Graefes Arch Clin Exp Ophthalmol DOI 10.1007/s00417-015-3099-7

BASIC SCIENCE

Retinal histopathology in eyes from patients with autosomal dominant retinitis pigmentosa caused by rhodopsin mutations Vera L. Bonilha 1,2 & Mary E. Rayborn 1 & Brent A. Bell 1 & Meghan J. Marino 1 & Craig D. Beight 1 & Gayle J. Pauer 1 & Elias I. Traboulsi 1,2 & Joe G. Hollyfield 1,2 & Stephanie A. Hagstrom 1,2

Received: 26 March 2015 / Revised: 31 May 2015 / Accepted: 23 June 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Purpose To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin (RHO) gene mutations. Methods Eyes from a 72-year-old male (donor 1), an 83-yearold female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells. Results DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to

* Vera L. Bonilha [email protected] 1

Cole Eye Institute, Cleveland Clinic, Ophthalmic Research - i31, 9500 Euclid Avenue, Cleveland, OH 44195, USA

2

Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA

the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea. Conclusions Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina. Keywords Retinitis pigmentosa . Rhodopsin mutations . Histopathology . Photoreceptors . Immunohistochemistry

Introduction Retinitis Pigmentosa (RP) is a genetically and phenotypically heterogeneous group of inherited retinal diseases, with a prevalence of approximately 1:4000 [1]. It is a progressive disease that affects photoreceptor cells, altering their function and leading to irreversible vision loss [2]. Characteristic clinical symptoms include night blindness, loss of peripheral vision, decreased visual acuity and a triad of ocular features, including waxy pallor of the optic nerve head, bone spicule pigmentation and attenuated retinal vessels [1]. Forty percent of RP cases display an autosomal dominant inheritance pattern, with approximately one-third of these attributed to mutations in the gene for rhodopsin (RHO), the photopigment molecule located in rod photoreceptor cells that initiates the phototransduction cascade [3]. Presently, there are more than 150 disease-causing mutations in RHO, and the vast majority are dominantly inherited (https://sph.uth.edu/retnet). Rhodopsin is a seven-transmembrane G-protein-coupled receptor that is densely packed into photoreceptor outer segment membranes. The identified mutations are located

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throughout the protein in the intradiscal, transmembrane and cytoplasmic domains. Understanding the pathogenic mechanism(s) leading to RHO-induced photoreceptor degeneration is vital for effective therapeutic intervention and has been studied extensively using tissue culture systems and transgenic animal models [4–6]. However, only a few studies have reported on the pathology of patient donor eyes with RHO mutations, specifically T17M, P23H, Q64X, C110R, R135W and E181L [7–15]. The most prevalent RHO mutation in patients with ADRP in the United States is the P23H mutation [5]. P23 is located in the N-terminus of rhodopsin, within the intradiscal space. The retinal histology of two related donor eyes carrying the p.P23H mutation revealed variable histological retinal findings [14, 15]. The eye from the older donor (87 years) showed a milder disease with sparse intraretinal bone spicule pigment, a normal appearing macula and up to three layers of cone nuclei in the fovea and parafovea. The eye from the younger donor (77 years) showed a more severe disease with extensive intraretinal peripheral pigment and scattered areas of only a single row of photoreceptors in the fovea and parafovea. A separate study evaluating a 68-year-old eye donor with a p.P23H mutation revealed extensive photoreceptor degeneration with only few foveal cones present [7, 16]. These results support the clinical variation in patients of comparable age with this P23H mutation [16]. Seven different point mutations of the P347 residue in RHO have been linked to RP (p.P347S, p.P347L, p.P347A, p.P347G, p.P347C, p.P347R and p.P347T). The most prevalent RHO mutation in patients with ADRP in Europe is p.P347L [17]. P347 is located in the Cterminal cytoplasmic tail of rhodopsin, located in the VxPx motif, which directs transport of the protein to the outer segment [18]. RP patients with the p.P347L mutation tend to have less visual function as a group, with early onset of night blindness, and lower visual function as monitored by visual field and electroretinogram amplitudes in comparison with patients with other rhodopsin mutations [19, 20]. More recently, patients with this mutation were reported to display cone-mediated vision, thinning or loss of the outer nuclear layer (ONL), thickening of the inner retina, and demelanization of the retinal pigment epithelium (RPE) in studies using optical coherence tomography (OCT) [21]. Interestingly, it has been reported that ADRP patients carrying a mutation at this residue have a less severe phenotype than those carrying a p.P23H mutation [19, 20]. To our knowledge, no histology of a donor eye known to carry a mutation at this residue has ever been published. Here, we report the ocular histopathology in eyes from three unrelated ADRP donors with point mutations in RHO.

