Prph2 mutant knock-in mice recapitulate all features of human central areolar choroidal dystrophy and reveals an aberrant synaptic remodelling and microglial activation

doi:10.21203/rs.3.rs-3221362/v1

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Prph2 mutant knock-in mice recapitulate all features of human central areolar choroidal dystrophy and reveals an aberrant synaptic remodelling and microglial activation

https://doi.org/10.21203/rs.3.rs-3221362/v1

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Journal Publication

published 01 Nov, 2023

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Purpose

Central areolar choroidal dystrophy is an inherited disorder characterized by progressive choriocapillaris atrophy and retinal degeneration, usually associated with mutations in the PRPH2 gene. We aimed to generate and characterize a mouse model with the p.Arg195Leu mutation previously described in patients.

Methods

Heterozygous (Prph2^WT/KI) and homozygous (Prph2^KI/KI) mice have been generated using the CRISPR/Cas9 system to introduce the Arg195Leu mutation. Retinal function was assessed by electroretinography and optomotor test at 1, 3, 6, 9, 12, and 20 months of age. The structural integrity of the retinas was evaluated at the same ages using optical coherence tomography. Immunofluorescence and transmission electron microscope images of the retina were also analyzed.

Results

Genetic sequencing confirmed that both Prph2^WT/KI and Prph2^KI/KI mice presented the p.Arg195Leu mutation. A progressive loss of retinal function was found in both mutant groups, with significantly reduced visual acuity from 3 months of age in Prph2^KI/KI and from 6 months of age in Prph2^WT/KI mice. Decreased amplitudes in the electroretinography responses were observed from 1 month of age in Prph2^KI/KI and from 6 months of age in Prph2^WT/KI. Morphological analysis of the retinas correlated with functional findings, showing a progressive decrease in retinal thickness of mutant mice, with earlier and more severe changes in the homozygous mutant mice. We corroborated the alteration of the outer segment structure, and we found changes in the synaptic connectivity in the outer plexiform layer as well as gliosis and signs of microglial activation.

Conclusions

The new Prph2^WT/KI and Prph2^KI/KI murine models show a pattern of retinal degeneration similar to that described in human patients with CACD and appear as good models to study the mechanisms involved in the onset and the progression of the disease, as well as to test the efficacy of new therapeutic strategies.

Animal model

central areolar choroidal dystrophy

CRISPR/Cas

peripherin

retinal dystrophy

Monogenic hereditary retinal dystrophies, in which only one gene carries a mutation, are an attractive target for developing new gene therapies and neuroprotection strategies, which have the potential to be further extrapolated to other retinal dystrophies. Central areolar choroidal dystrophy (CACD) is a monogenic hereditary disease that involves progressive retinal degeneration (Coco-Martin et al., 2020). CACD is considered a rare disease and its prevalence is 1-9 cases per 100,000 people (Orphanet). Patients with CACD show a gradual loss of visual acuity (VA) and color perception between the ages of 30 and 60 (Boon et al., 2009; Hoyng and Deutman, 1996). CACD is caused by different mutations in the peripherin-2 gene (PRPH2) usually with an autosomal dominant inheritance pattern (Smailhodzic et al., 2011). PRPH2 codifies the glycoprotein peripherin-2 (PRPH2), also known as retinal degeneration slow (RDS) protein, or tetraspanin-22 (Uniprot). PRPH2 is essential for the morphogenesis and structure of the outer segment (OS) discs of photoreceptor cells (Lewis et al., 2021). CACD presents a high genetic heterogeneity (Peeters et al., 2021), and at least five different mutations in PRPH2 have been histologically and functionally described as causing this disease (Boon et al., 2009; Hoyng and Deutman, 1996; Keilhauer et al., 2006a; Nakazawa et al., 1995; Piguet et al., 1996; Wroblewski et al., 1994; Yanagihashi et al., 2003). Each of these mutations was found in families from different countries, which suggests a founder effect in the transmission of CACD. In Spain, there is a large family with a PRPH2 gene mutation, p.Arg195Leu, that has been transmitted over at least 11 generations, with the first affected member dated in 1756. In this family, 119 relatives were reported to have retinal problems, and 56 living patients have been diagnosed with CACD, 10 of them further confirmed by genetic testing. The CACD phenotype of four members of this family was previously characterized by our group (Albertos-Arranz et al., 2021). To assess the efficiency of different therapeutic approaches, a deep knowledge of the pathophysiology of CACD and the progression of cellular and molecular changes in the retina is essential. Thus, we addressed, using the CRISPR/Cas9 system, the generation of a mouse model carrying the same point mutation as this Spanish family. With this mouse model, we characterized the progression of the retinal degeneration that affects this family with CACD.

