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Comparative Study
, 118, 145-53

Photoreceptors in Whirler Mice Show Defective Transducin Translocation and Are Susceptible to Short-Term Light/Dark Changes-Induced Degeneration

Affiliations
Comparative Study

Photoreceptors in Whirler Mice Show Defective Transducin Translocation and Are Susceptible to Short-Term Light/Dark Changes-Induced Degeneration

Mei Tian et al. Exp Eye Res.

Abstract

Usher syndrome combines congenital hearing loss and retinitis pigmentosa (RP). Mutations in the whirlin gene (DFNB31/WHRN) cause a subtype of Usher syndrome (USH2D). Whirler mice have a defective whirlin gene. They have inner ear defects but usually do not develop retinal degeneration. Here we report that, in whirler mouse photoreceptors, the light-activated rod transducin translocation is delayed and its activation threshold is shifted to a higher level. Rhodopsin mis-localization is observed in rod inner segments. Continuous moderate light exposure can induce significant rod photoreceptor degeneration. Whirler mice reared under a 1500 lux light/dark cycle also develop severe photoreceptor degeneration. Previously, we have reported that shaker1 mice, a USH1B model, show moderate light-induced photoreceptor degeneration with delayed transducin translocation. Here, we further show that, in both whirler and shaker1 mice, short-term moderate light/dark changes can induce rod degeneration as severe as that induced by continuous light exposure. The results from shaker1 and whirler mice suggest that defective transducin translocation may be functionally related to light-induced degeneration, and these two symptoms may be caused by defects in Usher protein function in rods. Furthermore, these results indicate that both Usher syndrome mouse models possess a light-induced retinal phenotype and may share a closely related pathobiological mechanism.

Keywords: Usher syndrome; light-induced degeneration; photoreceptor degeneration; retinitis pigmentosa; shaker1; transducin; whirler.

Figures

Figure 1
Figure 1. Activation threshold for rod α-transducin translocation in whirler photoreceptors has been shift to 700 lux
Immunostaining of α-transducin on wild type (A) and whirler (WHR) (B, C and D) retinas after light adaptation for 10 min of 500 lux (A and B), 10 min of 700 lux (C), and 1 hour for 1500 lux (D). Small arrows indicate labeling of transducin in rod inner segments. Large arrows indicate labeling of transducin in the rod synaptic terminals. Arrow heads indicate labeled blood vessels. Scale bar=25 μm. Fluorescence intensity of α transducin immunostaining in wild type (WT) and whirler (WHR) photoreceptors after dark adaptation (E) or exposure to different light intensities and durations (F-I). n=8 independent animals RPE=Retinal Pigment Epithelium; PRL=Photoreceptor Layer, OS=Outer Segments; IS=Inner Segments; ONL=Outer Nuclear Layer; OPL=Outer Plexiform Layer; DA=Dark Adaptation, AU=Arbitrary Units.
Figure 2
Figure 2. Tangential Serial Section Immunoblotting quantitative analysis of α-transducin and rhodopsin in wild type and whirler mouse photoreceptors
Upper panel: Western blots of tangential serial sections of a wild type mouse retina (left panel) and a whirler mouse retina (right panel) after being dark adapted for 8 hours and then light (500 lux) adapted for 10 min. The sections were cut starting from the top of the outer segments, and were immunolabeled by specific antibodies against rhodopsin (Rho) and transducin (Transd). R9AP was used as a marker for the location of rod outer segments. Centrin1 (Cen) was used as a marker for the connecting cilium. Cytochrome C (Cyt) was used as a marker for the locations of the rod inner segments. Lower panel: Densitometry profiles of the Western blots from the above lanes in which the densities of individual bands were expressed as a percentage of the total density of all bands representing each individual protein on the blot.
Figure 3
Figure 3. Mislocalization of rhodopsin in whirler photoreceptors
A–C: Immunostaining of rhodopsin (red) on wild type (A), whirler (WHR) (B) and shaker1 (C) mouse retinas. D–F: Double labeling of anti-R9AP (green) and anti-rhodopsin (Rho) (red) on a whirler mouse retina (focusing on the photoreceptor layer). Arrows indicate staining of rhodopsin in the inner segments. RPE=Retinal Pigment Epithelium; PRL= Photoreceptor Layer; OS=Outer Segments; IS=Inner Segments; ONL=Outer Nuclear Layer; OPL=Outer Plexiform Layer. Scale bar=25 μm.
Figure 4
Figure 4. A–C: Continuous light exposure induces more rod degeneration in whirler mice
A and B: Light micrographs of central retinal sections of a 3-month-old control wild type mouse (A) and a 3-month-old whirler mouse (B) after 6 days continuous 2,500 lux light exposure. RPE=Retinal Pigment Epithelium; PRL=Photoreceptor Layer; ONL=Outer Nuclear Layer; OPL=Outer Plexiform Layer; OS=Outer Segments; IS=Inner Segments. Scale bar=25 μm. C. Average densities (n=8) of photoreceptors in the ONL of central retinal cross sections of 3-month-old wild type (WT) and 3-month-old whirler mice after 6 days continuous 200 and 2500 lux light exposure (LA). *Statistically significant differences between wild typeL and whirlerL mice (P < 0.05). D: Kinetics of rod loss as a function of age in wild type and whirler mouse retinas reared under regular vivarium room light (< 200 lux at the cage level, WT, whirler) compared with that reared under 1500 lux light/dark cycle (WTL, whirlerL). Data points represent quantitative measures of rod numbers (average of 4 mice) in the central parts of the retinas. *Statistically significant differences between wild typeL and whirlerL mice (P < 0.05).
Figure 5
Figure 5. Short-term light/dark changes (1hour 2000lux light exposure and then 7 hours dark adaptation) induces more rod degeneration in shaker1 and whirler mice. A, B
and C: Light micrographs of central retinal sections of a 3-month-old control wild type mouse (A), a 3-month-old shaker1 mouse (B) and a 3-month-old whirler mouse (C) after 14 days alternative 1 hour light (2000 lux) exposure and then 7 hours dark adaptation. RPE=Retinal Pigment Epithelium; ONL=Outer Nuclear Layer; OPL=Outer Plexiform Layer; OS=Outer Segments; IS=Inner Segments Scale bar=25 μm. D. Average densities (n=8) of photoreceptors in the ONL of central retinal cross sections of 3-month-old wild type (WT), 3-month-old shaker1 and 3-month-old whirler mice after 14 days alternative light/dark adaptation. *Statistically significant differences between wild type (WT) and shaker1 and whirler mice (P < 0.05).

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