Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene

Abstract

We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Figure 1

Fundus appearance of various mouse strains at two months of age: rd1 (A), rd10 (B), nmf137 (C), and C57BL/6J wild type (D). The retinal degeneration in the rd10 and nmf137 strains was easily distinguished from wild type and rd1 retinal appearance at two months of age by fundoscopy.

Figure 2

Nuclei counts in the outer nuclear layer (ONL) of rd10 mouse retinas as a function of time. Plastic or paraffin sections of eyes from mice ranging in age from one week to three months were studied using light microscopy. Counts from rd10 sections are significantly lower than those from C57/Bl6 wild type (“C57”) at all stages expect for postnatal day15–16 samples (p<0.001). Data are means±SEM; numbers above bars are sampling sizes. Statistical analysis was by ANOVA with post-hoc Student-Newman-Keuls multiple comparisons testing.

Figure 3

Histology of rd10, rd1, and nmf137 mouse retina at 24 days of age showed comparative degrees of retinal degeneration. By this stage, rd10 outer nuclear layer (ONL) of cyclic light reared mice was about four nuclei thick, whereas nmf137 was only one nuclei thick and rd1 had no photoreceptor nuclei. Conversely, ONL of dark-reared rd10 mice showed no thinning at 24 days of age and had substantial inner segments (IS) and outer segments (OS). Retinal ganglion cell layer (GCL) and inner nuclear layer (INL) did not vary with strain or lighting regimen.

Figure 4

Electroretinograms of C57BL/6J and rd10 mice at P18 and P30 dark-adapted (A) and light-adapted (B) conditions. Dark-adapted responses were recorded after overnight dark-adaptation, while light-adapted responses were recorded with a background light of 1.46 log cd/m² following a 10 minutes exposure to the same intensity.

Figure 5

ERG a- and b-wave amplitude of rd10 mice declined with age. A) Rod dominated dark-adapted responses. B) Cone-isolated light-adapted responses. Error bars represent the standard error of the mean.

Figure 6

Apoptosis in P18 mouse retinas. Paraffin sections of P18 C57 wild type (A, C) and rd10 (B, D) mouse eyes were subjected to fluorescent TUNEL (A, B) or fluorescent immunochemistry using an antibody specific to activated caspase-3 (C, D). Sections were counterstained with propidium iodide. TUNEL-positive and activated caspase-3-positive cells fluoresced yellow-green. Wild type sections showed almost no TUNEL signal (A), whereas rd10 sections had abundant TUNEL signal in the outer nuclear layer and occasionally inner segments of photoreceptor cells, but not in other layers of the retina (B). Wild type sections showed almost no activated caspase-3 immunofluorescence (C), whereas rd10 sections had abundant signal in the inner segments of photoreceptor cells, but not in other layers of the retina (D). Control stainings lacking primary antibody had no signal (data not shown).

Figure 7

β-PDE immunoreactivity across development. Protein extracts from retinas removed from C57BL/6 (wild type; “wt”), rd1, and rd10 mice at postnatal days (P) 9 through 20 were analyzed by western immunoblotting using an antibody specific for the β subunit of rod cGMP PDE. Additionally, protein extract from retinas and whole brain of a P60 C57BL/6 were probed. Top Panel: Representative autoradiographs of western immunoblots. A band of approximately 86 kDa was present in the wt and rd10 retina protein extracts, but not in rd1 retina or wt brain extracts. Source for protein extracts is indicated above the lanes. “Std” is a “Magic Mark™” molecular weight standard (Invitrogen). “Peptide” is the synthetic peptide mix against which the primary antibody was raised (see text for details of this). As indicated by vertical lines, blots were taken from separate experiments and are representative. Middle Panel: Quantification of P9–20 data from western blots. Autoradiographs were scanned. Net intensities of bands were determined by subtracting background pixel densities from pixel densities of bands. Resulting net intensities were averaged for 3–4 mice per stage per strain. Bottom Panel: Immunohistochemistry shows specificity of β-PDE primary antibody. Cryosections of eyes from P22 rd1 (A), wt (B), and rd10 (D) mice were probed with the β-PDE primary antibody and FITC-conjugated secondary antibody. β-PDE antibody labeled the inner and outer segments of wt (B) and rd10 (D) photoreceptors, but no signal was apparent in rd1 sections (A) or in wt sections incubated with secondary antibody but not primary antibody (C).

