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 6

αA-crystallin, secreted by Müller glial cells, protects retinal neurons against serum deprivation-induced cell death.

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αA-crystallin, secreted by Müller glial cells, protects retinal neurons ...
(A) Representative Western blot images of whole-cell lysate and the media of Müller glial cells transfected with a vector overexpressing αA-crystallin and corresponding graphic representation. The serum-free culture media were recovered after 24 hours, concentrated, and treated or not with proteases (proteinase K) and permeabilizing agent Triton X-100 before the Western blot. (B and C) Protective effect of secreted αA-crystallins on retinal neuronal survival. Retinal neurons were treated for 4 hours with serum-free concentrated media, recovered from Müller cells, transfected with the empty vector (EV) or αA-crystallin WT (αA-WT), the phosphomimetic mutant (αA-T148D), or the nonphosphorylatable mutant (αA-T148A) of αA-crystallin on threonine 148 (B) or transfected with the EV and αA-T148D (C). (B) Representative Western blot images for αA-crystallin on the neuronal cell lysates, processed for the DNA fragmentation ELISA after 4-hour incubation with the different crystallin-enriched media (lysate) and Western blot of the concentrated media from transfected Müller cells (media). Relative amounts are normalized by volume. The graph depicts the DNA fragmentation ELISA data. (C) Representative Western blot images for αA-crystallin on the neuronal cell lysates, processed for the DNA fragmentation ELISA after 4 hours of incubation with αA-T148D crystallin-enriched media (lysate) with (+Ab) or without (-Ab) addition of an αA-crystallin–neutralizing antibody before the 4-hour incubation or neurons incubated with media from EV-transfected Müller cells and Western blot of the corresponding concentrated media from transfected Müller cells (media). The graph depicts the DNA fragmentation ELISA data (bottom panel). Each endpoint was measured on a minimum of 3 technical replicates in at least 2 independent experiments. ****P ≤ 0.0001, significantly different from untreated control cells; ##P ≤ 0.01; ####P ≤ 0.0001, significantly different from 4h EV media. Statistical analysis was by unpaired 2-tailed t test (A) and 1-way ANOVA followed by Student-Newman-Keuls test (B and C).