing cell death in the mutant retina had decreased to approximately half that occurring at 6.7 weeks (∼150 TUNEL-positive cells per 1 M μm2 of ONL). At any given time after 12 weeks of age, the proportion of dying photoreceptors was significantly reduced and close to 80 cells/1 M μm2 of ONL (Fig. 3) . TUNEL-labeled cells were equally distributed throughout the length of the retina, but it appeared that at the earlier ages, there were more dying cells located in the vitreous half of the ONL (Fig. 4A1) . Yet, although, TUNEL-positive photoreceptors were seen in the outer half of the ONL, it was extremely rare before 26 weeks of age to detect any labeling in the outermost row of ONL nuclei, where cone somas are located (Figs. 4A1 4A2) . In 26- and 40.6-week-old XLPRA2 retinas, it was frequent to observe, particularly in the retinal periphery, cone nuclei that were ectopically located in the IS (Fig. 4A3) . Double fluorescence labeling showed that a few displaced cone nuclei were TUNEL positive (Fig. 4A4) . Rare TUNEL-positive cells were also present in the INL and GCL in both mutant and normal young retinas (4–6 weeks), and this was therefore considered to be a normal finding not associated with disease. At later time points in the mutants, there was a relative absence of TUNEL-labeling in those layers. 
Immunohistochemical Analysis of Photoreceptors and Inner Retinal Changes with Disease
To further characterize the effects of the disease on the morphology, location, and density of several retinal cell populations, we tested a battery of antibodies that are commonly used as cell-specific markers (see Table 2 ) on retinas of mutants and normal age-matched control subjects. 
The integrity of the RPE was evaluated with an antibody directed against RPE65. There was no loss in RPE65-immunoreactivity in 16- and 26-week-old mutant retinas and the numerous phagocytic cells present in the subretinal space were not labeled by the RPE65 antibody (data not shown). Double-fluorescence immunolabeling with rod opsin and human cone arrestin antibodies showed partial mislocalization of the rod photopigment to the ONL as early as 2 weeks of age in mutant retinas (data not shown). Although rod opsin labeling was restricted to the OS in normal subjects (Fig. 4B1) , distinct staining of the plasma membrane around the rod somas also was visible throughout the entire length and thickness of the ONL in affected dogs at all ages (Fig. 4B2) . In the 7.9-week-old mutant retina, short rod-opsin–positive neurites originating from rod somas extended into the inner retina (Fig. 4B2) , and, in older animals, the sprouting was more prominent and extended deeper into the inner retina, reaching the inner plexiform layer (IPL; Fig. 4B3 ; 40.6 weeks of age). In contrast, cone neurite sprouting was not observed at any stages of the disease examined. 
To confirm further that these opsin-positive projections were rod neurites, we performed double immunofluorescence labeling with rod opsin and synaptophysin antibodies (Figs. 4C) . Anti-synaptophysin labeled both plexiform layers in normal subjects (Fig. 4C1) , but, in mutant retinas, outer plexiform layer (OPL) labeling was thinned, and punctuate staining in the INL colocalized with the rod-opsin–positive neurites (Fig. 4C2) . Colocalization occurred at beaded varicosities along the neurites and at their terminals. These had either a bulb-shaped appearance or that of a typical rod spherule (Fig. 4C3) . Although rod opsin and cone arrestin labeling persisted in the 40.6-week-old affected retina, we observed a decrease in cone arrestin immunoreactivity at the level of the cone axons and pedicles (Fig 4B3)that was first visible at 26 weeks of age. Because of the thinning of the ONL at that age (approximately three rows of nuclei), the lengths of the remaining rod and cone axons were significantly shorter than in a normal adults. Even though thinning and disruption of the photoreceptor layer caused occasional retinal separation artifacts during tissue fixation and processing in older retinas, distinct rod opsin and arrestin labeling was observed, respectively, in some of the remaining rod and cone OS (Fig. 4B3)
To characterize better the two subpopulations of cone photoreceptor cells during the course of disease, we used antibodies raised against short (S)- and medium (M)/long (L)-wavelength cone opsin on retinas at various ages and disease stages. Both S and M/L cone opsin labeling were present in young and older (40.9 weeks) affected dogs. In normal retinas, labeling was restricted to the cone OS (Figs. 4D1 4D3) , but in mutant retinas there was partial mislocalization of the two types of cone opsins to the IS, perinuclear area, axon, and pedicles (Figs. 4D2 4D4) . Mislocalization of S opsin was observed in some S cones, distributed throughout the entire length of the retina, as early as 3.9 weeks of age. By 6 weeks, S opsin mislocalization was found mainly in some peripheral cones (Fig. 4D2) . At later ages S opsin localization was normal. Although the M/L opsin antibody that we used caused some nonspecific background staining of the INL and faint labeling of cone somas and axons, we observed a similar transient and partial mislocalization of the photopigment, particularly in the peripheral retina of 3.9 and 6-week-old affected dogs (Fig. 4D4) . At 8 weeks of age, M/L opsin mislocalization was essentially restricted to the perinuclear area of the cones and did not extend into the axons and pedicles; in older animals, M/L opsin localization was normal. 
Because we observed OPL thinning and rod neurite sprouting, we decided to use several inner retina cell markers to determine whether photoreceptor disease and degeneration were also associated with inner retinal changes. A variety of antibodies that label subpopulations of horizontal, bipolar, and amacrine cells, as well as Müller cells were tested in either single or double immunofluorescence analysis. 
Anti-calbindin antibody labeled horizontal cells and, to a lesser extent, amacrine and RPE cells in both affected and normal retinas at 4 weeks of age. At later time points in disease, immunostaining was predominantly found in horizontal cells somas and processes. This pattern persisted throughout the course of the disease. Because of the variability in the intensity of calbindin-labeling in the dog, we were not able to quantify with certainty the number of horizontal cells present throughout the length of a retinal section and compare these counts at different stages of the disease. Nevertheless, we were able to observe in older affected retinas a flattening of their axonal arborization associated with the thinning of the OPL (Figs. 5A1 5A2 5A3)
PKCα staining of rod bipolar cells showed that these second-order neurons developed normally (Fig. 5B1)in the diseased retina (data not shown). At 11.9 weeks of age there was a mild reduction in the density of their dendritic arborization, which was followed by progressive shortening and total atrophy at later stages (Figs. 5B2 5B3) . By performing double-immunofluorescence experiments with PKCα and Goα (a cell-marker for ON bipolar cells) antibodies, we were able to distinguish rod bipolar cells that coexpressed PKCα and Goα from ON-cone bipolar cells that were only Goα immunoreactive (Fig. 5C1) . We confirmed that rod bipolar cell dendrites underwent retraction with time, but did not observe a similar change in ON-cone bipolar cells (Figs. 5C2 5C3 5C4) . Indeed, even at the latest time-point examined (40.6 weeks), there was distinct labeling of the dendrites of cone bipolar cells that appear as a continuous layer in the OPL (Fig. 5C4)
Although we tested a variety of antibodies (anti-ChAT, anti-TH, anti-Dab1, anti-γ-aminobutyric acid [GABA], see Table 2 ) reported to label different subpopulations of amacrine cells in rodents, we were successful only in detecting GABAergic amacrine cells in the canine retina. Labeling of amacrine cell bodies with the GABA antibody was limited to the central retina in the normal adult, yet intense staining of the IPL laminae was seen throughout the entire retina (Fig. 5D1) . A similar pattern was seen in the mutant retina until 11.9 weeks of age. Thereafter (26 and 40.6 weeks), there was a significant increase in the number of GABA-immunoreactive amacrine cells. These were located both at the inner border of the INL as well as displaced into the ganglion cell layer (GCL), and they were found all along the length of the retina. In addition, there was a thinning of the IPL and a loss of its normal lamination (Fig. 5D2 5D3)The GABA antibody also labeled the somas and processes of horizontal cells in the normal canine retina (Fig. 5D1) . This was also observed in young affected retinas until 8 weeks of age. In the 11.9-week-old mutant retina there was decrease in the intensity of the labeling, and, after 26 weeks, no staining of any horizontal cell was observed, although it was distinct in the normal retina (Fig. 5 , compare D1 with D2, D3). 
Because the cellular retinaldehyde-binding protein (CRALBP) antibody that we used to examine Müller cells did not label canine retina, we used instead an antibody directed against glutamine synthetase. With this antibody, we found a decrease in Müller cell length in mutant retinas associated with the thinning of the ONL; however, there was no apparent reduction in their density at 11.9 weeks of age. By 26 weeks of age, there was a significant reduction in Müller cell immunoreactivity, and by 40.6 weeks, immunolabeling was almost absent (data not shown). This precluded assessment of glial cell loss at later stages of the disease. GFAP immunolabeling was used to evaluate the level of glial reactivity in Müller cells. GFAP staining was limited to astrocytes and Müller cell end feet in normal retinas of all ages (Fig. 5E1)as well as in the youngest (3.9 weeks) retina. A gradual increase in GFAP immunoreactivity began at 5 weeks and peaked at 8–12 weeks of age (Fig. 5E2) . Minimal GFAP reactivity was seen in older retinas when outer retinal atrophy was more advanced (Fig. 5E3)
Discussion
XLPRA2 is a severe canine retinal degeneration of early onset that affects both rods and cones. The two nucleotide deletion in RPGR ORF15 causes a frameshift in the translation of the putative protein that changes the glutamic-acid–rich ORF15 domain to one containing many arginine residues. 10 This, presumably, causes a toxic gain of function in photoreceptor cells that results in early and severe disease. Although signs of disease are detected in both classes of photoreceptor cells before their complete maturation, rods start dying at a much earlier stage than do cones. Furthermore, early remodeling of the OPL and INL suggest that photoreceptor disease and death alter the synaptic connections with inner retinal neurons. 
Evaluation of mutant retinas showed that this was a very early-onset disease characterized by visible abnormalities in both rods and cones before their maturation. At 3.9 weeks of age, the earliest signs of OS disruption were detectable on 1-μm-thick plastic-embedded sections, and mislocalization of rod and cone opsins was seen by immunohistochemistry. At this same age, there was clear evidence of rod photoreceptors undergoing cell death. We confirmed that this differed from the normal developmental cell death process, which was found at 2 weeks of age in both normal and mutant retinas (data not shown). In the mutant retina the number of TUNEL-labeled photoreceptor cells was significantly higher than in age-matched control subjects. Over the ensuing weeks, there was an increase in the proportion of TUNEL-labeled photoreceptors that resulted in a peak of cell death between 6 and 7 weeks. After this early burst of cell death, the rate was significantly decreased after 12 weeks of age. Our findings are similar to that reported in the Rdy cat, a model of autosomal dominant rod–cone dysplasia, in which an early onset of photoreceptor death begins at 5 weeks of age and peaks at approximately 9 weeks of age. 22 Our results clearly suggest that the risk of death in a single photoreceptor cell is not the same at all ages and that the mathematical model of a constant or decreasing risk of photoreceptor death, as suggested by Clarke et al., 17 may not be applicable to this class of RPGR mutations, at least during the initial course of the disease. 