We focus on retinal pathology changes and the effect of the disease on the distribution of photoreceptors and other retinal cells. Eye donor 1 carries the p.P23H mutation, eye donor 2 carries the p.P347L mutation and eye donor 3 carries the p.P347T mutation. Given the limited molecular characterization of affected individuals whose eyes have been made available for postmortem examination, the opportunity to combine genetic, phenotypic and pathologic findings is unprecedented, especially in light of the prevalence of these RHO mutations within North America and Europe.

Methods Tissue acquisition and fixation: donor eyes were obtained through the Foundation Fighting Blindness (FFB) Eye Donor Program (Columbia, MD). Immunocytochemical analysis was performed with the approval of the Cleveland Clinic Institutional Review Board (IRB #14-057). The research adhered to the tenets of the Declaration of Helsinki. The analyzed tissue included FFB donations #696, #789, #872, #915, #922 and #929. Eyes were obtained from a 72-year-old male (donor 1, family 1, II-7, FFB #922), an 83-year-old female (donor 2, family 2, II2, FFB #915) and an 80-year-old female (donor 3, family 3 II-1, FFB #872) (Fig. 1a, c and e). Eyes were enucleated 5 to 17 h postmortem and fixed in 4 % paraformaldehyde and 0.5 % glutaraldehyde in phosphate buffer. The globes were stored in 2 % paraformaldehyde in D-PBS. Postmortem eyes from 65-year-old, 72-year-old, and 88-year-old donors without a history of retinal disease were used as controls. Genetic analysis Approximately 10 mL of peripheral blood was collected from family 1, donor 1 (family member II-7) and family member III-7. Blood was also collected from donor 2 (II-2) in family 2 and donor 3 (II-1) in family 3. DNA was extracted and purified from leukocytes by means of the Gentra Systems PUREGENE DNA Purification Kit (Qiagen). DNA from family 1 III-8 was extracted from a buccal sample using the same kit. Direct testing for mutations in the RHO gene was performed by PCR amplification and DNA sequencing, in two directions, of all coding exons and exon/intron boundaries including at least 30 nucleotides into the intron. Primer sequences and PCR conditions are available upon request. Ex-vivo imaging of eyes The combination of visible light fundus, confocal scanning laser ophthalmoscopy (SLO) and spectral domain optical coherence tomography (SDOCT) imaging systems was carried out within a D-PBSfilled plexiglass chamber that has been previously

Graefes Arch Clin Exp Ophthalmol Fig. 1 Mutational Analysis of individuals with ADRP due to RHO mutations a Pedigree of family 1. Slashed symbols reflect deceased family members. Affected family members are shown with filled symbols and unaffected family members are shown with unfilled symbols. Postmortem analysis was done on affected member II-7, referred to as donor 1 (★). b Sequence analysis of family 1 identified a heterozygous RHO mutation, c.68C>A and p.P23H. DNA analysis was performed on the donor’s two affected children (III7 and III-8). c Pedigree of family 2. Postmortem analysis was done on affected member II-2, referred to as donor 2 (★). d Sequence analysis of the donor in family 2 identified a heterozygous point mutation in RHO, c.1040C>T and p.P347L. e Pedigree of family 3. Postmortem analysis was done on affected member II-1, referred to as donor 3 (★). f Sequence analysis of the donor in family 3 identified a heterozygous point mutation in RHO, c.1039C>A and p.P347T

described and provides comprehensive characterization of retinal lesions in postmortem donor eyes prior to histopathology processing and analysis [22, 23].

Leica laser scanning confocal microscope (TCS-SP2, Leica, Exton, PA), as previously described [23].

