Regulation of retinal homeostasis and disease by Fic-mediated AMPylation

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PROJECT SUMMARY. The proper synthesis, folding, modification and degradation of proteins is vital to cellular health and function. These processes, known collectively as protein homeostasis/proteostasis, have evolved over time to have intricate mechanisms in place for careful regulation in the cell. The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). Activation of the UPR is critical for normal cellular function and health; however, a chronic or prolonged UPR results in elevated inflammation and the activation of apoptosis. If this occurs in post-mitotic cells, the tissue cannot be regenerated. Thus, when this occurs in the photoreceptor neurons of the retina, it causes irreversible blindness. Chronic or dysregulated UPR has been linked to a variety of retinal degenerative diseases; such as diabetic retinopathy, glaucoma, Leber congenital amaurosis, and retinitis pigmentosa (RP). Investigation into the role of the UPR that leads to photoreceptor degeneration can provide important insight into targets for novel therapeutic avenues to treat patients with retinal degenerative diseases. The UPR is known to be regulated by the ER chaperone BiP, which acts as both a molecular chaperone to clear misfolded proteins and as a regulator of the different branches of the UPR. We discovered, for first time, that the enzyme Fic can modulate the UPR via post-translational modification (AMPylation/deAMPylation) of BiP. This indicates that Fic-mediated AMPylation of BiP acts as a molecular rheostat for the UPR. In support of this, we found that a loss of fic in Drosophila leads to vision defects and altered UPR activation in the both the retina and lamina of the eye triggered by exposure to continuous light. We have generated a novel mouse model in which we can study the precise role of Fic-mediated BiP AMPylation in the mammalian retina. We hypothesize that the regulation of the UPR via Fic AMPylation of BiP is necessary to prevent photoreceptor death and vision loss. We will address the following questions: 1) do Fic-/- mice exhibit altered UPR activation in the retina under normal physiological conditions, and 2) are Fic-/- mice predisposed to UPR-associated damage under stress and disease states? The findings of this project will develop valuable tools for monitoring and defining the UPR in the absence of Fic in mammalian retinal cells, both during normal physiological aging and in retinal degenerative disease states. Discovering the role Fic plays in the regulation of ER homeostasis in the mammalian retina can provide insight into cellular targets for potential future therapeutics to treat or prevent ER stress-related photoreceptor cell death and vision loss.