Although the data on the ONL thickness (Fig. 2 , Table 4 ) are best fit with an exponentially declining curve that would be consistent with the kinetics of a constant or decreasing risk of photoreceptor death in XLPRA2, the TUNEL data (Fig. 3)do not support this model. We saw (Fig. 3)two distinct phases of cell death. An initial phase, from 3.9 to 7 weeks of age, showed an increased risk for photoreceptors to die (peak of TUNEL-positive cells at 6 to 7 weeks of age). This was followed by a second phase of a rapid (from 7 to 12 weeks) and then a more gradual decrease (after 12 weeks) in the number of TUNEL-positive cells per unit area of ONL. It is therefore possible that the limited number of observations between 3.9 and 7 weeks of age fails to show an early sigmoidal decline in photoreceptor cell number, which we would have expected to observe with an increased risk of cell death. This initial phase of photoreceptor death does not appear to be unique to XLPRA2. Indeed, a group of investigators used TUNEL assay to look at the kinetics of cell death in the rd mouse and recorded a similar increase in photoreceptor death that reached its peak by 16 days of age. 34 Not surprisingly, in their studies, Clarke et al. 17 35 reported that the rd mouse was the only animal model for which an increased risk of cell death could not be excluded, since the data fit equally well to mathematical models of constant and exponentially increasing risk. 
Several hypotheses may explain this biphasic pattern of cell death in XLPRA2: (1) the coexistence of two populations of photoreceptor cells that differ in their response to a same-death stimulus; (2) the existence of a single population of photoreceptors that acquire an increased resistance to death after the early degeneration of some cells. Supporting this hypothesis is evidence for the endogenous release of survival factors (bFGF, CNTF) in the degenerating retina that protect the remaining photoreceptors from undergoing cell death 36 37 ; and (3) the existence of a single population of photoreceptors that is affected at different ages by two distinct cell death stimuli. Based on the toxic gain-of-function hypothesis as being causal to the disease mechanism, 10 it may be that during photoreceptor maturation, rods are particularly sensitive to the toxicity of the mutant RPGR protein. This may lead to a burst of rod photoreceptor death, the release of survival factors, and the acquired resistance of the remaining photoreceptors to the endogenous mutant toxic protein. The second phase of death, after 12 weeks of age, could then be the result of either an incomplete resistance to the toxic mutant RPGR protein, or of sensitivity to the absence of the normal isoform. In this case, photoreceptor cells may be dying through a mechanism caused by the loss of function of the normal RPGR retinal isoform. If such is the case, this may open the possibility of rescuing photoreceptor cells that have survived the first phase of cell death in XLPRA2 through a gene replacement approach. 
The mislocalization of opsins in both rods and cones at the early stages of the disease suggests that RPGR is expressed in both populations of photoreceptors, and lends additional support to the hypothesis that RPGR is involved in the trafficking of proteins from the IS to the OS, or in their retention in the OS. 13 A similar mislocalization of both rod and cone opsins has been reported recently in a human carrier (Adamian M, et al. IOVS 2005;46:ARVO E-Abstract 3400), yet, in the mouse, only cone opsins appear to mislocalize. 8 In the present study, we found early signs of cone disease both in plastic-embedded sections and in cryosections treated for immunohistochemistry; however, we did not detect any features of cone cell death before the age of 26 weeks. This raises the question of whether the mechanism of cone death is directly caused by the RPGR mutation or is non–cell-autonomous and secondary to rod degeneration. In animal models of retinitis pigmentosa caused by mutations in rod-specific genes, the delayed cone loss is thought to be caused by the death of rods that induces structural alterations in the photoreceptor layer and ONL, the release of toxic byproducts, and/or a decrease in the secretion of cone survival factors. 38 39  
Contrary to implications of the results in immunocytochemical studies on human retinas with advanced stages of retinitis pigmentosa, 23 40 inner retinal remodeling is not, at least in the dog, a late response to photoreceptor degeneration. Our observations showed that synaptic connectivity in the OPL was altered at early stages of the disease. Rod neurite sprouting was first apparent within 1 to 2 weeks after the peak of photoreceptor cell death that occurred at approximately 7 weeks of age. Although this is the first report of rod neurite sprouting in the canine retina, it has been observed in two other animal models of retinitis pigmentosa: the rhodopsin transgenic pig 41 and the Rdy cat with autosomal dominant rod–cone dysplasia. 22 Neurite sprouting of cones has been described in the rd mouse as an early-onset change that starts at P8, when rod degeneration begins. 42 Neither this abnormality, nor elongation and branching of cone axons as previously described in humans 40 were observed in the XLPRA2 retina at any studied age, but these changes may occur in much older animals with more advanced disease. Concomitant to the onset of rod neurite sprouting, there was a loss in synaptophysin immunoreactivity of the OPL as early as 7.9 weeks of age. This illustrates the possible rapid and early disorganization in synaptic connectivity that could seriously hamper retinal function at a stage of disease when photoreceptor loss is limited. 
Another early change that occurred throughout the entire thickness of the neuroretina was the increased reactivity of Müller cells. An increase in GFAP labeling of Müller cells occurred approximately 2 weeks before the peak of photoreceptor cell death and reached its highest level in the following weeks. These glial cells, with somas located in the INL, have apical radial processes extending into the external limiting membrane (ELM). It is therefore possible that Müller cells detect early structural or chemical modifications in the photoreceptor layer and/or ONL caused by the degeneration of photoreceptor cells and relay this change in the outer retina environment to deeper retinal layers. GFAP reactivity in Müller cells had decreased at 26 weeks of age and was nearly normal by 40.6 weeks. These findings are different from those reported in human retinas with advanced disease in which GFAP reactivity persists. 43 This difference may be explained by the fact that we did not collect retinas from the very advanced stages of disease. Thus, it may be that GFAP reactivity in Müller cells occurs both at the onset of photoreceptor degeneration and during the terminal stages of retina atrophy. 
After these early changes in the inner retina, there was progressive retraction of rod bipolar cell dendrites that began by approximately 12 weeks of age. Contrary to what has been described in the rd mouse, 44 rod bipolar cells developed dendritic arborizations. This may be because in XLPRA2, unlike in the rd mouse, most photoreceptor cells reach a stage of functional and structural maturation, albeit abnormal, that allows the formation of synapses with second-order neurons. We did not observe any significant loss in the arborization of ON-cone bipolar cells, as occurs in the rd mouse at later stages of the disease, when cones undergo cell death. 25 The most probable explanation of this difference is that, at the latest time point that we examined ON cone bipolar cells (40.6 weeks), there was still a significant number of cone photoreceptors in the ONL that had not degenerated. This may suggest that the cone-mediated pathway remains functional at more advanced stages of the disease. Although more extensive electroretinographic testing is needed to verify this hypothesis, our previous study has shown the presence of robust cone signals at a time when there is significant loss of rod-mediated responses. 10  
Information on the histopathologic changes that occur in retinas of human patients with RPGR exon ORF15 mutations has currently only been reported in a carrier. 12 Yet, it appears that frameshift mutations in RPGR exon ORF15 have a comparably severe phenotype in both canine and human retinas. 
The findings reported in this study have several important implications for the development of therapeutic approaches for retinal degeneration in humans. Among these, the use of survival factors (e.g., ciliary neurotrophic factor [CNTF] and brain-derived neurotrophic factor [BDNF]) as well as a gene-silencing approach, via ribozymes or siRNA, are currently being investigated in our laboratory. The results of this present study suggest that a first strategy should consist in initiating therapy before the burst of photoreceptor cell death that occurs in the dog between 6 and 7 weeks of age. Delivering the therapeutic agent at approximately 4 weeks of age may prevent or delay the onset of photoreceptor degeneration. Intraocular injection of survival factors that are immediately biologically active is feasible at such a young age. Alternatively, another potential strategy would be to target the rescue of the photoreceptor cells that survived the initial burst of cell death. Indeed, we have shown that after this event, the rate of cell death is considerably slowed down, and that even in the most advanced stages of disease, the morphology of the remaining photoreceptor cells is reasonably preserved. We have observed that some photoreceptors maintain their IS and OS and continue to express proteins involved in the phototransduction pathway unlike what has been reported in humans with more advanced retinitis pigmentosa caused by rhodopsin gene mutations. 30 From this perspective, 12 weeks of age may be an optimal time-window to initiate therapy because, at that age, approximately 60% of photoreceptor cells remain. 
Another important aspect that future studies should address is whether rescuing photoreceptor cells also allows reversal of inner retinal changes that occur secondary to rod and cone disease and degeneration. Modification of the inner retina after photoreceptor degeneration does not appear to be dependent on the genetic cause of the disease. Indeed, in addition to the two-nucleotide deletion in RPGR ORF15 that occurs in the XLPRA2 dog, recent studies have shown inner retina remodeling in the rd and crx −/− mice, two models of retinal degeneration caused, respectively, by a mutation in the PDE6B and CRX genes. 24 44 In addition to these morphologic changes, a switch in neurotransmitter sensitivity of rod bipolar cells has been demonstrated in the rd mouse. 45 All these recent findings suggest that, if a similar phenomenon occurs in the human with early stages of retinal degeneration, then novel therapeutic approaches must be evaluated, not solely on their protective effect on photoreceptor cells, but also on their capacity in maintaining functional synaptic connections between photoreceptor cells and inner retinal neurons. 
In conclusion, we have characterized the structural changes that occur in the XLPRA2 retina, an early-onset canine model of X-linked retinitis pigmentosa caused by a microdeletion in RPGR exon ORF15 with resultant frameshift. This is a valuable spontaneous animal model that may provide a better understanding of the retinal function of the RPGR protein and the pathogenic mechanisms that lead to photoreceptor death. It also may provide a tool to assess the in vivo efficacy of novel therapies. 
 
