Abstract
Purpose :
P23H Rho mice, mimicking retinitis pigmentosa in humans, manifest chronic unfolded protein response activation during retinal degeneration (RD). GADD34, the UPR mediator and protein phosphatase 1 catalytic subunit, permits protein synthesis through dephosphorylation of eukaryotic initiation factor 2a (eIF2α) in cells. We hypothesized that further upregulation of phosphorylated (p) eIF2α modulates the rate of protein synthesis and affects vision loss in mice with RD.
Methods :
C57BL/6, P23H Rho, and P23H Rho GADD34-/- mice were used in this study. Mouse retinas were collected on postnatal (p) day 30 to study protein and RNA expression by western blot and RT-PCR analyses. Retinal function was assessed using electroretinography. The SUnSET method was applied for the in vivo assay of protein synthesis. Apoptotic cells were assessed via TUNEL staining of P22 retinas. Lipopolysaccharide (LPS; 10 mg/kg of body weight) was injected intraperitoneally into mice to induce a pro-inflammatory response at P30.
Results :
The P23H Rho mice demonstrated a decrease in protein synthesis associated with sustained phosphorylation of eukaryotic initiation factor 2a (p-eIF2a) compared to the C57BL/6 mice. Knocking out GADD34 resulted in further elevation of p-eIF2α in the degenerating retinas. Surprisingly, this elevation was not associated with a more pronounced decline in the rate of translation or restoration of the p-mTOR axis in P23H Rho GADD34-/- mice. Moreover, sustained upregulation of p-eIF2α was associated with a 30% decline in the a-wave amplitude of the scotopic ERG, while the b-wave was not affected. The ERG recordings were supported by a 50% increase in apoptotic cells in the P23H Rho GADD34-/- retinas compared to the P23H Rho retinas. Because GADD34 controls the expression of inflammatory cytokines in immune cells, we further analyzed IL6 and Tnfa gene expression in the retinas of LPS-treated mice. We found that a 4-fold reduction in Il-6 and a 2.7-fold upregulation in Tnfa expression in P23H Rho GADD34-/-retinas.
Conclusions :
Our results indicate that GADD34 deficiency does not affect the rate of protein synthesis during chronic ER stress, suggesting that p-eIF2α is not the major point of translational control in progressive RD. GADD34 may control the inflammatory response of P23H Rho retinas, and its deficiency may alter pro- and anti-inflammatory cytokine ratios, thus resulting in compromised photoreceptor homeostasis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.