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. 2007 Dec;48(12):5445-53.
doi: 10.1167/iovs.06-1402.

A Novel Form of Transducin-Dependent Retinal Degeneration: Accelerated Retinal Degeneration in the Absence of Rod Transducin

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Free PMC article

A Novel Form of Transducin-Dependent Retinal Degeneration: Accelerated Retinal Degeneration in the Absence of Rod Transducin

Elliott Brill et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: Rhodopsin mutations account for approximately 25% of human autosomal dominant retinal degenerations. However, the molecular mechanisms by which rhodopsin mutations cause photoreceptor cell death are unclear. Mutations in genes involved in the termination of rhodopsin signaling activity have been shown to cause degeneration by persistent activation of the phototransduction cascade. This study examined whether three disease-associated rhodopsin substitutions Pro347Ser, Lys296Glu, and the triple mutant Val20Gly, Pro23His, Pro27Leu (VPP) caused degeneration by persistent transducin-mediated signaling activity.

Methods: Transgenic mice expressing each of the rhodopsin mutants were crossed onto a transducin alpha-subunit null (Tr(alpha)(-/-)) background, and the rates of photoreceptor degeneration were compared with those of transgenic mice on a wild-type background.

Results: Mice expressing VPP-substituted rhodopsin had the same severity of degeneration in the presence or absence of Tr(alpha). Unexpectedly, mice expressing Pro347Ser- or Lys296Glu-substituted rhodopsins exhibited faster degeneration on a Tr(alpha)(-/-) background. To test whether the absence of alpha-transducin contributed to degeneration by favoring the formation of stable rhodopsin/arrestin complexes, mutant Pro347Ser(+), Tr(alpha)(-/-) mice lacking arrestin (Arr(-/-)) were analyzed. Rhodopsin/arrestin complexes were found not to contribute to degeneration.

Conclusions: The authors hypothesized that the decay of metarhodopsin to apo-opsin and free all-trans-retinaldehyde is faster with Pro347Ser-substituted rhodopsin than it is with wild-type rhodopsin. Consistent with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H(2), which form from retinaldehyde, were elevated in Pro347Ser transgenic mice.

Figures

FIGURE 1
FIGURE 1
Comparison of rhodopsin mutant mice on Trα+/+ and Trα−/− genetic backgrounds. (A) Time course of degeneration in Trα+/+ (○) and Trα−/− (△) mice (left); retinal morphology at 3 months (right). (B) VPP, Trα+/+ (○) and VPP, Trα−/− (△) degeneration kinetics (left); retinal morphology at 3-months (right). (C) Lys296Glu, Trα+/+ (○) and Lys296Glu, Trα−/− (△) degeneration kinetics (left); retinal morphology at 3 months (right). (D) Pro347Ser, Trα+/+ (○) and Pro347Ser, Trα−/− (△) degeneration kinetics (left); retinal morphology at 6 months (right). OS, outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer. n = number of animals sampled at each time point. Scale bar, 10 µm.
FIGURE 2
FIGURE 2
Role of rhodopsin/arrestin complexes in Pro347Ser rhodopsin mutant degeneration. The ONL thickness of 4-month-old Pro347Ser rhodopsin mutant mice in the presence or absence of arrestin and transducin was plotted. The absence of α-transducin or arrestin alone did not contribute to degeneration (compare 3 left columns). Loss of α-transducin in combination with the Pro347Ser rhodopsin mutation produced a statistically significant loss of ONL thickness (columns 4 and 5). The combined loss of α-transducin and arrestin did not protect from degeneration (last column). Error bars show SEM.
FIGURE 3
FIGURE 3
α-transducin stabilizes Pro347Ser metarhodopsin. (A) Retinal morphology of Pro347Ser rhodopsin mutant mice on a Trα+/+ or Trα−/− genetic background reared in cyclic light or dark reared. Pro347Ser, Trα+/+ mice showed similar degrees of degeneration whether reared in cyclic light or darkness. Dark rearing provided protection from degeneration but only in P347S, Trα−/− retinas. (B) Histogram comparing ONL thickness in cyclic light-reared (gray) and dark-reared (black) animals on the Trα+/+ or Trα−/− genetic background. Error bars show SEM. Scale bar, 10 µm.
FIGURE 4
FIGURE 4
Comparison of A2E and A2E-precursor levels in (A) 61-day-old WT mice (gray) and littermate Pro347Ser, Trα+/+ transgenic mice (black) show statistically significant differences for four bis-retinoids. A2E was present at 4.8 versus 1 pmol/eye in Pro347Ser, Trα+/+ and WT mice, respectively. (B) Lipofuscin granule density. Four mice of each genotype were surveyed by measuring square microns of lipofuscin granule per square micron of RPE cytoplasm. P347S mutant mice (black) had a higher density of granules than WT control mice (gray). Error bars represent SD.

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