Generation of PRPH2 p.Arg195Leu Murine Model Using CRISPR/Cas9

CRISPR/Cas9 System Design

The murine model C57BL/6J-Prph2^em1Sal carrying the mutation p.Arg195Leu was generated using CRISPR/Cas technology. One sgRNA (Prph2-sgRNA1) overlapping the intron1-exon2 genomic sequence of Prph2 was designed (http://bioinfogp.cnb.csic.es/tools/breakingcas/) to induce a double-strand break at the c.583 position. Two complementary oligos corresponding to Prph2-sgRNA1 were designed, including two 4 bp overhang sequences to clone both into pX458 vector (Addgene plasmid # 48138). The pX458 vector was linearized with BpiI (New England Biolabs Inc., Ipswich, MA, USA) and the Prph2-sgRNA1 complementary oligos were denatured at 95 °C for 5 min, ramp-cooled to 25 °C over 45 min to allow annealing, and were ligated with the linearized pX458. Competent cells were transformed with 2 μl of the ligated plasmid, and single colonies were grown before plasmid extraction (Qiagen, Hilden, Germany). A 200 nt single-strand DNA oligonucleotide (ssODN) (Integrated DNA Technologies Inc., Coralville, IA, USA) containing the mutated sequence c.584 G>T (CGC>CTC, Arg>Leu) was designed as a template for homologous recombination to integrate the point alteration. sgRNA sequence was PCR-amplified from pX458-based vector with primers carrying the T7 RNA polymerase promoter at the 5´ ends. PCR products were purified (NZYGelpure kit, NZYTech, Lisboa, Portugal) and used as a template for in vitro T7 RNA polymerase transcription (MEGAshortscript T7 Transcription Kit, Thermo Fisher, Waltham, MA, USA). Ribonucleocomplexes were formed by incubating 200 ng of sgRNAs and 200 ng of Cas9 nuclease (Integrated DNA Technologies Inc.) at 37 ºC for 10 min.

Mouse Embryo Microinjection

20 ng/µl of sgRNA-Cas9 protein and 25 ng/µl of single-strand DNA oligonucleotide (ssODN) template solution were microinjected into the pronucleus of one-cell stage embryos. These embryos were obtained from superovulated C57BL/6J females. Microinjected embryos were grown overnight on KSOM with non-essential amino acids (Millipore, Burlington, MA, USA), 37 °C, and 5% CO₂ atmosphere. After 12 h of culture, two-cell-stage embryos were recovered and transferred to foster CD1 females.

Analysis of CRISPR/Cas9 Target Sites

Founder mice were crossed with wild-type C57BL/6J animals to eliminate possible off-targets. The inheritance of the alleles followed Mendelian principles. Pure knock-in heterozygotes (Prph2^WT/KI) of the F3 were then crossed to obtain knock-in homozygous (Prph2^KI/KI) mice. To check that these animals carried the point mutation p.Arg195Leu, we genotyped the animals using PCR and Sanger sequencing from DNA obtained from a biopsy of the tail. Animals for this study were obtained by cross-breeding.

Animals

All animals, wild-type (Prph2^WT/WT), Prph2^WT/KI, and Prph2^KI/KI, were bred in the animal facility of the University of Alicante and maintained under the same environmental conditions: temperature of 23±1 ºC, humidity of 55-60%, photoperiod of 12/12 h light/dark, and light intensity of 300 lux. The retinal function of animals from the three genotypes was evaluated at 1, 3, 6, 9, 12, and 20 months of age using the optomotor test and electroretinography (ERG). Optical coherence tomography (OCT) was also performed at the same ages to evaluate the retinal structure. The number of animals used was N=12 for optomotor testing, N=11 for ERG, and N= 9 for OCT in each experimental group. We always ensure an equal number of females and males for each experiment. All procedures were approved by the Ethical Committee of the University of Alicante (2019/VSC/PEA/0266) and performed following the EU Directive 2010/63/EU. For all procedures where anesthetization was required, animals were anesthetized with 100 mg/kg of ketamine and 8 mg/kg of xylazine. Once procedures were finished, mice were euthanized by cervical dislocation.

Optomotor test

To evaluate the VA of the animals, Argos optomotor system was used (Instead Technologies, Elche, Spain). Mice were stimulated with a vertically oriented grating that rotates horizontally either in one sense or in the opposite, aleatory, for 5 s. Initial spatial frequency was 0.088 cycles/degree, and contrast was 100%. Smooth head movements in the direction of the rotating grating were evaluated by a trained observer. The minor spatial frequency to which the mouse responded was considered the VA threshold of the animal.

Electroretinography

ERG responses were recorded in total darkness, after overnight adaptation, using the HMsERG LAB System (OcuScience, Henderson, NV, USA). The animals were anesthetized and kept at 38 ºC on a thermal blanket. For pupil dilatation, 1% of tropicamide (Alcon Cusí, Barcelona, Spain) was used. Also, a 0.2% polyacrylic acid carbomer gel (Novartis, Barcelona, Spain) was used to prevent eye dehydration and assure the fitting of the recording electrodes. 14 stimuli with increasing luminance (-5.5 to 1 log cd s/m²) were presented to the animals. After a period of light adaptation, photopic responses were obtained using 8 increasing stimuli (-2.5 to 0.9 log cd s/m²). The amplitude of the a-wave was measured from the baseline to the most negative trough, and the amplitude of the b-wave was measured from the trough of the a-wave to the peak of the b-wave.

Optical coherence tomography

The retinal structure of the animal models was evaluated in vivo using Spectralis OCT system (version 6.9.4.0, Heidelberg Engineering, Heidelberg, Germany). After anesthesia induction, pupils were dilated with 1% of tropicamide (Alcon Cusí), and the dehydration of the cornea was prevented by applying a drop of physiological serum in each eye. A high-resolution dense scan at the superior central region of optic nerve was obtained at 1, 3, 6, 9, 12, and 20 months. The thickness of the total retina, including the retinal pigment epithelium (RPE), the outer nuclear layer (ONL), and the inner nuclear layer (INL) was measured at 400 μm from the optic nerve in ten different points separated 200 μm each other using ImageJ (Schneider et al., 2012). Two experimental researchers performed the analysis in a blind way.

