Light exacerbates Q344ter-induced retinal degeneration and activates Q344ter in the inner segment and outer nuclear layer.

<p>(<b>A</b>) Q344ter^rho+/− and nontransgenic littermate control mice or (<b>B</b>) Q344ter^rho−/− and nontransgenic littermates control mice that were either dark-reared only or exposed to continuous light (3000 lux with undilated pupils) for five days were sacrificed at p28–31. Retinal sections near the optic nerve were analyzed by retinal morphometry. The diagram displays the mean (± SD) ONL thickness along the entire span of the retina. We focused on a light-sensitive region in the superior half near the optic nerve marked by a green asterisk where a slight ONL thinning occurred in light-exposed nontransgenic mice when compared to their dark-reared counterparts. Under dark-rearing, Q344ter retinas showed a moderate level of degeneration when compared to their transgene-negative littermate controls (p≤0.05). Light-exposure induced a severe form of degeneration in Q344ter transgenic retinas when compared to both light-exposed nontransgenic retinas and dark-reared Q344ter transgenic retinas (p≤0.05). A representative light microscopy image within this region from each group is displayed to the right. Scale bar = 20 µm. (<b>C</b>) Isoelectric focusing gel of retinal extract from designated mice was blotted onto nitrocellulose and probed with the indicated antibodies against rhodopsin. The numbers to the right of each membrane image corresponds to the number of phosphates. In the left panel both Q344ter and WT rhodopsin molecules are detected by the anti-N-terminal rhodopsin mAb 4D2. Only non-phosphorylated rhodopsin (0) and apo-opsin (0*) species were detected in retinas from dark-reared mice. Light-exposure produced multiple phosphorylated rhodopsin species. Moreover, four extra bands (*) were detected in light-exposed Q344ter^rho+/− retinas. In the right panel, only WT rhodopsin molecules are detected by the anti-C-terminal rhodopsin mAb 1D4. Note that the four extra bands in the light-exposed Q344ter^rho+/− retinas detected by 4D2 are absent. (<b>D</b>) Mislocalized Q344ter molecules undergo light-dependent phosphorylation. With the rho−/− background, Q344ter molecules are mislocalized as shown by the absence of apparent ROS structures with R2-12N immunostaining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010904#pone-0010904-g003" target="_blank">Fig. 3E</a>). Retinal homogenates from light-exposed Q344ter^rho−/− mice were examined under similar IEF conditions described in (B). Light-dependent phosphorylation patterns of Q344ter molecules were detected by 4D2 (left panel) but not by 1D4 (right panel). This light-dependent phosphorylation pattern of Q344ter in these mice show that mislocalized Q344ter is capable of light-activation.