Table 1.
 
Status, Gender, and Age of Dogs Used in the Morphologic, TUNEL, and Immunohistochemical Studies
Table 1.
 
Status, Gender, and Age of Dogs Used in the Morphologic, TUNEL, and Immunohistochemical Studies
ID Gender Age (wk) Morphology TUNEL IHC
Affected
 Z215 F 2 +
 Z210 M 2 + +
 Z201 M 3.9 + + +
 Z266 F 4.1 +
 Z212 M 4.9 +
 Z202 M 5 + + +
 Z203 F 6 + + +
 Z253 F 6.1 +
 Z254 F 6.7 +
 Z255 F 6.7 +
 Z207 F 7.9 + + +
 Z250 M 8 +
 Z251 M 8 +
 Z216 M 8.3 +
 Z211 M 11.9 + + +
 Z219 F 12.1 +
 Z226 F 16 + + +
 Z194 M 17.3 +
 Z195 F 17.3 +
 Z193 M 17.7 +
 Z208 F 19.9 +
 Z204 M 19.9 +
 Z209 F 23.7 +
 Z181 F 26 + + +
 Z178 F 40.6 + + +
Normal
 7304 F 2 +
 2327-2 M 2.3 +
 7306 F 4 + +
 7307 M 5 +
 2297-1 F 5.4 +
 7308 F 6 + +
 7310 F 8.1 +
 2298-1 M 8.3 +
 7312 F 12 +
 7299 M 24 +
Table 2.
 
List of Primary Antibodies Tested and Used in the Study
Table 2.
 
List of Primary Antibodies Tested and Used in the Study
Antigen Host Source, Catalog No. or Name Working Concentration Normal Retinal Localization (Reported in Rodents) References
RPE65 Rabbit T. Michael Redmond 1:10,000 Retinal pigment epithelium 18 *
Human cone arrestin Rabbit Cheryl Craft, LUMIF 1:10,000 Cone photoreceptors 19 *
M/L cone opsin Rabbit polyclonal Chemicon, AB5405 1:10,000 OS of M/L cones 20 , †
S cone opsin Rabbit polyclonal Chemicon, AB5407 1:5,000 OS of S cones 20 , †
Rod opsin Mouse monoclonal Paul Hargrave, R2-12N 1:300 OS of rods 21 *
Synaptophysin Rabbit polyclonal DakoCytomation, A0010 1:100 Neuron synapses, OPL, IPL 22 * , 23, †
Calbindin D-28K Rabbit polyclonal Sigma, C2724 1:1,000 Horizontal, amacrine cells 24 * , 23 , †
Protein kinase C (PKCα) Mouse monoclonal IgG2b BD Biosciences, 610107 1:100 Rod bipolar cells 25 , † , 24 , †
Goα Mouse monoclonal IgG1 Chemicon, MAB3073 1:5,000 ON (rod and cone) bipolar cells 26 *
Tachykinin receptor 3 (NK3R, NKBR) Rabbit polyclonal Novus Biologicals, NLS4043 1:100 Cone bipolar cells 24 *
Rabbit polyclonal Abcam, ab13278 1:50
Metabotropic glutamate receptor (GRM6) Rabbit polyclonal Abcam, ab13362 1:10–1:200 Postsynaptic sites of ON bipolar cells 24 , † , 25 , †
γ-amino butyric acid (GABA) Rabbit polyclonal Chemicon, AB5016 1:50 GABAergic amacrine cells 27 , †
Choline acetyl transferase (ChAT) Rabbit polyclonal Chemicon, AB143 1:500 Cholinergic amacrine cells 28 * , 29 , †
Rat monoclonal Oncogene, NB05L 1:100
Tyrosine hydroxylase (TH) Rabbit polyclonal Chemicon, AB152 1:500 Dopaminergic amacrine cells 24 * , 25 *
Disabled 1 (Dab1) Rabbit polyclonal Chemicon, AB5840 1:50–1:100 All amacrine cells 25 , †
CRALBP Mouse monoclonal John Saari 1:5,000–1:40,000 Müller cells, RPE 9 *
Glial fibrillary acidic protein (GFAP) Rabbit polyclonal DakoCytomation, Z0334 1:1,000 Astrocytes, Müller cells (reactive) 30 *
Glutamine synthetase Mouse monoclonal Chemicon, MAB302 1:20,000 Müller cells 25 *
Figure 1.
 
Stages of development and photoreceptor degeneration in normal and mutant retinas. Images are from the midperiphery of the superior meridian. (AC) Normal retina. (A) By 2.3 weeks, photoreceptor nuclei were elongated, IS were visible, and short OS began to form. (B) By 5.4 weeks, OS were formed and elongated, and (C) photoreceptor maturation was complete by 8.3 weeks. (DI) XLPRA2 mutant retina. (D) Stage 0, 2.2 weeks. Photoreceptor development was normal and comparable to the control. (E) Stage 1, 5 weeks. OS were misaligned and partially fragmented (*). Pyknotic nuclei were visible in the ONL (arrow). (F) Stage 2, 7.9 weeks. OS were disintegrating (*), and there was pyknosis in the ONL (arrows). ONL, eight to nine rows of nuclei. (G) Stage 3, 16 weeks. The subretinal space was narrowed, rod IS were very short (arrow), and remaining but distorted rod and cone OS were visible. Phagocytic cells were present in the subretinal space (arrowhead). ONL, six rows of nuclei. (H) Stage 4, 26 weeks. The subretinal space was narrowed further, but distorted OS remained (arrow). Both rod and cone IS were shortened, and cone IS were broader than normal. Spaces were visible in the IS layer secondary to rod loss (arrowheads). ONL, five rows of nuclei, with increased spacing between photoreceptor somas (*). (I) Stage 5, 40.6 weeks. The interphotoreceptor space had open areas, yet some photoreceptors appeared to retain their distorted OS (arrows). ONL, two to three rows of nuclei. RPE, retinal pigment epithelium; PR, photoreceptor layer; OPL, outer plexiform layer. Scale bar, 20 μm.
Figure 1.
 
