Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin

Abstract

Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

Figure 1.

The light-dependent distribution of transducin α-subunit (Gα_t1) in WT mouse retina. In normal mouse retina, Gα_t1 distributes in the ROS layer in the dark and moves from the ROS to the IS, ONL, and OPL after light exposure (60 sc.cd/m²).

Figure 2.

Light-intensity-dependent transducin translocation in WT and Rs1–KO mice at P21 and P60. Animals were either dark adapted or exposed to 1 h of light of different intensities (indicated on the right). Comparison of Gα_t1 distribution in WT and Rs1–KO mice retinas shows that, in P21 and P60 WT retinas, Gα_t1 translocates from ROS and distributes into OPL at much lower light intensities compared with Rs1–KO retinas. Immunofluorescence of Gα_t1 in P21 Rs1–KO retinas shows persistent staining only in the ROS. Only exposure to very bright light (180–300 sc.cd/m²) caused Gα_t1 distribution into the OPL. The inset shows the light intensities at which movement of Gα_t1 out of the ROS and into the OPL was detected in P21 and P60 Rs1–KO and WT mice. All slides were visualized using the same microscope settings. Scale bar, 20 μm.

Figure 3.

Quantification of transducin translocation by measurement of Gα_t1 staining intensity. A, Plot profiles of stain intensity in retinal sections from the ROS tips to the INL in confocal images. Values are the DAPI (blue) and Gα_t1 (red) stain intensity on a scale of 0–255 averaged over at least 100 μm of retinal length. DAPI and Gα_t1 channels were measured separately, and the merged image is shown below the graph. B, Examples of plot profiles from WT and Rs1–KO mice replotted relative to the maximum ROS intensity in each section for comparison of OPL peak values. The threshold for transducin translocation (dashed line) was the combined average value of the OPL peak in retinas exposed to 0, 1, and 2 sc.cd/m². The OPL peak is clearly above threshold in P21 WT at 18 and 60 sc.cd/m², but 300 sc.cd/m² was required in P21 Rs1–KO mice. At P60, 12 and 30 sc.cd/m² is enough to elicit OPL peaks above threshold in WT and Rs1–KO mice, respectively. C, The average ± SE OPL peak stain intensity as a percentage of ROS peak stain intensity plotted for WT and Rs1–KO at P21 and P60 as a function of light exposure intensity. WT is significantly above threshold at 18 sc.cd/m² in P21 mice, but among Rs1–KO mice, only those exposed to 300 sc.cd/m² are significantly above threshold. Dashed line is OPL threshold as in B. Statistically, the OPL stain intensity in P60 Rs1–KO mice exceeds threshold at the same intensity as WT, 12 sc.cd/m². *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test corrected for multiple comparisons using the Holm–Sidak method (GraphPad Prism 6.0 for Windows).

Figure 4.

Arrestin movement in response to light in P21 WT and Rs1–KO mice. In the dark, arrestin immunostaining was confined to the OPL, ONL, and IS in WT mice. After exposure to light intensity of 1 sc.cd/m² for 1 h, arrestin was concentrated in the IS compartment, and very sparse staining was detected in the ROS. Light of 2 sc.cd/m² was able to mobilize arrestin from the IS to the ROS. In Rs1–KO retina, the threshold light intensity for arrestin translocation was the same as WT retina. However, at light intensity below the threshold, arrestin redistribution in the inner retina was confined mainly in the ONL and OPL, and none was detected in the IS of Rs1–KO mice. Scale bar, 15 μm.

Figure 5.

Re-translocation of Gα_t1 (rows A and B) and arrestin (rows C and D) in P21 WT and Rs1–KO mice retinas. Mice were dark adapted overnight (12 h), exposed to light (for 1 h), and then placed in the dark (for 4 h). Gα_t1 partially re-translocated into the ROS when mice were replaced in the dark for 4 h after exposure to 30 sc.cd/m² (WT, row A) or 300 sc.cd/m² (Rs1–KO, row B) sufficient to cause translocation from the ROS into the OPL. Arrestin moved from the IS in dark to the ROS in the light (6 sc.cd/m²) and then back into OPL in the dark in WT (row C) and Rs1–KO (row D) retinas. As in Figure 4, arrestin was not detected in the IS of Rs1–KO retina after 12 h dark adaptation. Scale bar, 22 μm.

Figure 6.

Age-related changes in photoreceptor morphology in WT and Rs1–KO mice. Top, Compared with WT retinas, ONL width was thinner in Rs1–KO retina at both P21 (p < 0.001) and P60 (p < 0.05). A significant (p < 0.05) thinning of the ONL occurred in WT mice from P21 to P60, which was not seen in Rs1–KO retinas. Bottom, ROS in Rs1–KO were shorter compared with WT at P21 (p < 0.05), but their length increased significantly between P21 and P60 (p < 0.05) when Rs1–KO and WT were not significantly different. Error bars indicate mean ± SE. n = 8 mice for all histological measurements. *p < 0.05, ***p < 0.001, two-way ANOVA and Sidak's multiple comparison test (GraphPad Prism 6.0 for Windows).

Figure 7.

Expression levels of photoreceptor-specific transcription factors. Retinal protein extracts (∼10 μg of total protein) from WT and Rs1–KO mice at ages P21 and P60 were Western blotted and probed for each of the transcription factor indicated on the left with specific antibodies, followed by anti-tubulin antibody. Expression levels were quantified using Odyssey software and normalized to α-tubulin. At P21, all of these transcription factor levels were lower (by 25–50%) in Rs1–KO mice. However, at P60, both NRL and CRX levels were comparable with that collected from WT retinas. Replicate gels were run for each antibody tested.

Figure 8.

Quantitative immunoblot analyses (Odyssey imaging system; LI-COR) of key phototransduction protein subunits, transducin α (Gα_t1), RGS9, PDE6α, andPDE6γ, in dark-adapted outer segment extracts from P21 WT and Rs1–KO mice (A–D). Transducin α levels relative to rhodopsin were 15–30% lower in Rs1–KO mice than in WT. RGS9, the GAP for Gα_t, was 1.7- to 2.5-fold higher in Rs1–KO than in WT. Both PDE6α and PDE6γ protein levels were marginally elevated in Rs1–KO retinas. Replicate gels were run for each antibody tested. The time course of phosphate formation during hydrolysis of [γ-³²P]GTP by G_t* in ROS of WT and Rs1–KO mice at P21 (E) and P60 (F). In single-turnover GTPase activity measurements in isolated ROS, GTP hydrolysis by transducin was nearly twofold higher in Rs1–KO than in WT at P21, resulting in a shorter lifetime of activated transducin in Rs1–KO ROS. The rates were not different at P60. The data were fitted to a single-phase exponential decay curve using GraphPad Prism (GraphPad Software).

Figure 9.

Intensity–response series of scotopic ERG a-wave amplitudes in P21 and P60 WT and Rs1–KO mice measured at 8 ms after the flash. Mean ± SE amplitudes (microvolts) were normalized by the average dark-adapted whole retina rhodopsin content for each category of mice. The response curves for all groups of mice were essentially superimposed up to the maximum stimulus intensity of +0.6 log cd · s/m². A linear correlation between the mean a-wave amplitude at 8 ms (+0.6 log cd · s/m²) and the product (ROS × ONL) calculated from the ONL widths and ROS lengths graphed in Figure 6 is displayed in the inset.