Abstract
Purpose :
The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) are the physical contact sites between the ER and the mitochondria and play a vital role in regulation of calcium trafficking and bioenergetics. The goal of the present study is to examine the impact of diabetes on retinal MAM protein profile and identify key pathways and proteins potentially implicated in the pathogenesis of diabetic retinopathy.
Methods :
Diabetes was induced with streptozotocin in Long-Evans rats. MAM was isolated from retinas of 10 month-diabetic rats and age- and sex-matched non-diabetic controls. A proteomic analysis was performed using an optimized nano reversed phase liquid chromatography mass spectrometry (RPLC)-MS system. In depth IPA pathway analysis and DAVID bioinformatics database were used to identify up- or downregulated biological processes, disease-relevant pathways and upstream regulators. Changes of selected proteins were validated by western blotting and immunostaining in diabetic retinas and cultured R28 cells.
Results :
A total of 2,664 non-redundant proteins were identified in rat retinal MAM, among which 179 proteins showed significant changes in diabetes. Functional annotation revealed that the 179 proteins are involved in important biological processes including cell survival, inflammatory response, cellular maintenance, developmental disorder, free radical scavenging, post-translational modification, cell signaling, cellular assembly and organization, amino acid metabolism, carbohydrate metabolism, lipid metabolism, and visual system development and function. Furthermore, the proteome changes predict multiple disease-relevant signaling pathways such as integrin signaling, leukocyte extravasation, actin cytoskeleton, PPAR, PTEN and RhoGDI signaling. Intriguingly, a number of proteins involved in retinal morphology, retinal degeneration, cone photoreceptor degeneration, and retinal function were found to be enriched in the MAM and altered by diabetes. The changes of MAM proteins Gpx4, Rp2, and Gal3 were confirmed in R28 cells treated with high glucose.
Conclusions :
Our study provides comprehensive and important information on MAM protein composition changes in diabetic retinas and this information is helpful for a better understanding of the mechanisms of metabolic dysfunction and injury of retinal cells in diabetic retinopathy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.