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Anne Ruebsam, Angela M. Myers, Patrice E Fort; αA-crystallins attenuate endoplasmic reticulum (ER) stress in retinal neurons but not in glia. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1751.
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© ARVO (1962-2015); The Authors (2016-present)
αA-crystallins are highly upregulated by ganglion cells and Müller glial cells in the retina of diabetic animals and humans, cell types in which they are protective in vitro. Increased ER stress, a pathway in which chaperones such as αA-crystallins play a central role, contributes to the pathophysiology of diabetes. We also showed that aA-crystallins phosphosphorylation on S/T148 is reduced during diabetes but its impact on the specific molecular mechanisms by which αA-crystallin promotes survival remain unclear. Thus we tested the hypothesis that phosphorylation of αA-crystallins on S/T148 regulates retinal cell survival by attenuation of serum starvation induced ER stress.
Differentiated R28 cells, a model of retinal neurons, as well as rMC-1, a Müller glial cell line, were transfected with plasmids encoding either wild-type (WT), phosphomimetic (148D), or non phosphorylatable mutants (148A) of αA-crystallin on the 148 residue in 3 independent experiments. Markers of the 3 unfolded protein response (UPR) branches, IRE1, ATF6 and PERK, were analyzed by a combination of transcriptomic, immunoblot and immunostaining methods under normal or metabolic stress conditions (1% FBS). Treatment with the ER stress inducer tunicamycin served as positive control.
Levels of phosphorylated eIF2α and total ATF4 were reduced in αA-crystallin-WT (285±12%;1059±20%) and further in αA-148D mutant (261±16%;684±18%) transfected neurons compared to the EV control (400±20%;2065±2%) upon serum starvation. This protective effect was not seen when cells were transfected with αA-148A (380±28%;1531±37%). While αA-crystallin and its phosphorylation regulates the PERK-associated branch, no effect could be detected in the 2 other branches. In glial cells an increase in ER stress upon serum starvation could be detected, but with no significant difference between the EV and αA-crystallin transfected cells (p-eIF2a level: 337±22% for EV; 302±19% αA-WT; 305±25% αA-148D; 330±40% αA-148A).
αA-crystallins protect retinal neurons through attenuation of the UPR, thus contributing to a novel mechanism of action of αA-crystallins. This effect is abolished by specific alteration of their phosphorylation profile mimicking what is seen during diabetes with a decrease in phosphorylation on the 148 phosphosite. αA-crystallins also protect Müller glial cells, but likely by a different mechanism.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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