Immunofluorescence

After sacrificing the animals, the dorsal margin of the limbus was marked by a suture (Lorca Marín, Murcia, Spain). The eyes were enucleated and fixed in 4% paraformaldehyde for 1 h, washed in 0.1 M phosphate buffer (PB) pH 7.4, and cryoprotected by sucrose gradient (15, 20, and 30%). The cornea, lens, and vitreous humor were removed, and the eyecups were embedded in Tissue-Tek OCT compound (SAKURA Finetech, Tokyo, Japan) and frozen in liquid nitrogen. Serial sections of 15 µm thick were obtained using a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany), mounted on microscope slides (Menzel GmbH & Co KG, Braunschweig, Germany), air-dried, and stored at -20 ºC.

For the immunostaining procedure, sections were thawed, washed in PB, and incubated at room temperature for 1 h in blocking solution, consisting of 10% (v/v) normal donkey serum in PB with 0.5% Triton X-100. Following, sections were incubated overnight at room temperature with the appropriate combinations of primary antibodies at different dilutions (listed in Table 1) in PB with 0.5% Triton X-100. Sections were then washed in PB and incubated for 1 h with the secondary antibodies. Secondary antibodies used were anti-rabbit-Alexa Fluor 488, and anti-mouse-Alexa Fluor 555, conjugates (1:100; Molecular Probes, Eugene, OR, USA). When indicated, nuclear marker TO-PRO-3 iodide (Molecular Probes) was added at 1:1000. Finally, sections were washed in PB and mounted with Citifluor (Citifluor Ltd, London, UK) under a coverslip. Images were taken with a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany).

Electron Microscopy

Prph2^WT/WT and Prph2^KI/KI mice at 1-month-old were perfused with a solution containing 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PB. Then, the eyes were enucleated and fixed for 2 h in the same solution. After rinsing in 0.1 M PB, the cornea, iris, lens, and vitreous body were removed and the remaining posterior part of the eye was cut into four quadrants. They were post-fixed in 1% osmium tetroxide (OsO₄) in 0.1 M PB for 1 h and then gradually dehydrated in growing concentrations of ethanol and acetone. The pieces were embedded overnight in EPON 812 and then blocks of EPON containing the pieces were made and polymerized at 60 ºC overnight. Ultrathin sections were obtained from blocks and contrasted using lead citrate and uranyl acetate. Images were acquired in a transmission electron microscope FEI Tecnai Spirit BioTwin (Thermo Fisher Scientific) using the Radius software v2.1 with a Xarosa digital camera (EMSIS GmbH, Münster, Germany).

Flow cytometry

Immune- and inflammatory-related cell populations in the retinas were analyzed by flow cytometry. After enucleating the eye, the retinas were dissected and disaggregated through a wide bore pipette tip in 1 ml of pH 7.4 phosphate-buffered saline by carefully pipetting up and down. The retinal cell suspensions were then filtered to prevent clumps with a 30 mm strainer (BD Biosciences, San Diego, Ca, USA). Samples were labeled with a cocktail of five antibodies acquired from e-Bioscience (San Diego, CA, USA): anti-CD11b-PE (Clone M/170), anti-CD45-FITC (Clone 30-F11) anti-CD11c-PerCpCy5.5 (Clone N418), anti-MHC class II (I-A/I-E)-PECy7 (Clone M5/114.15.2) and anti-CD169-eFluor 660 (Clone SER-4). After discarding doublets and debris events, the CD11b⁺ population (that stains macrophages and microglia cell populations or the CD45⁺ cells (marker of microglia and leucocyte populations were selected (Jurga et al., 2020; Martin et al., 2017; Ronning et al., 2019). The CD11b⁺ cell subpopulations were analyzed for their immunoreactivity against MHC class II (activated cells) (Chidlow et al., 2016; Lipski et al., 2017; Ronning et al., 2019), the dendritic cell marker CD11c (marker of dendritic and microglia cell populations) (Jiang et al., 1999; Lehmann et al., 2010; Tang et al., 2017) and sialoadhesin (CD169, that stains activated macrophages and microglial cell populations) (Bogie et al., 2018; Hughes et al., 2003; Linnartz-Gerlach et al., 2014). Each mouse retina was analyzed individually, and 3-12 retinas from different animals were assessed for each experimental group. Data were acquired on a LSR Fortessa cytometer (BD Biosciences) and analyzed using FCS 6 Flow Cytometry Software (De Novo, Los Angeles, CA, USA).

Statistics

Data were statistically analyzed using GraphPad Prism Analysis Software (GraphPad Software, Inc., San Diego, CA, USA). Analysis of variance (ANOVA) and Bonferroni’s Post Hoc test were applied to determine the significant differences between experimental groups. Differences were considered statistically significant at P < 0.05. Data in all graphs were represented as the mean value ± SEM.