Stages of development and photoreceptor degeneration in normal and mutant retinas. Images are from the midperiphery of the superior meridian. (AC) Normal retina. (A) By 2.3 weeks, photoreceptor nuclei were elongated, IS were visible, and short OS began to form. (B) By 5.4 weeks, OS were formed and elongated, and (C) photoreceptor maturation was complete by 8.3 weeks. (DI) XLPRA2 mutant retina. (D) Stage 0, 2.2 weeks. Photoreceptor development was normal and comparable to the control. (E) Stage 1, 5 weeks. OS were misaligned and partially fragmented (*). Pyknotic nuclei were visible in the ONL (arrow). (F) Stage 2, 7.9 weeks. OS were disintegrating (*), and there was pyknosis in the ONL (arrows). ONL, eight to nine rows of nuclei. (G) Stage 3, 16 weeks. The subretinal space was narrowed, rod IS were very short (arrow), and remaining but distorted rod and cone OS were visible. Phagocytic cells were present in the subretinal space (arrowhead). ONL, six rows of nuclei. (H) Stage 4, 26 weeks. The subretinal space was narrowed further, but distorted OS remained (arrow). Both rod and cone IS were shortened, and cone IS were broader than normal. Spaces were visible in the IS layer secondary to rod loss (arrowheads). ONL, five rows of nuclei, with increased spacing between photoreceptor somas (*). (I) Stage 5, 40.6 weeks. The interphotoreceptor space had open areas, yet some photoreceptors appeared to retain their distorted OS (arrows). ONL, two to three rows of nuclei. RPE, retinal pigment epithelium; PR, photoreceptor layer; OPL, outer plexiform layer. Scale bar, 20 μm.
Table 3.
 
Changes in the XLPRA2 Retina Associated with Disease Stages
Table 3.
 
Changes in the XLPRA2 Retina Associated with Disease Stages
Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
2 wk (Z215) 2 wk (Z210) 3.9 wk (Z201) 4.9 wk (Z212) 5 wk (Z202) 7.9 wk (Z207) 11.9 wk (Z211) 16 wk (Z226) 26 wk (Z181) 40.6 wk (Z178)
Superior retina
 Phagocytic cells* 0 0 0 0 0 0 3 13 31 27
 ONL thickness, †
  S1 10 10 14 10 10 7 7 7 5 3
  S2 10 11 12 9 11 9 6 6.5 5 3
  S3 11.5 11 15.5 9 14 8 5.5 5 3.5 2
 Pyknosis (ONL), ‡ 0 0 9 105 81 65 27 23 33 15
 INL thickness, †
  S1 4 5 6 4 4 3.5 3.5 3.5 3 3
  S2 5 5 5 3 3 3 3 2.5 2 2
  S3 5.5 6 5 3 4 3 2.5 1.5 2 1.5
Inferior retina
 Phagocytic cells* 0 0 0 0 0 0 6 28 31 17
 ONL thickness, †
  S1 10 11 15 9 11 9 6.5 6 4.5 3.5
  S2 10 11 14 8.5 9 8.5 6 6 3.5 3
  S3 10 11 15 9 9 10 5 5 3 2.5
 Pyknosis (ONL), ‡ 0 2 15 101 68 42 23 24 41 13
 INL thickness, †
  S1 5 5 5 4 4 4 3 2 3 3
  S2 5.5 4.5 5 3 3 3.5 3 2 2.5 2
  S3 6 4.5 5 3 3 4 2 1.5 2 2
Figure 2.
 
Rate of photoreceptor cell loss in mutant retinas as a function of age. ONL thickness was measured as the number of rows of photoreceptor nuclei (A, C) or in micrometers (B, D) along both the superior (A, B) and inferior (C, D) retinal meridians. Reported values are the mean of three measurements taken in the central, midperipheral, and peripheral region of the superior or inferior retinal meridian. The kinetics of photoreceptor cell loss are best fit by a model of constant or exponentially decreasing risk of cell death.
Figure 2.
 
Rate of photoreceptor cell loss in mutant retinas as a function of age. ONL thickness was measured as the number of rows of photoreceptor nuclei (A, C) or in micrometers (B, D) along both the superior (A, B) and inferior (C, D) retinal meridians. Reported values are the mean of three measurements taken in the central, midperipheral, and peripheral region of the superior or inferior retinal meridian. The kinetics of photoreceptor cell loss are best fit by a model of constant or exponentially decreasing risk of cell death.
Figure 3.
 
Number of TUNEL-labeled photoreceptor cell nuclei per unit area (1 M μm2) of ONL as a function of age in the superior retinal quadrant of affected and normal dogs. Symbols: the mean ± SD of three counts made on three sections from the superior retinal meridian of each animal.
Figure 3.
 
Number of TUNEL-labeled photoreceptor cell nuclei per unit area (1 M μm2) of ONL as a function of age in the superior retinal quadrant of affected and normal dogs. Symbols: the mean ± SD of three counts made on three sections from the superior retinal meridian of each animal.
Figure 4.
 
Characterization of photoreceptor cell death and disease in XLPRA2. (A) Photoreceptor cell death in XLPRA2. (A1) TUNEL-labeling (green) of rod photoreceptor cells in a 5-week-old affected retina with DAPI nuclear counterstain (blue). (A2) TUNEL labeling (green) of a cone nucleus in a 40.6-week-old retina (arrow). An ectopically located cone nucleus that was not TUNEL-positive is highlighted (arrowhead). Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (A3) Plastic section from peripheral retina of a 26-week-old retina shows a morphologically normal cone nucleus displaced in the IS (arrow and inset). (A4) TUNEL-labeling (green) of a fragmented ectopic cone nucleus (arrow) in a 40.6-week-old retina. Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (B) Double immunofluorescence labeling of rod and cones with, respectively, anti-rod opsin (green) and anti-human cone arrestin (red) in normal (B1, 8.1 weeks) and affected (B2, B3) retinas. (B2) In a 7.9-week affected retina, rod opsin was mislocalized to the IS and ONL, and there was early rod neurite sprouting (arrows). (B3) In a 40.6-week affected retina, rod opsin–positive neurites extended deep into the inner retina. Rod opsin and cone arrestin expression persisted at this stage of disease, but there was a decrease in cone arrestin immunoreactivity at the level of the cone axons and pedicles, even though the cone IS and OS were present. (C) Double immunofluorescent labeling with anti-rod opsin (green) and anti-synaptophysin (red) in normal (C1) and affected (C2, C3) retinas. (C1) In a 12-week normal retina, both the OPL and IPL were labeled with the synaptophysin antibody. (C2) In a 12-week affected retina, there was OPL thinning and punctuate synaptophysin-positive labeling that colocalized with rod opsin positive neurites (arrows). (C3) A 40.6-week affected retina. A higher-magnification view showing colocalization of synaptophysin and rod opsin along beaded varicosities of rod neurites (arrowheads) and at their terminal ends (arrows). (D) Immunofluorescent labeling of cones with anti-S opsin (D1, D2), and anti-M/L opsin (D3, D4). (D1) In a 4-week normal retina, S opsin labeling was restricted to the OS of S cones. (D2) In a 3.9-week affected retina, S opsin was mislocalized to the IS (horizontal arrows), soma (vertical arrows), and axons (arrowheads) of some S cones. (D3) In a 6-week normal retina, M/L opsin labeling was predominantly restricted to the OS of M/L cones, although faint background labeling of the M/L cone somas and axons was present. (D4) In a 6-week affected retina, M/L opsin was mislocalized to the somas (vertical arrows), axons (arrowheads), and pedicles (oblique arrows) of most M/L cones in the peripheral retina. Scale bars: (A1) 40 μm; (A3) 10 μm; (A2, A4, BD) 20 μm.
Figure 4.
 
