Neuroprotective effect of chaperone enhanced aA-crystallin recombinant protein in an in vitro model of diabetic retinopathy
Ameel, Quinn   (University of Michigan-Ann Arbor)
Mentor: Fort, Patrice
Diabetic retinopathy (DR) is a common complication of diabetes and is the leading cause of vision loss in the working-age population. While the mechanism remains unclear, retinal ganglion cell (RGCs) death is a well-recognized aspect of DR pathophysiology. Recent studies have pointed to the prosurvival role of aA-crystallin proteins in DR, and its regulation by its phosphorylation on residue 148. To test the hypothesis that aA-crystallin phosphorylation has a neuroprotective role in DR-associated neurodegeneration, primary cells were isolated from double-knockout mice for genes encoding aAcrystallin and aB-crystallin proteins (Ko-CryAA/AB). Wild-type (WT), phosphomimetic (T148D), and nonphosphorylatable (148A) aA-crystallin recombinant proteins were produced. Ko-CryAA/AB primary RGCs were isolated and exposed to diabetes-like conditions made of 100ng/mL TNF-a and 25mmol/L glucose (TNF-a/HG). To investigate the protective potential of A-crystallin recombinant proteins, cell survival and apoptotic rates were assessed in presence or absence of 500ng/mL of either one of the WT, T148A or T148D recombinant proteins. Following treatments, cells were immunohistochemically stained for RNA-binding protein with multiple splicing (RBPMS), a specific RGC cellular marker, along with terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), which labels DNA breaks to allow visualization of cells undergoing apoptosis. Cells were counted as RBPMS+ cells and RBPMS+/TUNEL+ to demonstrate total RGCs and proportion of these RGCs undergoing apoptosis, respectively. Mean RGC counts were lower in the TNF-a/HG group (58.66) than the control group (95.66), demonstrating diabetes-like milieu induced cell loss. The TNF-/HG/WT condition (87) and TNF- a/HG/148D condition (84.33) showed consistently higher RGC counts in comparison to the TNF- a/HG/148A condition (66.33) and TNF-a/HG condition. Inversely, the mean ratio of RBPMS+/TUNEL+ to only RBPMS+ was highest in TNF-a/HG/148A (0.32), demonstrating the greatest proportion of RGCs undergoing apoptosis. Apoptotic effects were much less significant in TNF-a/HG/WT (0.17) and TNF- a/HG/148D (0.15). These data suggest that phosphorylation on residue 148 of aA-crystallin promotes survival of RGCs in DR, observations consistent with our hypothesis. Due to the nonspecific mechanism of a-crystallin cell protection in the unfolded protein response, these findings have exciting implications for novel treatments of DR and other neurodegenerative diseases with the utilization of exogenous recombinant aA-crystallin proteins.