Influence of eye pigmentation on retinal degeneration in P23H and S334ter mutant rhodopsin transgenic rats.

Dark-rearing has been found to slow the rate of retinal degeneration in albino P23H but not S334ter mutant rhodopsin transgenic (Tg) rats. Since eye pigmentation has the same protective slowing effect as dark-rearing in RCS rats, we examined whether eye pigmentation has a comparable slowing effect in the different mutant rhodopsin Tg rats. Different lines of albino P23H and S334ter Tg rats on the Sprague-Dawley (SD) background were bred to Long-Evans (LE) rats to produce pigmented Tg rats. These were compared to albino Tg rats at postnatal days of different ages using the outer nuclear layer (ONL) as a morphological measure of photoreceptor number and electroretinogram (ERG) a- and b-wave amplitudes as a measure of retinal function. When compared to albino P23H rats, pigmented P23H rats had a slower rate of degeneration as measured by greater ONL thicknesses and greater ERG a- and b-wave amplitudes. By contrast, pigmented S334ter rats showed no difference in ONL thicknesses or ERG a- and b-wave amplitudes when compared to their albino equivalents. Thus, degeneration of photoreceptors in P23H Tg rats is slowed by eye pigmentation as measured by ONL thickness, while it is not in the S334ter Tg rats. Eye pigmentation also protects functional changes in ERG a- and b-waves for the P23H lines, but not for the S334ter lines.

Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration.

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

Long-term photoreceptor rescue in two rodent models of retinitis pigmentosa by adeno-associated virus delivery of Stanniocalcin-1.

Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P < 0.05) and the S334ter-4 (P < 0.005) models compared to controls. Additionally, retinal function was significantly improved in the P23H-1 model with overexpressed STC-1 as assessed by ERG analysis (scotopic b-wave P < 0.005 and photopic b-wave P < 0.05). Microarray analysis identified common downstream gene expression changes that occurred in both models. Genes of interest based on their function were selected for validation by quantitative real-time PCR and were significantly increased in the S334ter-4 model.