Immunohistochemistry All eyes were cut and processed similarly as described: first, the posterior retina-RPEchoroid was cut from the far periphery to the optic nerve head; next, a parallel cut was made through the middle of the optic nerve head and fovea (dashed line in Fig. 4a); and finally, a perpendicular cut was made ~1 mm away from the fovea, generating two smaller fragments named perifovea and peripheral. Fragments were processed for cryosectioning and immunofluorescence labeling as previously described [23]. Retinal sections were incubated with monoclonal antibodies 1D4 to rhodopsin (ab5417, 1:1000, from Abcam), 7G6 to cone arrestin (1:100, from Dr. P. MacLeish), and polyclonal antibodies to red/green cone opsin (AB5405, 1:1200, Chemicon). Labeled sections were analyzed using a

Results Clinical findings Three unrelated donors were evaluated. Donor 1 (FFB #922, family 1, II-7, p.P23H) was of English and German descent, but clinical records were not available for this donor (Fig. 1a, star). Donor 2 (FFB #915, family 2, II-2, p.P347L) was also of mixed European ancestry (Fig. 1c, star). She had a history of dementia, which made ophthalmic examinations difficult in her last few years of life. At the age of 76 years, her visual acuity was no light perception OU and her dilated fundus examination was consistent with advanced RP. Her pupils could not be checked because of corneal opacity and her intraocular pressures were 24 OD and 27 OS. Extraocular

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motility was full. She also had a history of narrow angle closure glaucoma and bilateral cataracts. Donor 3 (FFB #872, family 3, II-1, p.P347T) was of English and Scottish ancestry (Fig. 1e, star). She was last examined in 2001 at the age of 73 years. Her visual acuity measured 20/25 OD and 20/40 OS. She had cataracts in both eyes and had intraocular lenses. Goldmann visual fields showed severe constriction. Molecular genetic analysis In family 1, genetic analysis was performed on the affected donor (II-7), his affected daughter (III-7) and his affected son (III-8) (Fig. 1a). DNA sequence analysis revealed a heterozygous mutation in RHO, c.68C>A (p.P23H), in the donor (Fig. 1b). Segregation analysis revealed that both affected children carried the mutation. In family 2, the affected donor (II-2), prior to death, was identified by The Berman-Gund Laboratory for the Study of Retinal Degenerations as having a heterozygous mutation in RHO, c.1040C>T (p.P347L) (Fig. 1c and d) [19]. Upon her death, the FFB eye donor program received her eyes for histopathological analysis. No other family members were available for genetic analysis. In family 3, the affected donor (II-1), prior to death, was identified by the Laboratory for the Molecular Diagnosis of Inherited Eye Disease at The University of Texas-Houston as having a heterozygous mutation in RHO, c.1039C>A (p.P347T) (Fig. 1e and f). Upon her death, the FFB eye donor program received her eyes for histopathological analysis. Clinical records indicate that other affected family members also carried the p.P347T mutation; however, these family members were not available for confirmation by our laboratory. Ex-vivo imaging of donor eyes Fundus, SLO and SD-OCT imaging were performed on the donor eyes to qualitatively compare and contrast end-stage macromorphology prior to histological examination (Figs. 2 and 3). Anatomical landmarks, such as the optic disk and fovea, were clearly identified in the control (72-year-old) (Fig. 2a), p.P23H (Fig. 2b) and p.P347T (Fig. 2d) eyes. However, in the p.P347L eye, the optic nerve and macula were difficult to discern by fundus, SLO and SD-OCT imaging, respectively (Fig. 2c, g and k). All imaging modalities revealed the presence of bone spicules, to varying density and/ or coverage in the three donor eyes as compared to the control eye. Qualitatively, fundus images suggest that pigment density patterns are similar in the p.P23H and p.P347T eyes. In these two eyes, a region void of bone spicules encompasses both the perimacula and optic disk (Fig. 2b and d). In contrast, the spicule density and pattern in the p.P347L eye is noticeably different (Fig. 2c). Spicules in this donor have infiltrated the

