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 2

Cell-specific upregulation of α-crystallins in diabetic patients with and without diabetic retinopathy and in diabetic rodents.

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Cell-specific upregulation of α-crystallins in diabetic patients with an...
The expression of α-crystallins in the central and peripheral regions of the retina of human donors was analyzed by quantitative real-time PCR (A), immunoblot (B), and immunohistochemistry (C–F). Graphic representations and representative Western blot images of α-crystallins levels in human donors, nondiabetic (ND; n = 7) or diabetic without retinopathy (D; n = 9) or with diabetic retinopathy (DR; n = 9), are shown. Crystallin expression is presented normalized to actin levels and relative to the expression in nondiabetic donors (*P ≤ 0.05, significantly different from nondiabetic donors). Statistical analysis was performed by 1-way ANOVA followed by Student-Newman-Keuls test. Cellular localization of αA-crystallin (green) was assessed by immunofluorescent staining on retinal cross-sections from nondiabetic control and diabetic WT mice (C and D; n = 6) and nondiabetic and diabetic donors with diabetic retinopathy (E and F; n = 4). Coimmunostaining with specific markers of Müller glial cells (glutamine synthetase, C and D, or glial fibrillary acidic protein [GFAP], E and F, red) and ganglion cells (neurofilament-H, D and F, red) reveals colocalization of αA-crystallins with Müller glial cells and partially with ganglion cells in diabetic animals (D) and human donors with DR (F). Nuclei were counterstained with Hoechst (blue). Higher-magnification images of relevant regions (marked by a dash square) show increased expression of αA-crystallins in Müller glial cells (arrows) and ganglion cells (arrowheads) during diabetes (scale bar: 20 μm [C and E]; 4 μm [D and F]). ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; NFL, nerve fiber layer.