The effect of peripherin/rds haploinsufficiency on rod and cone photoreceptors

Abstract

Haploinsufficiency because of a null mutation in the gene encoding peripherin/rds has been thought to be the primary defect associated with the photoreceptor degeneration seen in the retinal degeneration slow (rds) mouse. We have compared the effects of this haploinsufficiency on rod and cone photoreceptors by measuring the levels of rod- and cone-specific gene expression, by determining the relative rates of rod and cone degeneration, and by electroretinography. These analyses were performed at ages before and after the onset of degeneration of the photoreceptor cells. The data were consistent in demonstrating that measures for cone photoreceptors are relatively spared in comparison to comparable measures for rod photoreceptors. Blue cones were retained in higher number than red/green cones for the first 3 months of the degeneration. Our results indicate that the haploinsufficiency present in rds/+ mice has a greater impact on the rod than on the cone photoreceptor, a finding that likely reflects the tight regulation of peripherin/rds and the need for two functional alleles to assemble the structure of the rod outer segment and/or differences between the ultrastructure of the rod and cone outer segments.

Fig. 1.

Northern blot analysis of rd/rdmice at the ages indicated. A, Blot probed for peripherin/rds transcripts. Note that a small signal is retained at P70, when all rods were lost and some cones are present. At 6–8 months of age, no peripherin/rds transcripts were detected because of the complete loss of both rods and cones. B, Blot probed for rod opsin transcripts. Note that the signal for the opsin transcripts decreases rapidly with age and is not discernible at P70 and older.

Fig. 2.

Light micrographs showing changes in the central parts of the retina of normal and rds/+ mice.A, Retinal cross section taken from a 2-month-old normal mouse. B–D, Retinal cross sections taken from 1-, 2-, and 3-month-old rds/+ mice. OS, Outer segments; IS, inner segments; ONL, outer nuclear layer. Scale bar, 20 μm. E, Counts of total photoreceptor nuclei. F, Thickness of outer segment layer. Each error bar represents the mean ± 1 SD for at least three measurements.

Fig. 3.

Cryostat sections of retina from normal andrds/+ mice stained for rod opsin (1D4). Shown are retinal cross sections from a normal 2-month-old mouse (A) and rds/+ mice at 1 month (B) and 3 months (C).D–F, Nomarski images of retinal sections shown inA–C, respectively. OS, Outer segments;IS, inner segments; ONL, outer nuclear layer. Scale bar, 20 μm.

Fig. 4.

Cryostat sections of retina from normal andrds/+ mice stained for cone opsins. Shown are retinal cross sections immunostained for blue cone opsin from a normal 2-month-old mouse (A) and rds/+mice at 1 month (B) and 3 months (C). D–F, Nomarski images of retinal sections shown in A–C, respectively.OS, Outer segments; IS, inner segments;ONL, outer nuclear layer. Scale bar, 20 μm.G, Counts of blue cone immunoreactive cells measured in the inferior central region of the retina. H, Counts of red/green cone immunoreactive cells measured in the superior central region of the retina. Each error bar represents the mean ± 1 SD for at least three measurements.

Fig. 5.

Relative retention of rod and cone photoreceptors in rds/+ mice. Each axis is expressed as a percentage of normal. The two cone subtypes are represented by different symbols (open circles, blue; filled circles, red/green). The solid diagonal linerepresents an equivalent change from normal. The areaabove the line indicates a relative preservation of cones; the area below theline indicates a relative preservation of rods.

Fig. 6.

Electroretinography. A, Representative ERGs obtained from a normal mouse (top trace) and from rds/+ heterozygotes at the ages indicated. Responses are to a high-intensity strobe flash (0.85 log cd sec/m²) presented to the dark-adapted eye (left) or superimposed on a 1.3 log cd/m² adapting field (right).B, Amplitude of cone ERG b-wave plotted as a function of rod ERG a-wave. Data are expressed relative to the normal mean. Eachsymbol represents an individual mouse. The solid diagonal line represents an equivalent decrease in the two response measures; points falling abovethis line indicate a relative sparing of cone responses; a relative sparing of rod responses would result inpoints falling below theline. C, Rod ERG a-waves obtained from a 1-month-old rds/+ mouse in response to high-intensity flash stimuli. The solid lines represent the response actually recorded, whereas dashed lines represent the least-squares fit of Equation 1. D, Value of amplification parameter S plotted as a function of stimulus intensity. Dashed lines indicate the range of normal, whereas the data points indicate the average (± 1 SD) value obtained from four rds/+ mice.

Fig. 7.

Northern blot analysis. A,Top panels show representative blots for peripherin/rds, rod opsin, and blue cone opsin, along with an actin control.Bottom panels show the corresponding average (± 1 SD) levels obtained for three different blots. For peripherin/rds, note the additional high-molecular-weight transcripts in therds/+ lanes; only the normal-sized transcripts were measured. B, Comparison of blue cone opsin signal plotted as a function of the corresponding rod opsin signal at each age analyzed. Data are expressed relative to normal. Each point represents the average (± 2 SD) value obtained from three assays, each using retinas obtained from four rds/+ mice at 1, 2, and 3 months of age. The solid diagonal line represents an equivalent decrease in the two measures; points fallingabove this line indicate a relative sparing of cone transcripts.