Generation of the animal model C57BL/6J-Prph2^em1Sal

The c.584 G>T knock-in mouse model was generated by homologous recombination using CRISPR/Cas9 system in mouse zygotes (Fig. 1A). Pure Prph2^WT/KIanimals were crossed to obtain Prph2^KI/KImice. At this point, all animals were genotyped and then cross-bred to obtain the needed animals for the study. The genotyping showed the insertion of the point mutation in c.584 (Fig. 1B). This mutation resulted in the change of a single amino acid. Arginine in position 195, with a positively charged side chain, was exchanged by the hydrophobic leucine. This mutation was localized in the helix D, which is localized in the peripherical area of the interface crevice, the domain involved in PRPH2 oligomerization (Fig. 1C). Retinal degeneration of both Prph2^WT/KIand Prph2^KI/KImice was characterized at 1, 3, 6, 9, 12, and 20 months of age.

Visual function decreases with age in peripherin mutant mice

To assess the effect of the peripherin gene mutation on visual function, the VA of Prph2^WT/WT, Prph2^WT/KI, and Prph2^KI/KI mice was analyzed by optomotor test (Figure 2A). As shown in Figure 2B, VA of both Prph2^KI/KI and Prph2^WT/KI peripherin mutantmice progressively decreased with age compared to Prph2^WT/WT mice, with higher decays in Prph2^KI/KI than in Prph2^WT/KI mice. Concretely, Prph2^KI/KI mutantmice showed VA values significantly lower than control mice from 3 months of age (28% of reduction, P < 0.0001), and VA scores were minimal in these animals from 12 months of age (78% of reduction, P < 0.0001). Meanwhile, Prph2^WT/KI mutantmice showed VA values significantly lower than that of Prph2^WT/WT mice from 6 months of age (24% of reduction, P < 0.0001), and VA scores were still relatively high at 20 months of age (32% of reduction, P < 0.0001).

Visual function in control and mutant mice was also assessed throughout ERG recording. Similarly to the optomotor test results, ERG responses in both Prph2^KI/KI and Prph2^WT/K^I mutantmice progressively diminished with age in comparison to that registered in Prph2^WT/WT mice, being ERG response more severely affected in Prph2^KI/KI than in Prph2^WT/KI mice (Fig. 3A, B). Specifically, maximum scotopic a-wave values recorded in Prph2^KI/KI mutant mice significantly fell from 1 month of age (34% of reduction, P < 0.001) and practically disappeared from 9 months of age (83% of reduction, P < 0.0001) (Fig. 3C). Meanwhile, the reduction in a-wave values in Prph2^WT/KI mice was significant from 6 months of age (33% of reduction, P < 0.05), and a-wave scores remained noticeable at 20 months of age (Fig. 3C). Scotopic b-wave responses showed similar results, even though the drop in b-wave values was later and smoother than that observed in a-wave responses in both Prph2^KI/KI and Prph2^WT/KI mice (Fig. 3D). This indicates that the loss of function caused by Prph2 mutation was earlier and more marked in the outer retina than in the inner retina. Photopic ERG responses also showed a progressive age-dependent diminution in a- and b-wave values of Prph2 mutant mice as compared to Prph2^WT/WT mice, with a more severe effect in Prph2^KI/KI than in Prph2^WT/KI mice, and a slightly smaller effect on the photopic than on the scotopic response (Fig. 3E, 3F). This means that the point mutation in Prph2 similarly affects the cone (photopic) response and the mixed (scotopic) response, with a slightly greater effect on the rod response than the cone response.

Structural and cellular alterations in the retina of peripherin mutant mice

OCT images were obtained from Prph2^WT/WT, Prph2^WT/KI, and Prph2^KI/KI peripherin mutantmice to study how the mutation affected the integrity of the retina. Figure 4 shows representative OCT images from each experimental group and Figure 5 shows the results of the measurement of total retinal, ONL, and INL thicknesses. In Prph2^WT/WT mice, there were no differences in the retinal structure over time. Although a decrease in the total retina and the ONL thickness was present in both Prph2 mutant mice over time, changes in the ONL thickness were more prominent. The INL remained stable among the experimental groups for up to 12 months (Fig. 4).

The total retinal thickness was significantly thinner at 9 months in Prph2^WT/KI mice (214 ± 3 µm, P < 0.05) and at 3 months in Prph2^KI/KI (203 ± 5 µm, P < 0.01) compared to Prph2^WT/WT (3 months: 231 ± 3 µm, P < 0.0001; 9 months: 233 ± 1 µm, P < 0.0001) (Fig. 5A). While the alteration of the total retinal thickness in Prph2^WT/KI mice was smooth, the retina of Prph2^KI/KIanimals showed a marked decreased at 9 and 12 months of age compared to Prph2^WT/WT mice. Differences in ONL thickness started to be significant from 6 months of age in Prph2^WT/KI mice and from 3 months in Prph2^KI/KI. Thus, these changes in total retinal thickness were mainly caused by the reduction of the ONL, since no changes were found in the INL among experimental groups, except for an increase in this layer at 20 months in the Prph2^KI/KI mice (27 ± 1 µm, P < 0.0001) compared to Prph2^WT/WT mice (21.5 ± 0.5 µm) (Fig. 5C). Unlike Prph2^WT/WT mice, the ONL of Prph2^KI/KI and Prph2^WT/KI mice continued the degenerative process over time. ONL thickness diminished progressively and smoothly in the Prph2^WT/KIretina with time (from 53.4 ± 0.8 µm to 32 ± 1 µm). Specifically, the ONL of Prph2^KI/KI mice showed marked changes at 3 (38 ± 2 µm, P < 0.0001), 9 (22 ± 3 µm, P < 0.0001), 12 (10.5 ± 0.8 µm, P < 0.0001) and 20 (2.4 ± 0.8 µm, P < 0.0001) months compared to Prph2^WT/WTanimals.

