A specific phosphorylation regulates the protective role of αA-crystallin in diabetes
Anne Ruebsam, Jennifer E. Dulle, Angela M. Myers, Dhananjay Sakrikar, Katelyn M. Green, Naheed W. Khan, Kevin Schey, Patrice E. Fort
Anne Ruebsam, Jennifer E. Dulle, Angela M. Myers, Dhananjay Sakrikar, Katelyn M. Green, Naheed W. Khan, Kevin Schey, Patrice E. Fort
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Research Article Ophthalmology

A specific phosphorylation regulates the protective role of αA-crystallin in diabetes

Abstract

Neurodegeneration is a central aspect of the early stages of diabetic retinopathy, the primary ocular complication associated with diabetes. While progress has been made to improve the vascular perturbations associated with diabetic retinopathy, there are still no treatment options to counteract the neuroretinal degeneration associated with diabetes. Our previous work suggested that the molecular chaperones α-crystallins could be involved in the pathophysiology of diabetic retinopathy; however, the role and regulation of α-crystallins remained unknown. In the present study, we demonstrated the neuroprotective role of αA-crystallin during diabetes and its regulation by its phosphorylation on residue 148. We further characterized the dual role of αA-crystallin in neurons and glia, its essential role for neuronal survival, and its direct dependence on phosphorylation on this residue. These findings support further evaluation of αA-crystallin as a treatment option to promote neuron survival in diabetic retinopathy and neurodegenerative diseases in general.

Authors

Anne Ruebsam, Jennifer E. Dulle, Angela M. Myers, Dhananjay Sakrikar, Katelyn M. Green, Naheed W. Khan, Kevin Schey, Patrice E. Fort

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Figure 5

αA-crystallin attenuates endoplasmic reticulum stress in retinal neurons, an effect regulated by its phosphorylation on the 148 residue.

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αA-crystallin attenuates endoplasmic reticulum stress in retinal neurons...
Retinal neurons were transfected with vectors overexpressing WT (αA-WT), the phosphomimetic mutant (αA-T148D), the nonphosphorylatable mutant (αA-T148A) of αA-crystallin on threonine 148, or the empty vector control (EV). The cells were then subjected to serum starvation (1% FBS) or tunicamycin (0.5 μg/ml, positive control), and the endoplasmic reticulum (ER) stress response was assessed by analysis of eukaryotic initiation factor α (eif2α) phosphorylation (A), activating transcription factor 4 (ATF4) nuclear translocation (B), binding immunoglobulin protein (BIP) and protein disulfide isomerase (PDI) induction (C and D), and X-box binding protein-1 (XBP-1) splicing (E). Representative images of the immunoblots and the corresponding graphic representation of the quantifications are shown for p-eIF2α, total-eIf2α, actin, and αA-crystallin (cryAA) (A) and ATF4, Histon H3, and α-tubulin (B). Representative images of the immunofluorescent staining obtained for BIP (green), PDI (red), and Hoechst (blue) (C) and the corresponding graphic representation of the relative quantification (D) (scale bar: 10 μm). Graphic representation of the quantitative real time PCR for spliced XBP-1 (E). Gene expression was normalized to the actin-encoding gene Actb. #P ≤ 0.05, ##P ≤ 0.01, ###P ≤ 0.001, ####P ≤ 0.0001, significantly different from serum deprived EV-transfected cells. Each endpoint was measured on a minimum of 3 technical replicates in 3 independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Student-Newman-Keuls test.