Characterization of photoreceptor cell death and disease in XLPRA2. (A) Photoreceptor cell death in XLPRA2. (A1) TUNEL-labeling (green) of rod photoreceptor cells in a 5-week-old affected retina with DAPI nuclear counterstain (blue). (A2) TUNEL labeling (green) of a cone nucleus in a 40.6-week-old retina (arrow). An ectopically located cone nucleus that was not TUNEL-positive is highlighted (arrowhead). Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (A3) Plastic section from peripheral retina of a 26-week-old retina shows a morphologically normal cone nucleus displaced in the IS (arrow and inset). (A4) TUNEL-labeling (green) of a fragmented ectopic cone nucleus (arrow) in a 40.6-week-old retina. Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (B) Double immunofluorescence labeling of rod and cones with, respectively, anti-rod opsin (green) and anti-human cone arrestin (red) in normal (B1, 8.1 weeks) and affected (B2, B3) retinas. (B2) In a 7.9-week affected retina, rod opsin was mislocalized to the IS and ONL, and there was early rod neurite sprouting (arrows). (B3) In a 40.6-week affected retina, rod opsin–positive neurites extended deep into the inner retina. Rod opsin and cone arrestin expression persisted at this stage of disease, but there was a decrease in cone arrestin immunoreactivity at the level of the cone axons and pedicles, even though the cone IS and OS were present. (C) Double immunofluorescent labeling with anti-rod opsin (green) and anti-synaptophysin (red) in normal (C1) and affected (C2, C3) retinas. (C1) In a 12-week normal retina, both the OPL and IPL were labeled with the synaptophysin antibody. (C2) In a 12-week affected retina, there was OPL thinning and punctuate synaptophysin-positive labeling that colocalized with rod opsin positive neurites (arrows). (C3) A 40.6-week affected retina. A higher-magnification view showing colocalization of synaptophysin and rod opsin along beaded varicosities of rod neurites (arrowheads) and at their terminal ends (arrows). (D) Immunofluorescent labeling of cones with anti-S opsin (D1, D2), and anti-M/L opsin (D3, D4). (D1) In a 4-week normal retina, S opsin labeling was restricted to the OS of S cones. (D2) In a 3.9-week affected retina, S opsin was mislocalized to the IS (horizontal arrows), soma (vertical arrows), and axons (arrowheads) of some S cones. (D3) In a 6-week normal retina, M/L opsin labeling was predominantly restricted to the OS of M/L cones, although faint background labeling of the M/L cone somas and axons was present. (D4) In a 6-week affected retina, M/L opsin was mislocalized to the somas (vertical arrows), axons (arrowheads), and pedicles (oblique arrows) of most M/L cones in the peripheral retina. Scale bars: (A1) 40 μm; (A3) 10 μm; (A2, A4, BD) 20 μm.
Figure 5.
 
Inner retina remodeling in XLPRA2. (A) Immunofluorescent labeling of horizontal cells with anti-calbindin (green) in normal (A1; 12 weeks) and affected (A2, A3) retinas. In the 12-week affected retina (A2), the horizontal cell processes were flattened by the narrowing of the OPL. This was more evident at 40.6 weeks of age, although there was persistent labeling of the horizontal cell somas (A3). DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescent labeling of rod bipolar cells with anti-PKCα (green) in normal (B1, 12 weeks) and affected (B2, B3) retinas. (B2) Twelve-week affected. There was early retraction of the dendritic arborization of rod bipolar cells (arrows); (B3) 40.6-week affected. Note the almost total loss of dendritic arborization in OPL (arrows). DAPI (blue) was used as a nuclear counterstain. (C) Double immunofluorescent labeling of ON cone bipolar cells with anti-Goα (red), and rod bipolar cells with anti-PKCα (green) in the normal (C1, 12 weeks) and affected (C2C4) retinas. Rod bipolar cells were colabeled with both Goα and PKCα and appeared yellow-orange, whereas ON cone bipolar cells were only labeled with Goα and appear red; (C1) Twelve-week normal. Note the extensive yellow-orange dendritic arborization of rod bipolar cells (arrowheads), and just below, the dendrites of ON cone bipolar cells that formed a continuous red layer (arrows). (C2) 12-week affected. Note the shortening of the rod bipolar cell dendrites (arrowheads) in comparison to the age-matched normal. (C3) In a 26-week affected retina, there was an overall decrease in PKCα immunoreactivity in rod bipolar cells and a significant retraction of dendritic arborization (arrowheads). (C4) In a 40.6-week affected retina, there was weak labeling of rod bipolar cell somas and axons with PKCα and complete retraction of dendrites. There was persistence of Goα-IR in rod bipolar and ON cone bipolar cells, as well as the presence of the cone bipolar cell dendrites (arrows). (D) Immunofluorescent labeling with anti-GABA in normal (D1, 24 weeks) and affected (D2, D3) retinas. (D1) GABA-IR was present in the midperipheral retina in several laminae of the IPL and in horizontal cells (arrowheads) and their processes. There was no labeling of amacrine cells, other than in the central region of the normal retina. (D2) In a 26-week affected midperipheral retina. There was loss of the IPL lamination, but increased immunoreactivity of putative amacrine cells located at the vitreous border of the INL and displaced in the GCL (arrows). There was also loss of horizontal cell labeling; (D3) In a 40.6-week affected midperipheral retina, IPL thickness was significantly reduced, and GABA-IR was increased in the amacrine cells (arrows). (E) Immunofluorescent labeling of Müller cells with GFAP (red) in normal (E1, 12 weeks) and affected (E2, E3) retinas. (E1) Twelve-week normal. GFAP labeling was weak and limited to astrocytes and Müller cell end feet (arrow); (E2) In a 12-week affected retina, intense GFAP-IR was present throughout the entire length of the Müller cells. (E3) In a 40.6-week affected retina, GFAP labeling was almost absent. Scale bars, 20 μm.
Figure 5.
 
Inner retina remodeling in XLPRA2. (A) Immunofluorescent labeling of horizontal cells with anti-calbindin (green) in normal (A1; 12 weeks) and affected (A2, A3) retinas. In the 12-week affected retina (A2), the horizontal cell processes were flattened by the narrowing of the OPL. This was more evident at 40.6 weeks of age, although there was persistent labeling of the horizontal cell somas (A3). DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescent labeling of rod bipolar cells with anti-PKCα (green) in normal (B1, 12 weeks) and affected (B2, B3) retinas. (B2) Twelve-week affected. There was early retraction of the dendritic arborization of rod bipolar cells (arrows); (B3) 40.6-week affected. Note the almost total loss of dendritic arborization in OPL (arrows). DAPI (blue) was used as a nuclear counterstain. (C) Double immunofluorescent labeling of ON cone bipolar cells with anti-Goα (red), and rod bipolar cells with anti-PKCα (green) in the normal (C1, 12 weeks) and affected (C2C4) retinas. Rod bipolar cells were colabeled with both Goα and PKCα and appeared yellow-orange, whereas ON cone bipolar cells were only labeled with Goα and appear red; (C1) Twelve-week normal. Note the extensive yellow-orange dendritic arborization of rod bipolar cells (arrowheads), and just below, the dendrites of ON cone bipolar cells that formed a continuous red layer (arrows). (C2) 12-week affected. Note the shortening of the rod bipolar cell dendrites (arrowheads) in comparison to the age-matched normal. (C3) In a 26-week affected retina, there was an overall decrease in PKCα immunoreactivity in rod bipolar cells and a significant retraction of dendritic arborization (arrowheads). (C4) In a 40.6-week affected retina, there was weak labeling of rod bipolar cell somas and axons with PKCα and complete retraction of dendrites. There was persistence of Goα-IR in rod bipolar and ON cone bipolar cells, as well as the presence of the cone bipolar cell dendrites (arrows). (D) Immunofluorescent labeling with anti-GABA in normal (D1, 24 weeks) and affected (D2, D3) retinas. (D1) GABA-IR was present in the midperipheral retina in several laminae of the IPL and in horizontal cells (arrowheads) and their processes. There was no labeling of amacrine cells, other than in the central region of the normal retina. (D2) In a 26-week affected midperipheral retina. There was loss of the IPL lamination, but increased immunoreactivity of putative amacrine cells located at the vitreous border of the INL and displaced in the GCL (arrows). There was also loss of horizontal cell labeling; (D3) In a 40.6-week affected midperipheral retina, IPL thickness was significantly reduced, and GABA-IR was increased in the amacrine cells (arrows). (E) Immunofluorescent labeling of Müller cells with GFAP (red) in normal (E1, 12 weeks) and affected (E2, E3) retinas. (E1) Twelve-week normal. GFAP labeling was weak and limited to astrocytes and Müller cell end feet (arrow); (E2) In a 12-week affected retina, intense GFAP-IR was present throughout the entire length of the Müller cells. (E3) In a 40.6-week affected retina, GFAP labeling was almost absent. Scale bars, 20 μm.
Table 4.
 
Parameter Estimates for the Kinetic Models Relating the ONL Thickness to Age
Table 4.
 
