Abstract
An integrated stress response (ISR), identified in several different animal models of inherited retinal degeneration (IRD), is activated following various cellular stresses. The ISR results in the phosphorylation of eIF2α (p-eIF2α) and a consequent halt in protein synthesis. Although generally protective, persistent elevations in p-eIF2α could lead to cell demise. Therefore, we aimed to determine whether ISR activation is associated with diminished translation rates in mice with IRD. Retinal protein extracts from rd16 mice at different time points were analyzed and the retinal levels of protein synthesis were assessed using the SUnSET method. We found that rd16 mice experience persistent ISR activation: p-eIF2α, ATF4, and CHOP were significantly upregulated at P15 and P20. In agreement with ISR activation, we found that rd16 mice experience translational attenuation at P15. Similar to rd16, other IRD models, T17M RHO, and rd10 also demonstrated a decline in protein synthesis, correlating with p-eIF2α elevation. We then assessed the role of PERK and eIF2α in translational attenuation in rd16 using a PERK inhibitor, GSK2606414. We found that while the treatment significantly reduced p-eIF2α, it did not cause a complete recovery in translation. This suggests that eIF2α is not the only or even the primary point of translational control in IRD, and a second node of translational regulation comprising AKT and mTOR should be evaluated. Surprisingly, we found that AKT-mTOR signaling was diminished in rd16 and rd10 retinas, suggesting a potential link between AKT-mTOR and translational inhibition. Therefore, for the first time, this study shows translation attenuation in IRD models, and highlights the potential roles of eIF2α kinases and AKT-mTOR signaling that could grant valuable insight into the potential treatments for IRD.
Highlights
Leber congenital amaurosis (LCA) is a class of inherited retinal dystrophies, in which there is currently no cure and generally diagnosed within the first few months of life[1,2]
activating transcription factor 4 (ATF4) regulates the expression of several stress response genes including the genes encoding C/EBP homologous protein (CHOP), growth arrest/DNA damage inducible 34 (GADD34), and Tribbles Homolog 3 (TRB3)[21]
We found that following PERK inactivation, phosphorylation of eIF2α (p-eIF2α) was reduced by ~38%, and protein synthesis was significantly restored (Fig. 4a); translation did not return to normal levels, meaning that PERK signaling is not the only factor contributing to diminishing protein synthesis rates, even in a model as specific as endoplasmic reticulum (ER)-stressed cells
Summary
Leber congenital amaurosis (LCA) is a class of inherited retinal dystrophies, in which there is currently no cure and generally diagnosed within the first few months of life[1,2]. In the United States, LCA cases most commonly involve mutations in the gene encoding the centrosomal protein of 290kD (CEP290)[2]. Various retinal degenerative disorders share cellular signaling pathways including the integrated stress response (ISR)[3,4,5]. We analyzed ISR signaling in the retinas of rd[16] mice, a model of LCA harboring a 5-exon deletion in Cep[290]. A variety stress, amino acid starvation, and viral infection lead to the activation of one or more kinases that phosphorylate the α-subunit of eukaryotic translation initiation factor 2 (eIF2α) at serine 51. EIF2 is responsible for bringing MettRNA to the 43S pre-initiation complex. When eIF2α is phosphorylated, eIF2 can no longer participate in translation, and protein synthesis rates go down. Persistent elevations in p-eIF2α have been shown to lead to cell demise[3,6,7,8]
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