perimacula and macular regions, making it difficult to definitively distinguish and pinpoint the fovea. The only region partially void of bone spicules in this donor is an area encompassing the optic disk. SLO imaging revealed areas of detached retina in the control eye (Fig. 2e and i, asterisks). We attribute the retinal detachments to postmortem enucleation or removal of the anterior segment in preparation for imaging and histological processing. Infrared (SLO-IR) and autofluorescence (SLO-AF) imaging showed bone spicule pigment patterns in agreement with fundus macroscopic imaging for p.P23H (Fig. 2f) and p.P347 (Fig. 2h) eyes. In the p.P347L mutation (Fig. 2g), SLO-IR showed even higher densities than observed by visible fundus imaging. These higher densities are likely attributed to IR light having a deeper depth of penetration than the visible light used for color fundus images. IR light used to probe the tissue extends beyond the retina and RPE pigment and into the choroid where additional melanopigment resides. This observation is further supported by SD-OCT data (Fig. 3g), which shows a degenerated retina with more backreflected signal originating from the choroid compared to the control and other two donor eyes. SLO-AF of the p.P347L donor (Fig. 2k) was substantially different than the visible fundus and SLO-IR images showing contrasting regions of hypo-fluorescent and hyper-fluorescent patches that do not seem to correlate identically to the bone spicule pattern. SLO-AF imaging also reveals a hyperfluorescent macula for P23H (Fig. 2j) and p.P347T (Fig. 2l) eyes, whereas the control is hypo-fluorescent. The area surrounding the optic nerve in the p.P23H showed evidence of RPE atrophy by SLO-AF, as choroidal vasculature could be visualized (Fig. 2j). SD-OCT imaging was performed on the control (72 years) and donor eyes to assess retinal layer morphology and integrity prior to sectioning (Fig. 3). The fovea and optic nerve could be identified in all samples by in-depth B-scans (Fig. 3e to h). SD-OCT B-scans also revealed structural differences in the retina of one of the donor eyes relative to the control. The p.P347L donor eye (Fig. 3g) exhibited a substantially degenerated retina compared to both the control (Fig. 3e) and the other two donor eyes (Figs. 3f and h). These data suggest that the pathology in the P347L donor is more severe than that of the p.P23H and p.P347T donor samples. Histopathology of donor retinas To evaluate the effect of RHO mutations on retinal structure, toluidine blue-stained plastic retinal sections of ADRP and control donor eyes were examined. All the analyzed eyes were cut and processed similarly. A schematic drawing depicts the regions harvested and processed for observation in both the morphological and immunohistological assays (Fig. 4a, dashed line). These include the perifovea (Fig. 4a, region 1) and periphery (Fig. 4a, region 2). Histology of the control

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Fig. 2 Ex-vivo imaging of ADRP donor eyes with RHO mutations. Fundus images (a-d) and SLO images (e-l) were collected from all ADRP and an age-similar control. In the control eye, detached retinas are apparent with SLO (e, i, *). Fundus images reveal bone spicule pigment density and pattern similar for p.P23H (b) and p.P347T (d), while p.P347L (c) includes spicules that, to some extent, impact the

periphery, macula and perimacula regions. SLO-IR imaging showed a lack of perimacular and optic disk hyper fluorescent ring in p.P347L (g), like the one observed in the p.P23H (f) and p.P347T (h) eyes. SLO-AF of p.P347L (k) also revealed a pattern that did not match the fundus (c) and SLO-IR (g) images. Scale bar in fundus image=0.5 cm

retina (88 years) in the periphery (Fig. 4b) and perifovea (Fig. 4f) displayed normal retinal lamination patterns. In all three ADRP donors, a highly degenerated retina with disorganization of the lamina and gliosis was evident in all peripheral areas analyzed (Fig. 4c to e). Furthermore, photoreceptor outer segments were also absent in all areas analyzed. Intraretinal bone spicule pigment was visible in the retinas of donors carrying p.P23H (Fig. 4c, asterisks) and p.P347T (Fig. 4e,

asterisk) mutations. A prominent nuclear lamina was present in the perifovea of the donor carrying the p.P23H mutation, possibly a combination of cells from the inner and outer nuclear layer (Fig. 4g). In addition, the RPE displayed lack of pigment and its height (from apical to basal surface) was severely reduced from normal in the perifovea in this eye. The retina of the donor carrying the P347L mutation was severely atrophic in the perifovea (Fig. 4h), and the RPE was