Parameter Estimates for the Kinetic Models Relating the ONL Thickness to Age
Constant Risk Model Exponentially Decreasing Risk Model Exponentially Increasing Risk Model
ONL(0) μ R 2 ONL(0) A μ0 R 2 ONL(0) A μ0 R 2
Superior retina thickness (cell count)
 Estimate 12.5 0.046 0.974 13.2 0.025 0.062 NA 13.2 −0.025 0.062 NA
 95% CI (10.6–14.4) (0.030–0.062) (10.2–16.2) (−0.048–0.098) (0.007–0.117) (10.2–16.2) (−0.098–0.048) (0.007–0.117)
P <0.0001 <0.0001 <0.0001 0.47 0.029 <0.0001 0.47 0.029
Superior retina thickness (μm)
 Estimate 90.9 0.062 0.965 115.7 0.088 0.150 0.978 115.7 −0.088 0.150 NA
 95% CI (73.9–107.8) (0.038–0.086) (80.7–150.8) (0.003–0.174) (0.035–0.265) (80.7–150.8) (−0.174–−0.003) (0.035–0.265)
P <0.0001 <0.0001 <0.0001 0.043 0.014 <0.0001 0.043 0.014
Inferior Retina Thickness (cell count)
 Estimate 12.6 0.053 0.967 13.5 0.031 0.074 NA 13.5 −0.031 0.074 NA
 95% CI (10.4–14.8) (0.033–0.072) (10.1–16.9) (−0.045–0.108) (0.010–0.138) (10.1–16.9) (−0.108–0.045) (0.010–0.138)
P <0.0001 <0.0001 <0.0001 0.39 0.027 <0.0001 0.39 0.027
Inferior retina thickness (μm)
 Estimate 86.4 0.068 0.954 124.1 0.120 0.207 0.975 124.1 −0.120 0.207 NA
 95% CI (67.5–105.4) (0.038–0.098) (81.1–167.1) (0.026–0.214) (0.057–0.358) (81.1–167.1) (−0.214–−0.026) (0.057–0.358)
P <0.001 0.0002 <0.0001 0.016 0.011 <0.0001 0.016 0.011
The authors thank T. Michael Redmond (National Eye Institute, Bethesda, MD), Cheryl Craft (University of Southern California, Los Angeles), Paul Hargrave (University of Florida, Gainesville), and John Saari (University of Washington, Seattle) for providing, respectively, the RPE65, human cone arrestin, rod opsin, and CRALBP antibodies; Benoît Grémaud (CNRS UMR 8552; Paris, France) for mathematical analyses; Roderick McInnes and Geoff Clarke (University of Toronto, Ontario, Canada) for comments on the kinetics of cell death; Julie Jordan for technical assistance; Sue Pearce-Kelling for valuable discussions; Gui-shuang Ying and Chencheng Liu for assistance with biostatistics; and Mary Leonard for providing the illustrations. 
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Figure 1.
 
Stages of development and photoreceptor degeneration in normal and mutant retinas. Images are from the midperiphery of the superior meridian. (AC) Normal retina. (A) By 2.3 weeks, photoreceptor nuclei were elongated, IS were visible, and short OS began to form. (B) By 5.4 weeks, OS were formed and elongated, and (C) photoreceptor maturation was complete by 8.3 weeks. (DI) XLPRA2 mutant retina. (D) Stage 0, 2.2 weeks. Photoreceptor development was normal and comparable to the control. (E) Stage 1, 5 weeks. OS were misaligned and partially fragmented (*). Pyknotic nuclei were visible in the ONL (arrow). (F) Stage 2, 7.9 weeks. OS were disintegrating (*), and there was pyknosis in the ONL (arrows). ONL, eight to nine rows of nuclei. (G) Stage 3, 16 weeks. The subretinal space was narrowed, rod IS were very short (arrow), and remaining but distorted rod and cone OS were visible. Phagocytic cells were present in the subretinal space (arrowhead). ONL, six rows of nuclei. (H) Stage 4, 26 weeks. The subretinal space was narrowed further, but distorted OS remained (arrow). Both rod and cone IS were shortened, and cone IS were broader than normal. Spaces were visible in the IS layer secondary to rod loss (arrowheads). ONL, five rows of nuclei, with increased spacing between photoreceptor somas (*). (I) Stage 5, 40.6 weeks. The interphotoreceptor space had open areas, yet some photoreceptors appeared to retain their distorted OS (arrows). ONL, two to three rows of nuclei. RPE, retinal pigment epithelium; PR, photoreceptor layer; OPL, outer plexiform layer. Scale bar, 20 μm.
Figure 1.
 
Stages of development and photoreceptor degeneration in normal and mutant retinas. Images are from the midperiphery of the superior meridian. (AC) Normal retina. (A) By 2.3 weeks, photoreceptor nuclei were elongated, IS were visible, and short OS began to form. (B) By 5.4 weeks, OS were formed and elongated, and (C) photoreceptor maturation was complete by 8.3 weeks. (DI) XLPRA2 mutant retina. (D) Stage 0, 2.2 weeks. Photoreceptor development was normal and comparable to the control. (E) Stage 1, 5 weeks. OS were misaligned and partially fragmented (*). Pyknotic nuclei were visible in the ONL (arrow). (F) Stage 2, 7.9 weeks. OS were disintegrating (*), and there was pyknosis in the ONL (arrows). ONL, eight to nine rows of nuclei. (G) Stage 3, 16 weeks. The subretinal space was narrowed, rod IS were very short (arrow), and remaining but distorted rod and cone OS were visible. Phagocytic cells were present in the subretinal space (arrowhead). ONL, six rows of nuclei. (H) Stage 4, 26 weeks. The subretinal space was narrowed further, but distorted OS remained (arrow). Both rod and cone IS were shortened, and cone IS were broader than normal. Spaces were visible in the IS layer secondary to rod loss (arrowheads). ONL, five rows of nuclei, with increased spacing between photoreceptor somas (*). (I) Stage 5, 40.6 weeks. The interphotoreceptor space had open areas, yet some photoreceptors appeared to retain their distorted OS (arrows). ONL, two to three rows of nuclei. RPE, retinal pigment epithelium; PR, photoreceptor layer; OPL, outer plexiform layer. Scale bar, 20 μm.
Figure 2.
 
Rate of photoreceptor cell loss in mutant retinas as a function of age. ONL thickness was measured as the number of rows of photoreceptor nuclei (A, C) or in micrometers (B, D) along both the superior (A, B) and inferior (C, D) retinal meridians. Reported values are the mean of three measurements taken in the central, midperipheral, and peripheral region of the superior or inferior retinal meridian. The kinetics of photoreceptor cell loss are best fit by a model of constant or exponentially decreasing risk of cell death.
Figure 2.
 
Rate of photoreceptor cell loss in mutant retinas as a function of age. ONL thickness was measured as the number of rows of photoreceptor nuclei (A, C) or in micrometers (B, D) along both the superior (A, B) and inferior (C, D) retinal meridians. Reported values are the mean of three measurements taken in the central, midperipheral, and peripheral region of the superior or inferior retinal meridian. The kinetics of photoreceptor cell loss are best fit by a model of constant or exponentially decreasing risk of cell death.
Figure 3.
 
Number of TUNEL-labeled photoreceptor cell nuclei per unit area (1 M μm2) of ONL as a function of age in the superior retinal quadrant of affected and normal dogs. Symbols: the mean ± SD of three counts made on three sections from the superior retinal meridian of each animal.
Figure 3.
 
Number of TUNEL-labeled photoreceptor cell nuclei per unit area (1 M μm2) of ONL as a function of age in the superior retinal quadrant of affected and normal dogs. Symbols: the mean ± SD of three counts made on three sections from the superior retinal meridian of each animal.
Figure 4.
 
Characterization of photoreceptor cell death and disease in XLPRA2. (A) Photoreceptor cell death in XLPRA2. (A1) TUNEL-labeling (green) of rod photoreceptor cells in a 5-week-old affected retina with DAPI nuclear counterstain (blue). (A2) TUNEL labeling (green) of a cone nucleus in a 40.6-week-old retina (arrow). An ectopically located cone nucleus that was not TUNEL-positive is highlighted (arrowhead). Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (A3) Plastic section from peripheral retina of a 26-week-old retina shows a morphologically normal cone nucleus displaced in the IS (arrow and inset). (A4) TUNEL-labeling (green) of a fragmented ectopic cone nucleus (arrow) in a 40.6-week-old retina. Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (B) Double immunofluorescence labeling of rod and cones with, respectively, anti-rod opsin (green) and anti-human cone arrestin (red) in normal (B1, 8.1 weeks) and affected (B2, B3) retinas. (B2) In a 7.9-week affected retina, rod opsin was mislocalized to the IS and ONL, and there was early rod neurite sprouting (arrows). (B3) In a 40.6-week affected retina, rod opsin–positive neurites extended deep into the inner retina. Rod opsin and cone arrestin expression persisted at this stage of disease, but there was a decrease in cone arrestin immunoreactivity at the level of the cone axons and pedicles, even though the cone IS and OS were present. (C) Double immunofluorescent labeling with anti-rod opsin (green) and anti-synaptophysin (red) in normal (C1) and affected (C2, C3) retinas. (C1) In a 12-week normal retina, both the OPL and IPL were labeled with the synaptophysin antibody. (C2) In a 12-week affected retina, there was OPL thinning and punctuate synaptophysin-positive labeling that colocalized with rod opsin positive neurites (arrows). (C3) A 40.6-week affected retina. A higher-magnification view showing colocalization of synaptophysin and rod opsin along beaded varicosities of rod neurites (arrowheads) and at their terminal ends (arrows). (D) Immunofluorescent labeling of cones with anti-S opsin (D1, D2), and anti-M/L opsin (D3, D4). (D1) In a 4-week normal retina, S opsin labeling was restricted to the OS of S cones. (D2) In a 3.9-week affected retina, S opsin was mislocalized to the IS (horizontal arrows), soma (vertical arrows), and axons (arrowheads) of some S cones. (D3) In a 6-week normal retina, M/L opsin labeling was predominantly restricted to the OS of M/L cones, although faint background labeling of the M/L cone somas and axons was present. (D4) In a 6-week affected retina, M/L opsin was mislocalized to the somas (vertical arrows), axons (arrowheads), and pedicles (oblique arrows) of most M/L cones in the peripheral retina. Scale bars: (A1) 40 μm; (A3) 10 μm; (A2, A4, BD) 20 μm.
Figure 4.
 