Figure 7

β-PDE immunoreactivity across development. Protein extracts from retinas removed from C57BL/6 (wild type; “wt”), rd1, and rd10 mice at postnatal days (P) 9 through 20 were analyzed by western immunoblotting using an antibody specific for the β subunit of rod cGMP PDE. Additionally, protein extract from retinas and whole brain of a P60 C57BL/6 were probed. Top Panel: Representative autoradiographs of western immunoblots. A band of approximately 86 kDa was present in the wt and rd10 retina protein extracts, but not in rd1 retina or wt brain extracts. Source for protein extracts is indicated above the lanes. “Std” is a “Magic Mark™” molecular weight standard (Invitrogen). “Peptide” is the synthetic peptide mix against which the primary antibody was raised (see text for details of this). As indicated by vertical lines, blots were taken from separate experiments and are representative. Middle Panel: Quantification of P9–20 data from western blots. Autoradiographs were scanned. Net intensities of bands were determined by subtracting background pixel densities from pixel densities of bands. Resulting net intensities were averaged for 3–4 mice per stage per strain. Bottom Panel: Immunohistochemistry shows specificity of β-PDE primary antibody. Cryosections of eyes from P22 rd1 (A), wt (B), and rd10 (D) mice were probed with the β-PDE primary antibody and FITC-conjugated secondary antibody. β-PDE antibody labeled the inner and outer segments of wt (B) and rd10 (D) photoreceptors, but no signal was apparent in rd1 sections (A) or in wt sections incubated with secondary antibody but not primary antibody (C).

Figure 7

β-PDE immunoreactivity across development. Protein extracts from retinas removed from C57BL/6 (wild type; “wt”), rd1, and rd10 mice at postnatal days (P) 9 through 20 were analyzed by western immunoblotting using an antibody specific for the β subunit of rod cGMP PDE. Additionally, protein extract from retinas and whole brain of a P60 C57BL/6 were probed. Top Panel: Representative autoradiographs of western immunoblots. A band of approximately 86 kDa was present in the wt and rd10 retina protein extracts, but not in rd1 retina or wt brain extracts. Source for protein extracts is indicated above the lanes. “Std” is a “Magic Mark™” molecular weight standard (Invitrogen). “Peptide” is the synthetic peptide mix against which the primary antibody was raised (see text for details of this). As indicated by vertical lines, blots were taken from separate experiments and are representative. Middle Panel: Quantification of P9–20 data from western blots. Autoradiographs were scanned. Net intensities of bands were determined by subtracting background pixel densities from pixel densities of bands. Resulting net intensities were averaged for 3–4 mice per stage per strain. Bottom Panel: Immunohistochemistry shows specificity of β-PDE primary antibody. Cryosections of eyes from P22 rd1 (A), wt (B), and rd10 (D) mice were probed with the β-PDE primary antibody and FITC-conjugated secondary antibody. β-PDE antibody labeled the inner and outer segments of wt (B) and rd10 (D) photoreceptors, but no signal was apparent in rd1 sections (A) or in wt sections incubated with secondary antibody but not primary antibody (C).

Figure 8

A. 96 mice from a backcross between rd10/rd10 and CAST/Ei were phenotyped and genotyped. Linkage to several markers on mouse Chr 5 was observed. The columns of squares represent haplotypes (filled boxes, rd10/rd10 allele; open boxes, CAST/Ei allele). The number of chromosomes with each haplotype is indicated below each column. B. Genetic map of Chr 5 in the rd10 region showing the closest markers and the region of human homology. Recombination estimates (± standard error) and order for the closest markers were D5Mit91 − 1.04±1.04 − rd10, D5Mit157 − 1.04±1.04 −D5Mit25.

Figure 9

The nucleotide sequences around the single base substitution at position 1678 (C to T) in exon 13 are shown for the wild type (WT) allele and the rd10 allele of Pde6b gene (panel A) and at position 1976 (T to C) in exon 16 are shown for the wild type allele and the nmf137 allele of Pde6b gene (panel B). The novel mutations changed codon 560 CGC to TGC (amino acid change: Arg560Cys) in the Pde6b gene in rd10 mice (panel A) and codon 659 CTC to CCC (amino acid change: Leu659Pro) in the Pde6b gene in nmf137 mice (panel B). Digestion of the PCR amplified products with CfoI from DNA of wild type C57BL/6J (lane 2), homozygous rd10/rd10 (lane 3) and heterozygous CXB-1xB6-rd10/+ F1 (lane 4) revealed the predicted RFLP pattern (panel C). Lane 1 is a 100 base pair (bp) DNA size marker. Numbers that flank the gel image are predicted fragment sizes in base pairs (bp).