Fig. 3 Ex-vivo OCT imaging of ADRP donor eyes with RHO mutations. OCT images were collected from all ADRP and an age-similar control. En face images reveal the location (dashed lines) of the in-depth, B-scan images of control (a) and ADRP donors (b–d). The fovea (arrowhead) and optic nerve (ON) were identified in all ADRP donor eyes. In-depth Bscans from the control eye (e) revealed a normal appearing retina with clearly defined fovea, some evidence of laminar architecture, and no appreciable evidence of retinal thinning or degeneration. Black spots (a)

are artifacts from air bubbles. Images from p.P23H (f) and p.P347T donors (h) revealed retinas of appreciable thickness but with less delineated outer retina architecture. In contrast, the p.P347L donor (g) showed clear evidence of thinned retina relative to the control (e) and other ADRP donor retinas (f, h), suggesting degeneration. The p.P347T donor’s retina (h) had a large macular (*) detachment, as indicated in the B-scans. B-scan scale is 0.5 mm

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Fig. 4 Histology of ADRP donor eyes with RHO mutations. a Fundus image of the control eye (OS) with a schematic drawing of the regions cut and processed for cryosectioning and immunolabeling. b–i Toluidine blue-stained plastic sections 1 μm (microm) of retinas from all ADRP donors and an age-similar control. Morphology of the control retina in the periphery (b) and perifoveal (f) regions displayed typical retinal lamina. Histology in the periphery of all three ADRP donors revealed a highly degenerated retina with disorganization of the lamina, gliosis, and absence of photoreceptor outer segments (c-e). Intraretinal bone spicule

pigments were visible in the retinas of p.P23H (c, asterisks) and p.P347T donors (e, asterisk). The perifovea of p.P23H (g) and p.P347T (i) donors displayed a prominent inner nuclear layer. The perifovea of p.P347L donor (h) was severely atrophic. Donor p.P23H RPE (g) displayed lack of pigment and was severely reduced from normal thickness as compared to the control donor (f). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, POS photoreceptor outer segments, RPE retinal pigment epithelium, Ch Choroid. Scale bar a=0.5 cm; b- i=50 μm (microm)

discontinuous in this region (Fig. 4h). A prominent inner nuclear layer was present in the perifovea of the donor carrying the p.P347T mutation (Fig. 4i), along with a few disorganized cell nuclei (Fig. 4i, arrows). These data suggest that all the rhodopsin mutations cause profound changes in the outer retina, leading to the loss of several retinal cell layers and the appearance of gliosis in the periphery. Analysis of the perifovea suggests that the pathology in the p.P347L donor is more severe than that of the p.P23H and p.P347T donor samples.

Next, we investigated the distribution of cone photoreceptors in retinas harboring RHO mutations. In the control retina (65 years), cone arrestin was distributed along the entire plasma membrane, from the tip of the outer segment to the synaptic base both in the periphery (Fig. 6a, green) and perifovea (Fig. 6e, green). Immunostaining of the control retina with antibodies against red-green cone opsin revealed that this protein is restricted to cone outer segments both in the periphery (Fig. 6a, red) and perifovea (Fig. 6e, red). Strikingly, cones were mostly absent from the periphery of all the ADRP donor retinas (Fig. 6b to d). In contrast, cones were present but highly disorganized in the perifovea of the donor carrying p.P23H (Fig. 6f) and p.P347T (Fig. 6h) mutations, and red-green cone opsin was distributed through the whole cell body. Specifically, a higher number of cones was present in the p.P23H retina as compared to the p.P347T retina. However, in both retinas, synaptic terminals were not visualized. The perifovea of the donor carrying the p.P347L (Fig. 6g, arrow) mutation revealed close to complete absence of cone arrestin-labeled or cone opsin-labeled cells. These data suggest that all the analyzed rhosopsin mutations cause a profound loss of cones in the periphery. The p.P23H and p.P347T rhodopsin mutations lead to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation also leads to near complete absence of cones in the perifovea.