Characterization of photoreceptor cell death and disease in XLPRA2. (A) Photoreceptor cell death in XLPRA2. (A1) TUNEL-labeling (green) of rod photoreceptor cells in a 5-week-old affected retina with DAPI nuclear counterstain (blue). (A2) TUNEL labeling (green) of a cone nucleus in a 40.6-week-old retina (arrow). An ectopically located cone nucleus that was not TUNEL-positive is highlighted (arrowhead). Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (A3) Plastic section from peripheral retina of a 26-week-old retina shows a morphologically normal cone nucleus displaced in the IS (arrow and inset). (A4) TUNEL-labeling (green) of a fragmented ectopic cone nucleus (arrow) in a 40.6-week-old retina. Cone photoreceptors were labeled with anti-human cone arrestin (red), and DAPI (blue) was used as a nuclear counterstain. (B) Double immunofluorescence labeling of rod and cones with, respectively, anti-rod opsin (green) and anti-human cone arrestin (red) in normal (B1, 8.1 weeks) and affected (B2, B3) retinas. (B2) In a 7.9-week affected retina, rod opsin was mislocalized to the IS and ONL, and there was early rod neurite sprouting (arrows). (B3) In a 40.6-week affected retina, rod opsin–positive neurites extended deep into the inner retina. Rod opsin and cone arrestin expression persisted at this stage of disease, but there was a decrease in cone arrestin immunoreactivity at the level of the cone axons and pedicles, even though the cone IS and OS were present. (C) Double immunofluorescent labeling with anti-rod opsin (green) and anti-synaptophysin (red) in normal (C1) and affected (C2, C3) retinas. (C1) In a 12-week normal retina, both the OPL and IPL were labeled with the synaptophysin antibody. (C2) In a 12-week affected retina, there was OPL thinning and punctuate synaptophysin-positive labeling that colocalized with rod opsin positive neurites (arrows). (C3) A 40.6-week affected retina. A higher-magnification view showing colocalization of synaptophysin and rod opsin along beaded varicosities of rod neurites (arrowheads) and at their terminal ends (arrows). (D) Immunofluorescent labeling of cones with anti-S opsin (D1, D2), and anti-M/L opsin (D3, D4). (D1) In a 4-week normal retina, S opsin labeling was restricted to the OS of S cones. (D2) In a 3.9-week affected retina, S opsin was mislocalized to the IS (horizontal arrows), soma (vertical arrows), and axons (arrowheads) of some S cones. (D3) In a 6-week normal retina, M/L opsin labeling was predominantly restricted to the OS of M/L cones, although faint background labeling of the M/L cone somas and axons was present. (D4) In a 6-week affected retina, M/L opsin was mislocalized to the somas (vertical arrows), axons (arrowheads), and pedicles (oblique arrows) of most M/L cones in the peripheral retina. Scale bars: (A1) 40 μm; (A3) 10 μm; (A2, A4, BD) 20 μm.
Figure 5.
 
Inner retina remodeling in XLPRA2. (A) Immunofluorescent labeling of horizontal cells with anti-calbindin (green) in normal (A1; 12 weeks) and affected (A2, A3) retinas. In the 12-week affected retina (A2), the horizontal cell processes were flattened by the narrowing of the OPL. This was more evident at 40.6 weeks of age, although there was persistent labeling of the horizontal cell somas (A3). DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescent labeling of rod bipolar cells with anti-PKCα (green) in normal (B1, 12 weeks) and affected (B2, B3) retinas. (B2) Twelve-week affected. There was early retraction of the dendritic arborization of rod bipolar cells (arrows); (B3) 40.6-week affected. Note the almost total loss of dendritic arborization in OPL (arrows). DAPI (blue) was used as a nuclear counterstain. (C) Double immunofluorescent labeling of ON cone bipolar cells with anti-Goα (red), and rod bipolar cells with anti-PKCα (green) in the normal (C1, 12 weeks) and affected (C2C4) retinas. Rod bipolar cells were colabeled with both Goα and PKCα and appeared yellow-orange, whereas ON cone bipolar cells were only labeled with Goα and appear red; (C1) Twelve-week normal. Note the extensive yellow-orange dendritic arborization of rod bipolar cells (arrowheads), and just below, the dendrites of ON cone bipolar cells that formed a continuous red layer (arrows). (C2) 12-week affected. Note the shortening of the rod bipolar cell dendrites (arrowheads) in comparison to the age-matched normal. (C3) In a 26-week affected retina, there was an overall decrease in PKCα immunoreactivity in rod bipolar cells and a significant retraction of dendritic arborization (arrowheads). (C4) In a 40.6-week affected retina, there was weak labeling of rod bipolar cell somas and axons with PKCα and complete retraction of dendrites. There was persistence of Goα-IR in rod bipolar and ON cone bipolar cells, as well as the presence of the cone bipolar cell dendrites (arrows). (D) Immunofluorescent labeling with anti-GABA in normal (D1, 24 weeks) and affected (D2, D3) retinas. (D1) GABA-IR was present in the midperipheral retina in several laminae of the IPL and in horizontal cells (arrowheads) and their processes. There was no labeling of amacrine cells, other than in the central region of the normal retina. (D2) In a 26-week affected midperipheral retina. There was loss of the IPL lamination, but increased immunoreactivity of putative amacrine cells located at the vitreous border of the INL and displaced in the GCL (arrows). There was also loss of horizontal cell labeling; (D3) In a 40.6-week affected midperipheral retina, IPL thickness was significantly reduced, and GABA-IR was increased in the amacrine cells (arrows). (E) Immunofluorescent labeling of Müller cells with GFAP (red) in normal (E1, 12 weeks) and affected (E2, E3) retinas. (E1) Twelve-week normal. GFAP labeling was weak and limited to astrocytes and Müller cell end feet (arrow); (E2) In a 12-week affected retina, intense GFAP-IR was present throughout the entire length of the Müller cells. (E3) In a 40.6-week affected retina, GFAP labeling was almost absent. Scale bars, 20 μm.
Figure 5.
 
Inner retina remodeling in XLPRA2. (A) Immunofluorescent labeling of horizontal cells with anti-calbindin (green) in normal (A1; 12 weeks) and affected (A2, A3) retinas. In the 12-week affected retina (A2), the horizontal cell processes were flattened by the narrowing of the OPL. This was more evident at 40.6 weeks of age, although there was persistent labeling of the horizontal cell somas (A3). DAPI (blue) was used as a nuclear counterstain. (B) Immunofluorescent labeling of rod bipolar cells with anti-PKCα (green) in normal (B1, 12 weeks) and affected (B2, B3) retinas. (B2) Twelve-week affected. There was early retraction of the dendritic arborization of rod bipolar cells (arrows); (B3) 40.6-week affected. Note the almost total loss of dendritic arborization in OPL (arrows). DAPI (blue) was used as a nuclear counterstain. (C) Double immunofluorescent labeling of ON cone bipolar cells with anti-Goα (red), and rod bipolar cells with anti-PKCα (green) in the normal (C1, 12 weeks) and affected (C2C4) retinas. Rod bipolar cells were colabeled with both Goα and PKCα and appeared yellow-orange, whereas ON cone bipolar cells were only labeled with Goα and appear red; (C1) Twelve-week normal. Note the extensive yellow-orange dendritic arborization of rod bipolar cells (arrowheads), and just below, the dendrites of ON cone bipolar cells that formed a continuous red layer (arrows). (C2) 12-week affected. Note the shortening of the rod bipolar cell dendrites (arrowheads) in comparison to the age-matched normal. (C3) In a 26-week affected retina, there was an overall decrease in PKCα immunoreactivity in rod bipolar cells and a significant retraction of dendritic arborization (arrowheads). (C4) In a 40.6-week affected retina, there was weak labeling of rod bipolar cell somas and axons with PKCα and complete retraction of dendrites. There was persistence of Goα-IR in rod bipolar and ON cone bipolar cells, as well as the presence of the cone bipolar cell dendrites (arrows). (D) Immunofluorescent labeling with anti-GABA in normal (D1, 24 weeks) and affected (D2, D3) retinas. (D1) GABA-IR was present in the midperipheral retina in several laminae of the IPL and in horizontal cells (arrowheads) and their processes. There was no labeling of amacrine cells, other than in the central region of the normal retina. (D2) In a 26-week affected midperipheral retina. There was loss of the IPL lamination, but increased immunoreactivity of putative amacrine cells located at the vitreous border of the INL and displaced in the GCL (arrows). There was also loss of horizontal cell labeling; (D3) In a 40.6-week affected midperipheral retina, IPL thickness was significantly reduced, and GABA-IR was increased in the amacrine cells (arrows). (E) Immunofluorescent labeling of Müller cells with GFAP (red) in normal (E1, 12 weeks) and affected (E2, E3) retinas. (E1) Twelve-week normal. GFAP labeling was weak and limited to astrocytes and Müller cell end feet (arrow); (E2) In a 12-week affected retina, intense GFAP-IR was present throughout the entire length of the Müller cells. (E3) In a 40.6-week affected retina, GFAP labeling was almost absent. Scale bars, 20 μm.
Table 1.
 
