Retinal degeneration in mice expressing the constitutively active G90D rhodopsin mutant

Abstract

Rhodopsin is the G protein-coupled receptor in rod photoreceptor cells that initiates vision upon photon capture. The light receptor is normally locked in an inactive state in the dark by the covalently bound inverse agonist 11-cis retinal. Mutations can render the receptor active even in the absence of light. This constitutive activity can desensitize rod photoreceptor cells and lead to night blindness. A G90D mutation in rhodopsin causes the receptor to be constitutively active and leads to congenital stationary night blindness, which is generally thought to be devoid of retinal degeneration. The constitutively active species responsible for the night blindness phenotype is unclear. Moreover, the classification as a stationary disease devoid of retinal degeneration is also misleading. A transgenic mouse model for congenital stationary night blindness that expresses the G90D rhodopsin mutant was examined to better understand the origin of constitutive activity and the potential for retinal degeneration. Heterozygous mice for the G90D mutation did not exhibit retinal degeneration whereas homozygous mice exhibited progressive retinal degeneration. Only a modest reversal of retinal degeneration was observed when transducin signaling was eliminated genetically, indicating that some of the retinal degeneration occurred in a transducin-independent manner. Biochemical studies on purified rhodopsin from mice indicated that multiple species can potentially contribute to the constitutive activity causing night blindness.

Figure 1

Histology of retina from WT, Rho^TgG90D/+ and Rho^{TgG90D/TgG90D} mice. Retina sections were prepared from 1- to 6-month old mice. Shown is the inferior region of the retina containing the OS, inner segment (IS) and ONL. Scale bar, 15 μm.

Figure 2

Retinal degeneration in Rho^{TgG90D/TgG90D} mice. (A) and (B) The number of nuclei spanning the ONL in the superior (A) or inferior (B) region of the retina in WT, Rho^TgG90D/+ and Rho^{TgG90D/TgG90D} mice housed under cyclic lighting conditions and Rho^{TgG90D/TgG90D} mice housed under constant dark conditions at different ages is shown. (C) Spider plot of the number of nuclei spanning the ONL in the retina of 1-month and 6-month-old WT and Rho^{TgG90D/TgG90D} mice. (D) The number of nuclei spanning the ONL in the superior and inferior regions of the retina in 6-month-old WT, Gnat1^−/−, Rho^{TgG90D/TgG90D} and Rho^{TgG90D/TgG90D}; Gnat1^−/− mice is shown. Mean values along with the standard deviation is shown. Retinal sections from 3 to 6 mice were analyzed. Statistical analyses of the data are presented in the Supplementary Materials.

Figure 3

UV/Vis absorbance spectroscopy, immunohistochemistry and western blot analysis. (A) Absorbance spectra of purified rhodopsin from WT (WT, blue), Rho^TgG90D/+ (WT/G90D, black) and Rho^{TgG90D/TgG90D} (G90D, red) mice. The λ_max for WT rhodopsin was 498.5 ± 0.4 nm (n = 5), the λ_max for G90D rhodopsin was 484.6 ± 0.4 nm (n = 5) and the λ_max for WT/G90D rhodopsin was 492.9 ± 0.7 nm (n = 3). (B) Absorbance spectra of purified WT or G90D rhodopsin from the same number of mice. Rhodopsin was purified from the same number of WT or Rho^{TgG90D/TgG90D} (G90D) mice and the absorbance spectra collected in parallel. The absorbance spectra reflect the relative level of rhodopsin present in each mouse line. (C) Absorbance spectra of purified WT and G90D rhodopsin purified from Rho^TgG90D/+ mice. Absorbance spectra were obtained from untreated samples (black line) and after treatment with 20 mm hydroxylamine for 20 min (grey line). The absorbance spectrum from untreated samples exhibited a λ_max of 493 nm, indicating a mixture of WT and G90D rhodopsin. The absorbance spectrum from hydroxylamine-treated samples exhibited a λ_max of 499 nm, corresponding to WT rhodopsin. The absorbance spectrum from hydroxylamine-treated samples was subtracted from the absorbance spectrum from untreated samples, which resulted in a spectrum with a λ_max of 485 nm (dashed black line), which corresponds to G90D rhodopsin. The vertical dotted lines in all absorbance spectra correspond to a wavelength of 485 and 499 nm. (D) Immunohistochemistry of retina from WT and Rho^{TgG90D/TgG90D} mice. Rhodopsin was labeled with the anti-1D4 antibody (red) and nuclei were labeled with DAPI (blue). The OS and ONL are labeled. Scale bar, 100 μm. (E) Western blot analysis of extracts of WT and G90D rhodopsin. Retinal extracts of WT rhodopsin from WT mice and G90D rhodopsin from Rho^{TgG90D/TgG90D} mice were quantified by UV/Vis absorbance spectroscopy. Equal amounts (5 pmol) of WT rhodopsin (lane 1) and G90D rhodopsin (lane 2), as assessed from absorbance spectra, were loaded on the gels. Molecular weight markers are indicated in kDa. Rhodopsin was detected by the anti-1D4 antibody, and the intensity of the bands were quantified. Three different samples (n = 3) containing WT or G90D rhodopsin were run on the same gel and the band intensity quantified. The mean band intensity along with the standard deviation is shown in the graph. The band intensity was normalized to the average band intensity for WT rhodopsin. A t-test showed a significant difference in band intensities for WT and G90D rhodopsin (P = 0.01).

Figure 4

UV/Vis absorbance spectroscopy of purified samples treated with hydroxylamine. Rhodopsin was purified from WT (A), Rho^{TgG90D/TgG90D} (B), or Rho^TgG90D/+ (C) mice. Purified WT rhodopsin from WT mice and purified G90D rhodopsin from Rho^{TgG90D/TgG90D} mice were mixed in equal proportion (D). Absorbance spectra were obtained on untreated samples (black line) and after treatment with 20 mm hydroxylamine for 1, 5 and 20 min (dashed and dotted black lines). After hydroxylamine treatment for 20 min, samples were bleached with light for 1 min (grey line). The vertical dotted lines in all absorbance spectra correspond to a wavelength of 485 and 499 nm.

Figure 5

Thermal stability and MII decay of purified rhodopsin. (A) and (B) UV/Vis absorbance spectroscopy of purified WT rhodopsin from WT mice (A) or purified G90D rhodopsin from Rho^{TgG90D/TgG90D} mice (B). Absorbance spectra were recorded at 55°C at the different time intervals indicated. (C) Thermal decay of WT and G90D rhodopsin. The absorbance at 499 nm for purified WT rhodopsin and 485 nm for purified G90D rhodopsin is plotted for different incubation times at 55°C. The data were fit by non-linear regression to determine the time constant τ. WT rhodopsin decayed with a τ equal to 38.8 ± 2.8 min (n = 3) and G90D rhodopsin decayed with a τ equal to 13.2 ± 1.2 min (n = 3). (D) MII decay of WT and G90D rhodopsin. The decay of the active MII state was determined by monitoring the dequenching of tryptophan fluorescence at 330 nm. The data were fit by non-linear regression to determine the time constant τ. The MII state of WT rhodopsin decayed with a τ equal to 24.7 ± 2.9 min (n = 9) and the MII state of G90D rhodopsin decayed with a τ equal to 13.3 ± 2.5 min (n = 12).