Since this mutation in Prph2 affects the visual function and the structural integrity of the retina, specific markers were used to study morphological changes in photoreceptor cells associated with the mutation. As PRPH2 is a structural protein required for the organization of the disc membranes, we expected that OSs were unstructured and, herein, photoreceptor morphology altered. To check out this hypothesis, we performed immunolabelling in cryosections of the retina of Prph2^WT/WT, Prph2^WT/KI, and Prph2^KI/KImice at 9 months of age with anti-PRPH2 antibody, to visualize OS morphology and possible mislocalization of the protein; and anti-cone arrestin (CAR) antibody, that allowed us to visualize the entire morphology of cones; together with anti-rhodopsin (RHO) antibody, that immunolabels OS of rods. The immunolabeling with anti-PRPH2 and anti-RHO antibodies showed the well-structured OSs in the retina of Prph2^WT/WTmice (Fig. 6A, B, G, H, M, N). PRPH2 immunostaining surrounds the immunolabelling of anti-RHO, due to PRPH2 localization being restricted to the rim region of the discs (Fig. 6B), the specialized hairpin loop that circumscribed the two closely spaced membranes of each disc. In the retina of Prph2^WT/KI animals, OSs looked swollen (Fig. 6C, D, I, J, O, P). Nevertheless, in the retina of Prph2^KI/KIanimals, there were membranous aggregations of OSs that were completely disorganized (Fig 6E, F, K, L, Q, R). Also, RHO was mislocalized, which could be indicating a loss of compartmentalization of the disc membranes (Fig. 6F, L, R). The immunolabeling with anti-PRPH2 also confirmed that mutant PRPH2 was not mislocalized towards the inner segment (IS) or the cell body in the photoreceptor cells during the curse of neurodegeneration and the mutant protein remained confined in the OSs (Fig 6D, J, P; F, L, R). Cone morphology was affected in the retina of Prph2^WT/KIand Prph2^KI/KImice, as shown in CAR immunostaining (Fig. 7). In the Prph2^WT/KI retina, OSs of cones showed an unusual twisted appearance, losing the straight organization that is present in the OSs showed of the Prph2^WT/WTmouseretina (Fig. 7J). Pedicles also showed some morphological alteration in the Prph2^WT/KI retina, showing unusual telodendria. Cones in the retina of Prph2^KI/KI animals were shortened and their OSs practically missing in contrast to Prph2^WT/WT(Fig. 7E, F, K, L). ISs of cones were swollen in the retina of the Prph2^KI/KImodel. (Fig. 7E, F, K, L). Nuclei staining with TO-PRO-3 iodide confirmed the marked decline of ONL thickness in the retina of Prph2^KI/KI mice observed with OCT, revealing a decreased number of photoreceptor rows (Fig. 7).

To deeply study the morphological changes in the OSs of photoreceptor cells in the C57BL/6J-Prph2^em1Sal model and also to detect if there were any structural alterations in the photoreceptor cells before detectable changes in the ERG or optomotor test, we performed transmission electron microscopy (TEM) in the retina of Prph2^WT/WT and Prph2^KI/KIat 1 month of age. At this age, OSs are still conserved in the Prph2^KI/KI. Because PRPH2 is necessary for the formation of photoreceptor discs and retinal immunostaining revealed a considerable impairment in OS structure, we decided to evaluate the ultrastructure of the OS discs. The Prph2^KI/KImice displayed disorganized and shortened OSs and ISs compared with the Prph2^WT/WT mice (Fig. 8A, B). The normal parallel distribution of the OSs discs enclosure in a membrane in Prph2^WT/WT mice (Fig. 8C) switched to major structural abnormalities of the OSs in Prph2^KI/KImice (Fig. 8D, E). One of the most abnormal structures found in the OSs was a pattern of spirals or concentric circles that contained darker stained membranes (Fig. 8D, E, arrowheads). These structures were similar to those described in other peripherin mutations leading to the loss of visual function and photoreceptor degeneration found in this model (Lewis et al., 2020; Tebbe et al., 2020). Additionally, we observed unusual intracellular vacuoles in the RPE (Fig. 8B, asterisk), and an increase in the paracellular space in the interphotoreceptor matrix (Fig. 8B, arrows). Both anomalies were also described by Tebbe and collaborators (Tebbe et al., 2022).

Considering the significant reduction in ONL thickness and photoreceptor nuclei in Prph2 mutant mice, we proceeded to examine the synaptic connections in the retinas of both normal (Prph2^WT/WT) and dystrophic homozygous (Prph2^KI/KI) animals. Retinal sections were subjected to immunostaining using anti-calbindin antibody, which specifically labels horizontal cells (Fig. 9A-F). Double labeling was performed with antibodies against bassoon, which highlights synaptic ribbons of photoreceptors (Fig. 9A-D, G, H). Images revealed a striking decrease in synaptic contacts between photoreceptors and horizontal cells. This decline in synaptic connectivity indicates that the mutation in Prph2 not only affected the OSs of photoreceptors but also disrupts the interactions between photoreceptors and the second-order neural cells in the visual pathways. Consequently, this loss of synaptic connectivity in the OPL contributes to the overall deterioration of the visual system in Prph2 mutant mice.

