Abstract
Purpose: :
To test for altered rhodopsin signaling in rod outer segments from heterozygous knock–in mice expressing a human rhodopsin–EGFP fusion (+/hrhoG), and to determine whether light–independent degeneration seen in homozygotes (hrhoG/hrhoG) is due to abnormal outer segment membrane formation.
Methods: :
Rod outer segments were isolated from dark–adapted +/+ and +/hrhoG mouse retinas under IR illumination and assayed for activation of the downstream effector, cGMP phosphodiesterase (PDE), by dim light flashes of varying intensities. In addition, eyes from +/+, +/hrhoG, and hrhoG/hrhoG, and hrhoG(H)/hrhoG(H) knock–in mice, which express low levels of human rhodopsin–EGFP, and from rhodopsin knock–out (null) mice were fixed, embedded, sliced in ultrathin sections, stained and imaged using transmission electron microscopy at ages ranging from 10 days to 23 weeks postnatal.
Results: :
Light–induced PDE activities in both +/+ and +/hrhoG mice were indistinguishable over light levels ranging from 4.5 X 10–6 R*/R (photoactivated/[total rhodopsin + rhodopsin–EGFP]) to saturating intensities. Retinas from hrhoG/hrhoG mice displayed abnormal morphology in rod outer and inner segment regions as early in development as 10 days postnatal. Membrane whorls, tubes and/or vesicles were seen in hrhoG/hrhoG mouse rod outer segment regions, as well as in the hrhoG(H)/hrhoG(H) mice, which have only 20% of the rhodopsin–EGFP levels of hrhoG/hrhoG mice. Extensive proliferation of lysozomes was seen in the RPE and choroids of hrhoG(H)/hrhoG(H), which have low levels of visual pigment, and also in null mice, which have no visual pigment.
Conclusions: :
1. The activation phase of phototransduction in +/hrhoG rods is indistinguishable from that in wildtype rods. 2. The earliest detectable defect in either hrhoG(H)/hrhoG(H) or hrhoG/hrhoG mice is abnormal outer segment membrane morphogenesis, which likely underlies the cell death that occurs later. 3. This defect is caused by the absence of wildtype rhodopsin, rather than by excessive levels of rhodopsin–EGFP, because mice with very little rhodopsin–EGFP (hrhoG(H)/hrhoG(H) mice) suffer from this defect, while mice with 2.5 times as much rhodopsin–EGFP (+/hrhoG) do not. 4. Very low levels of visual pigment lead to lysosome proliferation in neighboring cells.
Keywords: retinal degenerations: cell biology • photoreceptors • signal transduction