Increased Light Exposure Alleviates One Form of Photoreceptor Degeneration Marked by Elevated Calcium in the Dark

Background: In one group of gene mutations that cause photoreceptor degeneration in human patients, guanylyl cyclase is overactive in the dark. The ensuing excess opening of cGMP-gated cation channels causes intracellular calcium to rise to toxic levels. The Y99C mutation in guanylate cyclase-activating protein 1 (GCAP1) has been shown to act this way. We determined whether prolonged light exposure, which lowers cGMP levels through activation of phototransduction, might protect photoreceptors in a line of transgenic mice carrying the GCAP1-Y99C. Methodology/Principal Findings: We reared cohorts of GCAP1-Y99C transgenic mice under standard cyclic, constant dark and constant light conditions. Mouse eyes were analyzed by histology and by immunofluorescence for GFAP upregulation, a non-specific marker for photoreceptor degeneration. Full-field electroretinograms (ERGs) were recorded to assess retinal function. Consistent with our hypothesis, constant darkness accelerated disease, while continuous lighting arrested photoreceptor degeneration. Conclusions/Significance: In contrast to most forms of retinal degeneration, which are exacerbated by increased exposure to ambient light, a subset with mutations that cause overly active guanylyl cyclase and high intracellular calcium benefitted from prolonged light exposure. These findings may have therapeutic implications for patients with these types of genetic defects

Preservation of retinal function by constant light.

<p>(A) The L52H line of mutant mice were raised from birth under cyclic lighting until 3 weeks of age, when they were switched to constant light or constant darkness and kept for an additional three months. Dark-adapted, full-field ERGs with 10 µsec flashes of white light (4.3 log ft-L) were elicited in a Ganzfeld dome. ERG a- and b-waves represent the activities of photoreceptors and inner retinal neurons, respectively. In mutant mice kept under constant light, the a-wave amplitude of 271±29 µV (mean±SEM) was well within the range for age-matched cyclic light reared WT mice (258±15 µV), while the b-wave amplitude of 654±68 µV was slightly below the WT average of 837±68 µV (n = 6 each). Under dark-rearing conditions, the a- and b-wave amplitudes declined to 72±6 µV and 318±40 µV (n = 5), respectively. The ERG a- and b-wave amplitudes in mice kept in constant light were significantly higher than those kept in the dark (<i>P<0.003</i>). (B) Representative dark-adapted, rod dominant ERG waveforms recorded from GCAP1 mutant mice reared in dark or light for 3 months, as well as waveforms from an age matched WT mouse.</p

Model for the disease mechanism in the GCAP1 mutant mice and the impact of light exposure.

<p>Intracellular calcium is elevated in the dark, but falls to the normal minimum in the light. Hence the dark state presents a pathogenic condition but the light state does not. Prolongation of the light phase at the expense of the dark phase is predicted to enhance photoreceptor survival.</p

Preservation of photoreceptor cells in the GCAP1 mutant mice by constant light.

<p>(A) Wild-type mouse (B) Degeneration had already begun in GCAP1 mutant mice raised from birth under standard cyclic lighting for only 3 weeks of age as indicated by the presence of pyknotic nuclei as well as disorganized and shortened outer segments (n = 3). (C) After three months of constant darkness only approx. 2 rows of photoreceptors remained in mutant mice, and the inner and outer segments were severely shortened. (D) In contrast, mutant mice kept in constant light showed no further loss of photoreceptors and outer segment organization actually improved, as compared to the earlier time when they initially entered the constant light condition. (E) Even after 10 months in constant light, photoreceptors continued to show excellent preservation (n = 3). (F) Wild-type retina staining for GFAP shows the typical, very mild signal restricted to the GCL. (H) Similar to wild-type retina, only mild GFAP immunostaining was seen in mutant retina after three months under constant light whereas widespread GFAP immunostaining in the dark-reared mutant retina (G) was consistent with severe photoreceptor degeneration. GFAP was stained yellow. Cell nuclei were stained blue with Hoechst dye 33342. RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer (photoreceptor) nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.</p

Morphometric analyses of mutant eyes after 3 months in constant darkness or constant light.

<p>A. Light micrographs of inferior (I) and superior (S) hemispheres of a representative pair of dark- and light-reared mutant retinas. B. Rescue of photoreceptor cells in different regions of the mutant retina. Shown are morphometric measurements of outer nuclear layer thickness (left) and combined inner and outer segment lengths (right) along the vertical meridian from dark and light reared mutant retinas (mean±SEM; n = 6 each group, <i>P<0.0001</i> for both parameters).</p