Abstract
Purpose: :
Rats with inherited retinal degenerations express high levels of retinal crystallins prior to the onset of visual cell loss. In this study we examined rod outer segment (ROS) crystallins and opsin/rhodopsin levels in young rats with genetic retinal degenerations to determine their time course of expression.
Methods: :
Royal College of Surgeons (RCS) rats and P23H rhodopsin transgenic rats (line 3) were born and reared in dim cyclic light. Heterozygous and homozygous animals were sacrificed at P 18-36 and ROS prepared by sucrose density gradient ultracentrifugation. Western analysis of ROS and retinal proteins was performed after 1D- or 2D- gel electrophoresis and transfer to PVDF membranes. Anti -, β-, and γ-crystallin antibodies, N-and C-terminal opsin antibodies and those against GAPDH were used. Protein immunoreactivity was detected by chemiluminesence and relative density determined with an on-line gel viewing and annotation program (GelScape, Univ. Alberta, Canada). Whole eye rhodopsin levels were measured in an attempt to correlate intact visual pigment with ROS opsin levels.
Results: :
All 3 classes of crystallins were markedly elevated in ROS from homozygous RCS rats at P 24, while the phenotypically normal heterozygous RCS rats had approximately half those levels. In homozygous RCS rats, -and β-crystallins were highest in both ROS and retina at P 30. In heterozygous RCS rats opsin contained within ROS nearly doubled from P 18 to P33. ROS opsin in homozygous rats increased by only 10-20% whereas whole eye rhodopsin, present in ROS and extracellular locations, increased by almost 100%. Heterozygous P23H rats also expressed high levels of ROS crystallins at P 24, while crystallins were essentially absent in ROS from homozygous P23H rats. At both P 18 and P 21 ROS crystallin and opsin levels were higher in homozygous P23H rats than in heterozygous animals, whereas the P23H homozygous-rhodopsin level was 50% lower than in heterozygous rats.
Conclusions: :
ROS crystallins are elevated during periods of stress associated with genetic retinal degenerations. The elevated crystallin levels in ROS generally reflect changes seen in retinal extracts and may precede photoreceptor cell apoptosis in both RCS and P23H rats. Opsin accumulation within ROS reflects rod cell development in young heterozygous rats, but appears to be abnormally high in homozygous P23H rats. The P23H mutation may affect the formation or regeneration of rhodopsin leading to higher levels of mutated opsin in ROS membranes.
Keywords: retinal degenerations: hereditary • crystallins • opsins