Immunocytochemistry of donor retinas To investigate the effect of RHO mutations on retinal architecture, immunofluorescent studies with antibodies against rhodopsin were performed. In the control retina (88 years), rhodopsin distribution was restricted to the rod outer segments in the periphery (Fig. 5a) and perifovea (Fig. 5e). A few highly disorganized rhodopsin-labeled cells were present in the periphery of the donor carrying the p.P23H mutation (Fig. 5b, arrows), but were nearly absent in the retinas from donors carrying p.P347L (Fig. 5c) and p.P347T (Fig. 5d) mutations. In the perifovea, donors carrying p.P23H (Fig. 5f) and p.P347T (Fig. 5h) mutations displayed a few disorganized rhodopsin-labeled cells; however, even less rhodopsinlabeled cells were detected in the perifovea of the donor carrying the p.P347L mutation. These data suggest that the p.P23H and p.P347T rhodopsin mutations analyzed cause a profound loss of rods in the retina both in the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both the periphery and perifovea.

Discussion Rhodopsin is the primary protein component of photoreceptor rod outer segments and is the light-sensing molecule of the

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Fig. 5 Immunocytochemistry of ADRP retinal sections with RHO mutations stained with rhodopsin antibodies. Immunofluorescence of ADRP retinal sections labeled with antibodies to rhodopsin (Alexa488, green) showed significantly decreased staining when compared to control. The control retina showed that rhodopsin was restricted to the rod outer segments in both the periphery (a) and perifovea (e). A few highly disorganized rhodopsin-labeled cells (arrows) were still present in the periphery of donors p.P23H (b), but were nearly absent in the retinas

from donors p.P347L (c) and p.P347T (d). In the perifovea, donors p.P23H (f) and p.P347T (h) also displayed a few disorganized rhodopsin-labeled cells; however, close to no rhodopsin- labeled cells were detected in the perifovea of donor p.P347L (g). Bruch’s membrane is indicated by the hashed white line. Nuclei were labeled with TO-PRO-3. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, POS photoreceptor outer segments. Scale bar=40 μm (microm)

retina. Thus, it is not surprising that mutations within the gene encoding for rhodospin (RHO) account for approximately 25 % of autosomal dominant retinal degenerations known as retinitis pigmentosa (RP). Since its discovery as the first RHO mutation directly linked to RP, p.P23H, in the N-terminus of rhodopsin within the intradiscal space, has been documented as the most prevalent RHO mutation in North America [5]. For this reason, the p.P23H mutation is one of the best studied and most characterized of RHO mutations. On the other hand, the C-terminal residue P347 is considered a mutational hot spot due to mutations occurring at a CG dinucleotide, with

seven RP-causing variants identified at this locus [20, 24, 25]. It has been documented that the most prevalent RHO mutation in Europeans is the p.P347L mutation [17]. A classification system sorting rhodopsin mutations places p.P23H into Class 2 [26, 27]. This category of mutants causes opsin to be retained in the endoplasmic reticulum and may cause photoreceptor cell death by continued activation of the unfolded protein response; these mutants retain their ability to join in vitro to II-cis retinal to form a photo labile visual pigment. Class 2 mutants, and specifically the p.P23H mutation, tend to result in a milder disease phenotype [28, 29]. In

Fig. 6 Immunocytochemistry of ADRP retinal sections with RHO mutations stained with cone-specific antibodies. Immunofluorescence of ADRP retinal sections labeled with antibodies to cone arrestin (Alexa488, green) and red-green cone opsin (Alexa568, red) showed significantly decreased staining in the periphery when compared to control. In the periphery (a) and perifovea (e) of the control retina, cone arrestin was distributed along the entire plasma membrane, from the tip of the outer segment to the synaptic base, while the red-green cone opsin was restricted to the outer segments. Cones were mostly absent from the

periphery of all the ADRP donor retinas (b-d). In contrast, cones were present but highly disorganized in the perifovea of the donors p.P23H (f) and p.P347T (h) and red-green cone opsin was distributed through the whole cell body. Donor p.P347L (g, arrow) displayed close to complete absence of cone arrestin-labeled or cone opsin-labeled cells in the perifovea. Bruch’s membrane is indicated by hashed white line. Nuclei were labeled with TO-PRO-3. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer, POS photoreceptor outer segments. Scale bar=40 μm (microm)

