Activation Of JNK Signaling Promotes All-trans-retinal-induced Photoreceptor Apoptosis

Objective:To explore the mechanism of photoreceptor cell death caused by atRAL and to investigate the role of JNK signaling pathway in retinal degeneration and photoreceptor cell death.Methods:First,the cell viability of 661W photoreceptor cells was detected by MTS method and apoptosis cells were detected by TUNEL staining.The related protein levels were detected by Western blotting and verified by immunofluorescence.And then we use the JNK specific inhibitor JNK-in-8 and the Jnk1-/-Jnk2-/-661W to further study.Flow cytometry and staining were used to analyze the levels of reactive oxygen species in 661W cells after treatment of atRAL.Antioxidant NAC was used to further analyze the production of ROS,cell viability and the related protein levels.Finally,HE staining was used to analyze the histological morphology of the retina of WT and Abca4-/-Rdh8-/-mice after light expose.TUNEL staining was used to analyze apoptotic cells and western blotting to detect the protein levels.JNK-specific inhibitor JNK-IN-8 was intraperitoneal administration to further study.Results:We reported that atRAL activated JNK signaling at least partially through reactive oxygen species(ROS)production to promote mitochondria-mediated caspase-/DNA damage-dependent apoptosis in photoreceptor cells.Damage to mitochondria in atRAL-exposed photoreceptor cells resulted from JNK activation leading to decreased expression of Bcl2 protein,increased levels of Bak protein,decreased of Δψm and release of the cytosol cytochrome c(Cyt c).Cytosolic Cyt c specifically provoked caspase-9 and caspase-3 activation to initiate apoptosis.Alternatively,JNK phosphorylation in atRAL-loaded photoreceptor cells induced the appearance of γH2AX,a sensitive marker for DNA damage correlated with induction of apoptotic cell death.Suppressing JNK signaling protected photoreceptor cells against apoptosis caused by atRAL.Moreover,Knockout of Jnk1 and Jnk2 genes rescues 661W photoreceptor cells from atRAL-induced apoptosis via blocking JNK signaling.Abca4-/-Rdh8-/-mouse displays defects in atRAL clearance which are characteristic of STGD1 and dry AMD.We found that JNK signaling was activated in neural retina of light-exposed Abca4-/-Rdh8-/-mice.More importantly,intraperitoneal administration of JNK-IN-8 effectively ameliorated photoreceptor degeneration and apoptosis in Abca4-/-Rdh8-/-mice after light exposure.Conclusion:atRAL activated JNK signaling at least partially through reactive oxygen species(ROS)production to promote mitochondria-mediated caspase-/DNA damage-dependent apoptosis in photoreceptor cells.Activation of JNK signaling involves light-induced retinal degeneration and photoreceptor cell apoptosis in Abca4-/-Rdh8-/-mice.Suppression of JNK signaling by JNK-IN-8 ameliorates photoreceptor degeneration and apoptosis in light-exposed Abca4-/-Rdh8-/-mice.