Animals deficient in C2Orf71, an autosomal recessive retinitis pigmentosa-associated locus, develop severe early-onset retinal degeneration

Abstract

Genetic mapping was recently used to identify the underlying cause for a previously uncharacterized cohort of autosomal recessive retinitis pigmentosa cases. Genetic mapping of affected individuals resulted in the identification of an uncharacterized gene, C2Orf71, as the causative locus. However, initial homology searches failed to reveal similarities to any previously characterized protein or domain. To address this issue, we characterized the mouse homolog, BC027072. Immunohistochemistry with a custom polyclonal antibody showed staining localized to the inner segments (IS) of photoreceptor cells, as well as the outer segments (OS) of cone cells. A knockout mouse line (BC(-/-)) was generated and demonstrated that loss of this gene results in a severe, early-onset retinal degeneration. Histology and electron microscopy (EM) revealed disorganized OS as early as 3 weeks with complete loss by 24 weeks of age. EM micrographs displayed packets of cellular material containing OS discs or IS organelles in the OS region and abnormal retinal pigmented epithelium cells. Analyses of retinoids and rhodopsin levels showed <20% in BC(-/-) versus wild-type mice early in development. Electroretinograms demonstrated that affected mice were virtually non-responsive to light by 8 weeks of age. Lastly, RNAseq analysis of ocular gene expression in BC(-/-) mice revealed clues to the causes of the progressive retinal degenerations. Although its function remains unknown, this protein appears essential for normal OS development/maintenance and vision in humans and mice. RNAseq data are available in the GEO database under accession: GSE63810.

Figure 1.

Sequence alignment of C2Orf71 and its homologs. (A) Sequences from human, cow, mouse and chicken were aligned with ClustalW alignment software. Asterisks indicate amino acids conserved in all four species. Gray-shaded areas represent domains that are potentially homologous between species and were chosen solely based on high levels of sequence identity between all four species. (B) Phylogenetic tree including C2Orf71 sequences from 26 species that include mammals, birds and fish.

Figure 2.

Gene expression analysis by qRT-PCR. Gene-specific primers/probes were used to probe gene expression in various mouse organs (A) or eye tissues (B) with Taqman quantitative real-time PCR. Results demonstrate a nearly exclusive expression in the neural retina. The 18S rRNA band was used as an internal control.

Figure 3.

Immunoblot and IHC localization of BC027072 in mouse eye with a custom pAb. (A) Tissues were dissected and proteins were extracted with RIPA buffer. Immunoblot analysis confirmed the qRT-PCR analysis and demonstrated that expression is confined to the neural retina within the eye. rBC027072 is a truncated recombinant protein (amino acids 1–360) expressed in E. coli which was not used for immunization. (B) Eyes from 3-week-old WT and BC^−/− mice were fixed in paraformaldehyde and embedded in OCT compound. Cryosections (12 µm) were probed with a BC027072 pAb and PNA (for cone cell staining). Staining of BC027072 is consistent with localization to the IS of both cone and rod photoreceptors as well as the OS of cone cells.

Figure 4.

Targeting strategy and genotypic analysis for BC^−/− mice. (A) Diagram of the knockout strategy. Blue and red regions correspond to short and long homology arms, respectively. One-sided arrows represent location of primers used for genotypic analysis. Nmr, neomycin resistance gene. (B) Genotypic analysis of knockout using PCR. The 6 kb band corresponds to a fragment including a WT copy of exon 1 and the 2.5 kb band represents a fragment that includes the neomycin resistance gene. (C) qRT-PCR was used to probe total RNA isolated from eyes of both WT and BC^−/− mice. The 18S rRNA band was used as an internal control.

Figure 5.

Analysis of mouse retinal integrity by OCT and histology. (A) SD-OCT imaging of WT and BC^−/− retinas from mice at indicated ages shows age-related degeneration of the outer retina. (B) Light microscopic images of plastic-embedded retinas from WT and BC^−/− mice at indicated ages demonstrate the outer retinal degeneration imaged by OCT represents a loss of photoreceptor cells as noted by the diminished ONL. (C) Line graph mapping of the time-dependent degeneration of ONL thickness analyzed by SD-OCT in BC^−/− versus WT and BC^+/− mice. (D) Graph representing ONL thickness from the superior to inferior retina in BC^−/− and WT mice at indicated ages analyzed by histology of plastic-embedded eyes.

Figure 6.

IHC of retinas from BC^−/− mice. (A) Cryosections from 4-, 8- and 24-week-old animals were probed with anti-rhodopsin antibody for rod cells (1D4; red) and DAPI for nuclei (blue). Staining shows a highly disorganized OS region as well as rhodopsin staining in the inner nuclear layer of BC^−/− mice. (B) Cryosections from 8-week-old WT and BC^−/− mice were probed with anti-RPE65 (red), for RPE cells, or anti-OPN1SW (red), for blue cones, and DAPI for nuclei (blue). Staining is similar for RPE65 but aberrant for OPN1SW, consistent with a defect in both rods and cones. (C) IHC with anti-Iba-1 Ab (red) and DAPI (blue) is shown. Infiltration by activated microglia can be seen as early as 3 weeks in the BC^−/− mice, with increasing numbers as the animal ages.

Figure 7.

Retinoid and rhodopsin analysis of BC^−/− mice. (A) Total retinoids were extracted from 3-week-old dark-adapted animals and analyzed by normal phase HPLC. Quantification of areas under the peak of 11-cis-retinal based on our standard curve determined WT and BC^−/−levels to be 277.8 ± 11 and 63.8 ± 0.9 pmol/eye, respectively. (B) Isolated rhodopsin was analyzed spectrophotometrically to determine the amount of bound chromophore, results showed 8.6 µg of rhodopsin/retina for WT while only 1.4 µg of rhodopsin/retina in BC^−/−. Both rhodopsin and retinoid analyses showed that BC^−/− mice contained <20% of the WT amounts.

Figure 8.

EM analysis of BC^−/− mouse retinas. Eyes from 3-week-old WT (A) and BC^−/− (B) mice were embedded in plastic and 70 µm sections were analyzed by EM. Micrographs of BC^−/− retinal cross sections show a highly disorganized OS region which is significantly shorter than seen in WT retinas. (C) BC^−/− mice exhibited packets of the IS material immediately adjacent to the RPE. (D) BC^−/− photoreceptor cells had CC which were intact but lacked a connection to the OS (asterisks). BC^−/− RPE cells (F) showed accumulation of massive amounts of undigested material when compared with WT (E) RPE cells. Necrotic RPE cells can be periodically found in 8-week-old BC^−/− (H) which are dramatically different than WT RPE cells (G) of similar age.

Figure 9.

ERG analysis of BC^−/− mice. (A) Representative full-field scotopic and photopic ERG responses of anesthetized WT and BC^−/− mice at 4 and 8 weeks of age show reduced scotopic and photopic responses in BC^−/−when compared with WT animals. Whereas WT mice showed a robust response to increasing intensities of light, BC^−/− mice evidenced a nearly flat response by 8 weeks of age. (B) Amplitudes of both rod photoreceptor cell-evoked scotopic (left) a- and b-waves and cone photoreceptor cell-dependent photopic (right) a- and b-waves were reduced in 8-week-old BC^−/− mice (n > 4).