Abstract
Purpose :
Intrinsic protective mechanisms including those involving αA-crystallin play an important role in retinal neuroprotection. We previously reported that rodent models of diabetes presented with high levels of αA-crystallin and reduced phosphorylation on residue 148. This study tested the hypothesis that phosphorylation of αA-crystallin on T148 is negatively regulated by diabetes in human retina and that this phosphorylation site affects αA-crystallin protective function.
Methods :
Ocular tissues from 8 non-diabetic, 10 diabetic without retinopathy and 10 diabetic with retinopathy donors were used in this study. One eye was rapidly fixed in formaldehyde to assess regional changes by histology and immunohistochemistry. The contralateral eye was rapidly dissected to isolate and analyze the expression, phosphorylation and subcellular localization of αA-crystallin, Bax and other targets in the different regions of the retina using gene expression, biochemical, and proteomic-based methods.
Results :
αA-crystallin expression was significantly increased in peripheral and central retinal regions of donors with diabetes but without signs of retinopathy (44 and 84% increase respectively; p<0.03). While similar increases were detected in donors with diabetic retinopathy, we also found by mass spectrometry that phosphorylation of αA-crystallin on T148 was reduced by 83% in these donors (n=3; p=0.08). Immunolocalization analysis demonstrated expression of αA-crystallin in both neurons and glia shown by a strong colocalization with GFAP and glutamine synthetase (Müller glia), and Neurofilament-H (ganglion cells). Overexpression of the αA-crystallin 148D phosphomimetic mutant protects neurons (differentiated R28) and glia (MIOM1) from serum deprivation while the non-phosphorylatable 148A mutant does not (p<0.01).
Conclusions :
Our results strongly support our hypothesis that phosphorylation on T148 controls the protective function of αA-crystallin. They also show that while αA-crystallin is induced in both diabetic donors with and without retinopathy, its phosphorylation is highly reduced in those with retinopathy, consistent with its necessity for the protective function of αA-crystallin. The impact of this change is currently being investigated, including through RNA-seq examination of human donor tissues with and without retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.