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contrast, mutations involving the C-terminus of rhodopsin result in a more severe form of RP. Residue p.P347 belongs to the VxPx motif located in the cytoplasmic tail of rhodopsin, targeting transport of rhodopsin along the connecting cilium into the outer segment [18, 30]. Mutations affecting this residue are categorized as Class 1 mutations, and as such, these types of mutations translocate to the plasma membrane, fold correctly, and can form a functional chromophore with 11-cisretinal. Class 1 mutants disrupt the eight C-terminal amino acids, which constitute an outer segments localization signal [31] and an ADP ribosylation factor 4 (arf-4) binding site [32]. Therefore, the loss of polarized distribution of these Class 1 mutants may lead to photoreceptor death and retinal degeneration [33]. Both imaging and histological examinations presented here involved individuals with both Class 2 (p.P23H) and Class 1 (p.P347L and p.P347T) RHO mutations. In the periphery, these retinas were degenerated with little evidence of stratified nuclear layers. In contrast, the perifoveal region maintained a prominent inner nuclear layer in the p.P23H and p.P347T retinas. Previous reports described the phenotype of rhodopsinrelated RP from mutations involving codon 347 as being more severe than those involving codon 23 [28, 29]. Our data is consistent with both histological and electrophysiological data of animal models expressing the p.P347L mutation and with the clinical phenotype of patients carrying this mutation. Morphological analysis of a transgenic pig model that expresses p.P347L mutation revealed an initial phase of rapid and extensive degeneration of the rod cells in the first 6 weeks of age, followed by an acute phase of cone cell degeneration involving approximately half of the population and lingering rod degeneration from 6 to 12 weeks of age; and finally a partial cone recovery to be followed by a chronic degenerative phase of the remaining cones cells from 12 to 33 weeks of age [34]. Alternatively, histologic and ERG studies of transgenic p.P347L rabbit retinas showed early loss of rod function associated with relatively preserved cone function, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming [35–37]. These findings are very similar to the clinical findings of RP patients with the p.P347L mutation who demonstrate an RP phenotype characterized by early onset and severe disease course, with subtle interfamilial and intrafamilial variability [17, 19, 20]. Future analysis of additional human eyes carrying the p.P347L and p.P347T mutations is needed to improve our understanding of the increased severity of the p.P347L mutation. Since the different rhodopsin mutations associated with RP lead to different disease phenotypes including characteristic differences in the rate and regional distribution of photoreceptor degeneration, it is also important to characterize the histopathologic changes in donor retinas with the different RHO genotypes. We expect that continued identification of specific gene mutations in patient donors

and also in the existing collection of preserved RP eyes will increase our understanding of the pathologic mechanism of retinal degeneration in this disease. This information is essential for developing effective therapies for this important group of blinding disorders. It should be noted that in all three ADRP retinas analyzed, there was advanced retinal degeneration with near-absence of rods both in the periphery and in the perifovea and an absence of cones in the periphery. Degeneration of the outer retina resulting in vision loss due to the progressive death of photoreceptor cells is usually accepted to represent the end-stage of RP and several other retinal degenerations. According to this criterion, the donated human eyes analyzed in this study are at the end-stage of the disease. However, the value of analyzing human retinas in this retinal degeneration stage is emphasized by recent human experiments using subretinal electronic implants that helped previously blind patients to read after stimulation of the implanted electrodes [38, 39]. Acknowledgments The authors thank Dr. Peter MacLeish (Morehouse School of Medicine, Atlanta, GA) for providing us with the antibody to cone arrestin (7G6). Pedigree courtesy of the Berman-Gund Lab (Massachusetts Eye and Ear Infirmary, Boston, MA). This work was supported by The Foundation Fighting Blindness Histopathology Grant F-OH011102-0231 (JGH), Research Center Grants from The Foundation Fighting Blindness (JGH), Research to Prevent Blindness Unrestricted Grant and National Institutes of Health grant R01EY014240-08 (JGH) and the Llura and Gordon Gund Foundation. Conflict of interest statement The authors declare that they have no financial interest in the subject matter or materials discussed in this manuscript.

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