To investigate the synaptic contacts between photoreceptor cells and ON rod bipolar cells in the OPL, we conducted immunostaining of retinal sections using antibodies against synaptophysin, a specific marker of presynaptic vesicles. Additionally, double immunostaining with PKCα was performed to visualize the contacts between the axon terminals of photoreceptors and the dendrites of bipolar cells. In the retina of Prph2^KI/KImice, a noticeable reduction in synaptophysin-positive dots (Fig. 10B, D, H) and a loss of bipolar cell dendrites labeled with PKCα (Fig. 10B, D, E) were observed compared to normal mouse retinas (Fig. 10A, C, E, G). Moreover, upon closer examination at high magnification of the double immunolabelling, fewer synaptophysin immunoreactive spots paired with bipolar cell dendritic tips were observed in the peripherin mice compared to the control (Fig.10C, D).

We also investigated the inflammatory process and the involvement of activated microglial cells in the C57BL/6J-Prph2^em1Sal model. For this purpose, we assessed the activity of Iba1⁺ cell populations in retinal sections (Fig. 11). Immunofluorescence images revealed an elevated presence of Iba1⁺ cells (shown in green) in the retina of Prph2^KI/KI mice (Fig. 11B, D), compared to Prph2^WT/WT animals (Fig. 11A, C). Furthermore, these cells exhibited migration towards the photoreceptor layers, including the ONL and OSs, specifically in the retinas of Prph2^KI/KI mice. Conversely, in Prph2^WT/WT animals, Iba1⁺ cells were not detected above the OPL. The Prph2^KI/KI mice retinas exhibited an increased number of Iba1+ cells with an ameboid shape, indicating microglia activation in this particular model. To analyze the reactive gliosis of macroglial cells (astrocytes and Müller cells), we observed GFAP immunoreactivity. The retina of Prph2^KI/KI mice demonstrated an elevated GFAP immunoreactivity (Fig. 11B, F), that was higher than in Prph2^WT/WT animals (Fig. 11A, E). Interestingly, the retinas of Prph2^KI/KI mice exhibited GFAP immunoreactivity not only at the inner margin but also throughout the entire length of Müller cells.

To analyse the inflammation- and immune-related cell populations in the different stages of the degenerative process, we examined the expression of several surface markers by flow cytometry in mice retinas at different ages. After excluding doublets and cellular debris (Fig. 12A) CD45 or CD11b immunopositive populations were selected (Fig. 12A, B). Among the CD45 immunoreactive cells (CD45^all), two different populations were observed, one with medium reactivity (CD45^med) and another one highly immunopositive (CD45^high) (Figure 12B, C). CD45^high population has previously described as corresponding to an activated phenotype of microglia and also to macrophages, consistent with the invasion of peripheral cells (Jurga et al., 2020; Martin et al., 2017; Rangaraju et al., 2018; Ronning et al., 2019). In Prph2^KI/KI mouse retinas, the percentage of population with this phenotype was increased in advanced ages (9 m and 12 m), and reached significance at 12 months of age (2 ± 1% in Prph2^WT/W , 3.2 ± 0.7% and 11.00 ± 5% in Prph2^WT/KI and Prph2^KI/KI respectively). In Prph2^WT/KI mouse retinas, the population was also inscreased with respect to the wild type strain although no significance was reached (Fig. 12D). On the other side, CD11b⁺ cells were analyzed for their immunoreactivity against CD11c and MHC-class II (Fig. 12E) or against CD169 (Fig. 12F) antigens. A significant increase of activated immune-related cells (CD11b⁺CD11c^highMHC class II⁺) was observed in Prph2^KI/KI mice compared to control mice at all conditions tested (4 ± 2% to 1.0 ± 0.6% in age 3 months, 3 ± 2% to 1.0 ± 0.6% in age 6 months, 5 ± 2% to 1.00 ± 1% in age 9 months, 4.4 ± 0.6% to 1.0 ± 0.6% in age 12 months) (Figure 12E, G). In Prph2^WT/KI mouse retinas, CD11b⁺CD11c⁺MHC class II⁺ population was increased, although no statistical significance was reached. CD169 immunopositive population was significantly increased in Prph2^KI/KI mice at 12 months of age (7.53 ± 1.80%) with respect to Prph2^WT/WT and Prph2^WT/KI (4 ± 2% and 6 ± 1% respectively) (Fi. 12F, H), a marker of pathogenic phagocytes (Hughes et al., 2003; Linnartz-Gerlach et al., 2014).