Status, Gender, and Age of Dogs Used in the Morphologic, TUNEL, and Immunohistochemical Studies
Table 1.
 
Status, Gender, and Age of Dogs Used in the Morphologic, TUNEL, and Immunohistochemical Studies
ID Gender Age (wk) Morphology TUNEL IHC
Affected
 Z215 F 2 +
 Z210 M 2 + +
 Z201 M 3.9 + + +
 Z266 F 4.1 +
 Z212 M 4.9 +
 Z202 M 5 + + +
 Z203 F 6 + + +
 Z253 F 6.1 +
 Z254 F 6.7 +
 Z255 F 6.7 +
 Z207 F 7.9 + + +
 Z250 M 8 +
 Z251 M 8 +
 Z216 M 8.3 +
 Z211 M 11.9 + + +
 Z219 F 12.1 +
 Z226 F 16 + + +
 Z194 M 17.3 +
 Z195 F 17.3 +
 Z193 M 17.7 +
 Z208 F 19.9 +
 Z204 M 19.9 +
 Z209 F 23.7 +
 Z181 F 26 + + +
 Z178 F 40.6 + + +
Normal
 7304 F 2 +
 2327-2 M 2.3 +
 7306 F 4 + +
 7307 M 5 +
 2297-1 F 5.4 +
 7308 F 6 + +
 7310 F 8.1 +
 2298-1 M 8.3 +
 7312 F 12 +
 7299 M 24 +
Table 2.
 
List of Primary Antibodies Tested and Used in the Study
Table 2.
 
List of Primary Antibodies Tested and Used in the Study
Antigen Host Source, Catalog No. or Name Working Concentration Normal Retinal Localization (Reported in Rodents) References
RPE65 Rabbit T. Michael Redmond 1:10,000 Retinal pigment epithelium 18 *
Human cone arrestin Rabbit Cheryl Craft, LUMIF 1:10,000 Cone photoreceptors 19 *
M/L cone opsin Rabbit polyclonal Chemicon, AB5405 1:10,000 OS of M/L cones 20 , †
S cone opsin Rabbit polyclonal Chemicon, AB5407 1:5,000 OS of S cones 20 , †
Rod opsin Mouse monoclonal Paul Hargrave, R2-12N 1:300 OS of rods 21 *
Synaptophysin Rabbit polyclonal DakoCytomation, A0010 1:100 Neuron synapses, OPL, IPL 22 * , 23, †
Calbindin D-28K Rabbit polyclonal Sigma, C2724 1:1,000 Horizontal, amacrine cells 24 * , 23 , †
Protein kinase C (PKCα) Mouse monoclonal IgG2b BD Biosciences, 610107 1:100 Rod bipolar cells 25 , † , 24 , †
Goα Mouse monoclonal IgG1 Chemicon, MAB3073 1:5,000 ON (rod and cone) bipolar cells 26 *
Tachykinin receptor 3 (NK3R, NKBR) Rabbit polyclonal Novus Biologicals, NLS4043 1:100 Cone bipolar cells 24 *
Rabbit polyclonal Abcam, ab13278 1:50
Metabotropic glutamate receptor (GRM6) Rabbit polyclonal Abcam, ab13362 1:10–1:200 Postsynaptic sites of ON bipolar cells 24 , † , 25 , †
γ-amino butyric acid (GABA) Rabbit polyclonal Chemicon, AB5016 1:50 GABAergic amacrine cells 27 , †
Choline acetyl transferase (ChAT) Rabbit polyclonal Chemicon, AB143 1:500 Cholinergic amacrine cells 28 * , 29 , †
Rat monoclonal Oncogene, NB05L 1:100
Tyrosine hydroxylase (TH) Rabbit polyclonal Chemicon, AB152 1:500 Dopaminergic amacrine cells 24 * , 25 *
Disabled 1 (Dab1) Rabbit polyclonal Chemicon, AB5840 1:50–1:100 All amacrine cells 25 , †
CRALBP Mouse monoclonal John Saari 1:5,000–1:40,000 Müller cells, RPE 9 *
Glial fibrillary acidic protein (GFAP) Rabbit polyclonal DakoCytomation, Z0334 1:1,000 Astrocytes, Müller cells (reactive) 30 *
Glutamine synthetase Mouse monoclonal Chemicon, MAB302 1:20,000 Müller cells 25 *
Table 3.
 
Changes in the XLPRA2 Retina Associated with Disease Stages
Table 3.
 
Changes in the XLPRA2 Retina Associated with Disease Stages
Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5
2 wk (Z215) 2 wk (Z210) 3.9 wk (Z201) 4.9 wk (Z212) 5 wk (Z202) 7.9 wk (Z207) 11.9 wk (Z211) 16 wk (Z226) 26 wk (Z181) 40.6 wk (Z178)
Superior retina
 Phagocytic cells* 0 0 0 0 0 0 3 13 31 27
 ONL thickness, †
  S1 10 10 14 10 10 7 7 7 5 3
  S2 10 11 12 9 11 9 6 6.5 5 3
  S3 11.5 11 15.5 9 14 8 5.5 5 3.5 2
 Pyknosis (ONL), ‡ 0 0 9 105 81 65 27 23 33 15
 INL thickness, †
  S1 4 5 6 4 4 3.5 3.5 3.5 3 3
  S2 5 5 5 3 3 3 3 2.5 2 2
  S3 5.5 6 5 3 4 3 2.5 1.5 2 1.5
Inferior retina
 Phagocytic cells* 0 0 0 0 0 0 6 28 31 17
 ONL thickness, †
  S1 10 11 15 9 11 9 6.5 6 4.5 3.5
  S2 10 11 14 8.5 9 8.5 6 6 3.5 3
  S3 10 11 15 9 9 10 5 5 3 2.5
 Pyknosis (ONL), ‡ 0 2 15 101 68 42 23 24 41 13
 INL thickness, †
  S1 5 5 5 4 4 4 3 2 3 3
  S2 5.5 4.5 5 3 3 3.5 3 2 2.5 2
  S3 6 4.5 5 3 3 4 2 1.5 2 2
Table 4.
 
Parameter Estimates for the Kinetic Models Relating the ONL Thickness to Age
Table 4.
 
Parameter Estimates for the Kinetic Models Relating the ONL Thickness to Age
Constant Risk Model Exponentially Decreasing Risk Model Exponentially Increasing Risk Model
ONL(0) μ R 2 ONL(0) A μ0 R 2 ONL(0) A μ0 R 2
Superior retina thickness (cell count)
 Estimate 12.5 0.046 0.974 13.2 0.025 0.062 NA 13.2 −0.025 0.062 NA
 95% CI (10.6–14.4) (0.030–0.062) (10.2–16.2) (−0.048–0.098) (0.007–0.117) (10.2–16.2) (−0.098–0.048) (0.007–0.117)
P <0.0001 <0.0001 <0.0001 0.47 0.029 <0.0001 0.47 0.029
Superior retina thickness (μm)
 Estimate 90.9 0.062 0.965 115.7 0.088 0.150 0.978 115.7 −0.088 0.150 NA
 95% CI (73.9–107.8) (0.038–0.086) (80.7–150.8) (0.003–0.174) (0.035–0.265) (80.7–150.8) (−0.174–−0.003) (0.035–0.265)
P <0.0001 <0.0001 <0.0001 0.043 0.014 <0.0001 0.043 0.014
Inferior Retina Thickness (cell count)
 Estimate 12.6 0.053 0.967 13.5 0.031 0.074 NA 13.5 −0.031 0.074 NA
 95% CI (10.4–14.8) (0.033–0.072) (10.1–16.9) (−0.045–0.108) (0.010–0.138) (10.1–16.9) (−0.108–0.045) (0.010–0.138)
P <0.0001 <0.0001 <0.0001 0.39 0.027 <0.0001 0.39 0.027
Inferior retina thickness (μm)
 Estimate 86.4 0.068 0.954 124.1 0.120 0.207 0.975 124.1 −0.120 0.207 NA
 95% CI (67.5–105.4) (0.038–0.098) (81.1–167.1) (0.026–0.214) (0.057–0.358) (81.1–167.1) (−0.214–−0.026) (0.057–0.358)
P <0.001 0.0002 <0.0001 0.016 0.011 <0.0001 0.016 0.011
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