The Retinal Proteome in Experimental Diabetic Retinopathy

Abstract

Diabetic retinopathy is the leading cause of blindness in working age persons. Targeted studies have uncovered several components of the pathophysiology of the disease without unveiling the basic mechanisms. This study describes the use of complementary proteomic and genomic discovery methods that revealed that the proteins of the crystallin superfamily are increased dramatically in early diabetic retinopathy. Orthogonal methods confirmed that the amplitude of the up-regulation is greater than other changes described so far in diabetic retinopathy. A detailed time course study during diabetes showed differential up-regulation of the different isoforms of the crystallins superfamily. ␣-and ␤-crystallins were regulated primarily at the translation level, whereas ␥-crystallins were also regulated transcriptionally. We also demonstrated cell-specific patterns of expression of the different crystallins in normal and diabetic rat retinas. Diabetic retinopathy is the leading cause of blindness in working age persons, and despite numerous studies, the pathophysiological mechanisms, especially during the early stages of diabetes, remain to be elucidated. Diabetic retinopathy develops, to some degree, in nearly all patients with diabetes and is the most common cause of new cases of blindness among adults. The predominant causes of vision loss are clinically significant macular edema and proliferative diabetic retinopathy, but vision impairment can be prevented or minimized if the retinopathy is identified in its early stages. Diabetic retinopathy includes microvascular and neuronal, glial, and microglial cell defects early in the course of the disease before clinically visible vascular lesions. Photoreceptor and ganglion cell death occurs as early as 2-4 weeks after the onset of diabetes (1-3).Because of the difficulty of studying the mechanisms of the early stages of diabetes in humans, rodent models of type 1 and type 2 diabetes have been developed. Those models have been used to study various specific aspects of early stages of diabetic retinopathy including blood retinal barrier leakage (4), growth factor signaling such as the insulin/insulinlike growth factor receptor and vascular endothelial cell receptor pathways (5, 6), and oxidative stress mechanisms such as reactive oxygen species and advanced glycation end product production (7,8). These targeted studies provide valuable information on different aspects of the pathology and are useful in new therapeutic development, but more detailed discovery research is also needed to understand the full range of metabolic dysregulation that leads to diabetes complications. Proteome profiling using two-dimensional (2D) 1 DIGE and/or isobaric tag for relative and absolute quantitation (iTRAQ) are methods that can characterize new pathophysiological components and potential therapeutic targets.Several studies have used 2D DIGE to gain a better understanding of diabetic complications in the heart and retina (9 -12). These global approaches revealed biochemical changes in the ret...

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

“…This pattern of expression is consistent with previous reports in animal models of diabetes (6,26) as well as other neurodegenerative models primarily affecting the inner retina, such as ischemic neuropathies, optic nerve crush, and glaucoma (27). This expression pattern is consistent with studies suggesting a central role for αA-and αB-crystallin in the neuroglial unit in neurodegenerative diseases.…”

“…In addition to our data in animal models (6,17), recent reports have suggested that α-crystallins also accumulate in ocular tissues of diabetic patients, supporting an important role for this stress response in the pathology of DR (18,19). We showed that overexpression of α-crystallins protects retinal neurons in culture and that diabetes dramatically reduces the solubility and alters the subcellular localization of Neurodegeneration is a central aspect of the early stages of diabetic retinopathy, the primary ocular complication associated with diabetes.…”

“…Phosphorylation of αA-crystallin on residue 148 is dramatically reduced by diabetes. αΒ-crystallin function is highly regulated by PTM, and our previous studies showed that both αΑ-and αΒ-crystallins are targeted by PTMs in the context of diabetes (6). Using tandem mass spectrometry, we detected phosphorylation of serine residues 122, 148, 155, and 173 of αΑ-crystallin in normal rodent retina.…”

“…To better understand the role of α-crystallin proteins, which accumulate in the retina during diabetes (6,17), we assessed the impact of diabetes in mice lacking expression of αΑ-crystallin (Ko-CryAA), αΒ-crystallin (Ko-CryAB), or both (Ko-CryAA/AB). αΑ-and αΒ-crystallin-KO mice have only been partially characterized previously (22,23), so we analyzed the main tissues in which α-crystallins have been reported and found no obvious defects in heart, brain, and retina (Supplemental Figure 1, A-C; supplemental material available online with this article; https://doi.…”

“…Several groups, including ours (6,7), have demonstrated that retinal neurodegeneration in animal models of diabetes is consistently associated with increased levels of several members of the crystallin protein family. Crystallin proteins are grouped in 3 major classes: α-, β-, and γ-crystallins.…”