CACD is an inherited dystrophy that affects photoreceptors of the macula but also causes the atrophy of choroidal vessels and RPE. Currently, there is no treatment for this disease, and the high heterogeneity of the phenotypes observed in patients, even in those who share the same mutation, makes it difficult not only the diagnosis but also to establish the characteristics of the different stages of the disease (Albertos-Arranz et al., 2021). The existence in the northern region of Spain of a family with 56 living patients with an Arg195Leu mutation in the PRPH2 gene and with diagnosed CACD, suggested that this mutation could serve as a good candidate for generating an animal model for this disease which would serve us to effectively compare the evolution of the disease in the animal model and the human patients. Hence, the generation of the newC57BL/6J-Prph2^em1Sal mouse allowed us to better understand the onset and progression of retinal degeneration in CACD. Moreover, having an animal model that reproduces the phenotype of CACD patients will permit us to assay suitable neuroprotective therapies that could hopefully be extrapolated to humans in the future. Other animals with point mutations in Prph2 were generated by other researchers (Chakraborty et al., 2020; Ding et al., 2004; Stuck et al., 2014; Zulliger et al., 2018) but, to our knowledge, this is the first animal model carrying the mutation p.Arg196Leu, present in three large families, the above mentioned Spanish one, and also in a Japanese and a German family (Keilhauer et al., 2006b; Yanagihashi et al., 2003). Even more, this is the first study in which the onset and progression of a PRPH2-associated retinal degeneration have been characterized over time by different techniques, and also this is the first time that synaptic remodeling of the OPL is described in a CACD model. We have characterized the decay of the retinal responsiveness and the VA of this model in both heterozygosis and homozygosis. We also analyzed the alterations in the retinal structure of this model in vivo with OCT. As well, we studied the morphological alterations in the photoreceptor cells and their OSs, since PRPH2 is a key protein for the formation and organization of disc membranes. Additionally, we studied the loss of synaptic contacts with rod bipolar and horizontal cell dendrites using proper cell markers. Finally, we have assessed the inflammatory state of the retina by studying Iba1⁺ cells and the gliosis in Müller cells. We have found a change in the phenotype of populations related with immune and inflammatory responses. In Prph2^KI/KI mouse retinas at 12 months of age, an increase in CD45^high, CD11b and CD169 immunopositive populations was observed, showing signs of microglial inflammation and/or recruitment of monocytes and macrophages (Hughes et al., 2003; Linnartz-Gerlach et al., 2014). We also found an increase in CD11b⁺CD11c^highMHC class II⁺ population in Prph2^KI/KI mouse retinas at all ages tested. The expression of both, integrin CD11c and MHC class II are increased in degenerating retinas (Chidlow et al., 2016; Jiang et al., 1999; Karlen et al., 2018; Lehmann et al., 2010; Lipski et al., 2017; Ronning et al., 2019; Tang et al., 2017). The fact that this population is present even at young ages indicates that signals of inflammation are already present even when no signs of the disease are present, pointing to the potential benefits of an early onset of the therapy, and so encourages to deepen in the study of early molecular changes that could be targeted therapeutically.

PRPH2 was first described as the product of the retinal degeneration slow (rds) gene. This gene is exclusively expressed in photoreceptor cells, and mice homozygous for rds show abnormal photoreceptor morphology and slow degeneration (Travis et al., 1991). PRPH2 controls the precision of disc morphology, as it is key for the formation and organization of the discs (Lewis et al., 2021). PRPH2 forms non-covalent homodimers and heterodimers, by interacting with rod outer segment membrane protein 1 (ROM-1), in a head-to-head assembly. These dimers can be linked to form higher-order oligomers. Three dimers make up the scaffold of the rim region that circumscribes the disc membranes and, herein, orientates and stabilizes the OS discs (Conley et al., 2019; El Mazouni and Gros, 2022). How these complexes assemble in the presence of mutated PRPH2 has a deep impact on the disease onset and its phenotype (Tebbe et al., 2020), which remarks the importance of deep knowledge of what changes in the tertiary and quaternary conformation it implies. According to other authors, incorrect oligomerization leads to the formation of too-long discs and membranous accumulations that were denominated as “whorls” (Lewis et al., 2020; Lobanova et al., 2019; Tebbe et al., 2020). In this new model, we observed a marked disruption of photoreceptor OS morphology that affected both rods and cones, which is consistent with the decrease of both scotopic and photopic ERG responses in our mice. TEM images revealed the “whorls” previously described by other authors, which consist of an accumulation of membranes in a concentric circular pattern (Conley et al., 2019; Lewis et al., 2021; Lewis et al., 2020; Nour et al., 2004; Tebbe et al., 2020).

The p.Arg195Leu point mutation of PRPH2 is located in helix D, in the peripheral area of the interface crevice, which is implied in the assembly of the PRPPH2-PRPH2/ROM1 dimers. This mutation implies the change of a positively charged amino acid by a hydrophobic residue, which could be provoking incorrect interactions of the side chains with the helix, like alterations of the intrachain hydrogen bonding, that could cause changes on the dimer assembly at the interface crevice provoking an imperfect arrange of the rim scaffold. It has been reported that some point mutations of PRPH2 provoke the mislocalization of these proteins into the IS of photoreceptor cells, presumably due to retention in the endoplasmic reticulum (Loewen et al., 2003; Salinas et al., 2013). However, we did not observe PRPH2 accumulation in photoreceptor ISs or cell bodies. Herein, we hypothesized that the p.Arg195Leu mutation does not block PRPH2 transportation to the OSs of photoreceptor cells and its oligomerization. Thereby, disc membranes can be formed, although abnormally, as observed by immunolabeling of Prph2^WT/KI retinas. With this, we agree with previous researchers that different combinations of mutated and non-mutated PRPH2 oligomers can be formed in heterozygous models and patients (Böhm et al., 2017). Indeed, the protease cleavage assay performed by Böhm and collaborators showed that p.Arg195Leu mutation does not disturb the overall folding of PRPH2 to a big degree (Böhm et al., 2017). Other models with point mutations in the region that participates in the dimerization of PRPH2 also showed the ability to initiate disc formation, but their morphology was aberrant (Lewis et al., 2020). In Prph2^WT/KI mice, there may be a mix of dimers formed by non-mutated and mutated proteins. Meanwhile, in Prph2^KI/KI animals only aberrant dimers would exist. Herein, this could explain the marked alteration of the morphological and the visual function of the Prph2^KI/KI model compared to the Prph2^WT/KI. Nevertheless, further experiments are needed to confirm this hypothesis. In any case, the morphological alteration in the disc membranes could trigger diverse processes of oxidative stress and inflammation, which can worsen the disease and lead to neurodegeneration with age (Cuenca et al., 2014; Martínez-Gil et al., 2022; Pinilla et al., 2022).

