Abstract
Purpose:
IRBP-deficient mice exhibit rod and cone degeneration. The mechanisms underlying photoreceptor degeneration remains poorly understood. The purpose of this study is to identify cell death signaling pathway(s) involved in the photoreceptor degeneration in Irbp-/- retina.
Methods:
Wild-type (WT), Irbp-/-, and rd12 RPE65-deficient mice at postnatal day 14 (P14) and 4 weeks were used. These mice have homologous Leu450 alleles for Rpe65 gene. Contents of 11-cis retinal in these mice retinas were compared by HPLC. Numbers and morphology of photoreceptors stained with PNA and antibodies against M- or S-opsin were analyzed by confocal microscopy. Apoptosis of photoreceptors was assessed by TUNEL assay and activation of caspase-3 and apoptosis-inducing factor (AIF). Upregulation of RIP1 and RIP3 in retinas was determined by immunoblot analysis and quantitative RT-PCR. Necrostatin-1, an inhibitor of RIP1 kinase was intraperitoneally injected to Irbp-/- mice to estimate whether RIP-mediated necrosis is involved in the Irbp-/- photoreceptor degeneration.
Results:
Compared to WT mice, Irbp -/- mice had 30% lower 11-cis retinal content at 30 minutes of dark-adaptation after photobleaching. In contrast, the content of 11-cis retinal in rd12 retinas was less than 2% of that in WT retinas. Numbers of cone photoreceptors stained with PNA, M- or S-opsin were significantly reduced in Irbp-/- mice at P14 whereas PNA-positive cells were not significantly reduced in rd12 mice at the same age. Moreover, the outer nuclear layers (ONL) of Irbp-/- mice at 4-weeks old were reduced in thickness to 6-8 nuclei in versus 10-11 nuclei in rd12 and WT mice. In addition, the number of TUNEL positive photoreceptor was increased in Irbp-/- retina but not in rd12 and WT retinas. Although caspase-3 activation and AIF nuclear translocation were induced in Irbp-/- retina, RIP1 and RIP3, were more dramatically up-regulated in Irbp-/- retina. Consistent with this result, treatment with necrostatin-1 significantly protected cone and rod photoreceptors from degeneration in Irbp-/- mice.
Conclusions:
Our results indicate that IRBP plays an essential role in photoreceptor survival beyond its role in the visual cycle. In addition to apoptosis, RIP-mediated necroptosis also contribute to the photoreceptor degeneration in Irbp-/- mice.
Keywords: 695 retinal degenerations: cell biology •
705 retinoids/retinoid binding proteins