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Ishaq Ahmed Viringipurampeer, Zeinabsadat Mohammadi, Xianghong Shan, Kevin Gregory-Evans, Cheryl Gregory-Evans; Rip3 knockdown rescues photoreceptor cell death in pde6c zebrafish model of achromatopsia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5955.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal recessive achromatopsia is characterized by loss of colour discrimination, photophobia and poor visual acuity. Mutations in four genes (CNGA3, CNGB3, GNAT2, PDE6C) account for 90% of cases, and these mutations cause cone photoreceptor cell death. Mutation-independent treatment strategies aimed at preventing cell death are very attractive for photoreceptor disorders that are genetically heterogeneous. The aim of this study was to determine the mechanism of photoreceptor cell death in the pde6c zebrafish model and then test treatment options targeted to the specific pathways identified.
Cell death in the pde6cw59 mutant zebrafish model was analyzed by histology, TUNEL staining and immunohistochemistry for the following cell death-associated markers: cleaved caspase-3, PARP, calpain, RIP1 and RIP3. FACs sorting of cone photoreceptors coupled to qRT-PCR was used for gene expression analysis. Antisense oligonucleotide morpholinos were used to knockdown the rip3 gene in pde6c mutant embryos. Daily doses of either zVAD-FMK, necrostatin-1 or necrosulfonamide small molecule drugs were tested in vivo and analyzed by histology.
Rapid photoreceptor cell death detected by TUNEL staining began at 4 days post-fertilization (dpf). Activated caspase-3 was detected in rods, but not the degenerating cone photoreceptor cells. Immunohistochemistry and western blot demonstrated high levels of RIP1 in the degenerating cones. Quantitative RT-PCR revealed a 4-5 fold increase in rip3 expression in the sorted cone photoreceptor cells, confirming activation of the necroptosis form of cell death. Morpholino knockdown of rip3 inhibited cone photoreceptor cell death. Pharmacological treatment with necrostatin-1 (RIP1-specific inhibitor) and necrosulfonamide (RIP3-specific inhibitor) delayed cone degeneration.
These results demonstrate in the pde6c mutant retina that cone and rod photoreceptors die by different cell death mechanisms. Elevated levels of RIP1and RIP3 confirm that the necroptosis cell death pathway is activated in cone photoreceptors, whereas caspase-3 activation in rods results in bystander cell death. This suggests that a combinatorial approach of inhibitors for both necroptosis and caspase-3 might be an important principal in order to treat photoreceptor cell death in achromatopsia.
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