In peripherin diseases, it is becoming increasingly evident that a single mutation can result in a range of different phenotypes and, conversely, it was observed that different mutations can lead to the manifestation of the same phenotype. This results in a strong heterogeneity of phenotypes, even within a family that shares the same mutation, with clinical manifestations ranging from retinitis pigmentosa to various forms of macular dystrophy (Antonelli et al., 2022; Bianco et al., 2023). It was described that the presence of ROM1 variants (Strayve et al., 2020), the existence of different PRPH2 haplotypes (Shankar et al., 2016), and/or the interactions with other genes/proteins (Xiao et al., 2022) can modulate the patient phenotype. This intriguing pattern highlights the intricate relationship between specific genetic alterations and the resulting phenotypic expression. Consequently, a comprehensive investigation of these genotype-phenotype associations is imperative for gaining a deeper understanding of the underlying molecular mechanisms of the pathologies associated with PRPH2. Such research holds significant promise in unraveling the complex nature of these diseases and paving the way for more targeted and effective therapeutic interventions.

In a previous study of our group characterizing tne phenotype of patients with the p.Arg195Leu mutation in Prph2, we demonstrated that, even though different phenotypes were observed, all the patients showed a reduction of the ONL at the foveola and a disruption of the third outer retinal band, related to the outer segment tips (Albertos-Arranz et al., 2021). These results agree with those obtained in our animal model, where we also observed the decrease of the ONL and the alteration of the OSs in both rods and cones. Remarkably, the temporal progression of degeneration observed in human patients correlates with the time course of degeneration observed in mice, aligning the years of degeneration in a meaningful manner (6-9 mouse months are equivalent to 30-40 human years (Flurkey et al., 2007) when clear symptoms of the disease appear). Having an animal model that recapitulates the retinal degenerative process of the mutation can help to elucidate the cell death pathways involved in answering the question of how structural alterations of the OSs can activate cell death pathways, such as apoptosis, inflammation, or oxidative stress. This information is crucial for the development of targeted and effective therapies aimed at preventing or mitigating retinal degeneration.

In summary, since both functional and structural signs observed in patients with the PRPH2 mutation p.Arg195Leu were also found in the C57BL/6J-Prph2^em1Sal mice, this animal model becomes the best approach to understanding the degenerative process of this retinal dystrophy and will permit us in the future to analyze the efficacy of different

In our investigation, the presence of the p.Arg195Leu point mutation in PRPH2 resulted in the atrophy of outer segment disc membranes. Our study establishes a strong correlation between functional and morphological changes in the retinas of mutant mice. We observed a gradual decline in retinal thickness, with more pronounced and earlier alterations evident in homozygous mutants. Furthermore, novel findings from our study reveal impaired synaptic connectivity within the outer plexiform layer, along with the occurrence of Müller gliosis and microglial activation. These remarkable outcomes closely resemble the observed retinal degeneration pattern in human patients with CACD. As a result, the newly developed Prph2WT/KI and Prph2KI/KI murine models not only enhance our understanding of the mechanisms driving disease onset and progression but also offer reliable platforms for evaluating the effectiveness of innovative therapeutic strategies.

CACD central areolar choroidal dystrophy, ERG electroretinography, INL inner nuclear layer, IS inner segment, OCT optical coherence tomography, ONL outer nuclear layer, OS outer segment, PB phosphate buffer, RPE retinal pigment epithelium, ssODN single-strand DNA oligonucleotide, VA visual acuity.

Authors' contributions:

The hypothesis and the study design were conceived by NC, PL, VM, MS-M. Methodology was performed by MJR-P, XS-S, OK, IO-L, VM-F. CRISPR methodology was performed by MS-M, LM. Image analysis and statistical analysis was done by HA-A, CS-C, XS-S, NM-G, LV-G, OK, IO-L. The manuscript was written by MJR-P, MS-M, PL, VM-F, NC. All authors read and approved the final manuscript.

Competing interests:

The authors declare no competing interests.

SUPPORT

The study was supported by grants funded by the Spanish Ministry of Science and Innovation [FEDER-PID2019-106230RB-I00, 2019]; Spanish Ministry of Universities [FPU16/04114, 2016, FPU18/02964, 2018 and Union Europea-Next Generation MARSALAS21-35], Generalitat Valenciana [IDIFEDER/2017/064, 2017, ACIF/2020/203, 2020, PROMETEO/2021/024, 2021], Es Retina Asturias [1-22I], Begisare-Araba [1-22I], and FARPE-FUNDALUCE [FUNDALUCE18-01, 2019].

ACKNOWLEDGMENTS

The authors would like to thank the association of patients Retina Asturias and Retina Cantabria. We especially thank Jesús Delgado, Andrés Mayor, and all the patients and their relatives that promoted the initiative of this project. Also, we thank Marina Pastor Mas and Miguel Angel Compañy for their technical support and Ignacio García-Tuñon for his assistance in CRISPR technology.

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