Abstract
Purpose :
The α-crystallins molecular chaperones are involved in the pathophysiology of diabetic retinopathy (DR), however, the role and regulation of these proteins remained unclear. We recently demonstrated that the αA-crystallin exerts its intrinsic neuroprotective role during diabetes, by its phosphorylation on residue 148. We also reported that αA-crystallin is highly expressed by glial cells. Because of the growing interest of the potential causative role of low-grade inflammation in the DR pathophysiology, this study was carried out to delineate the regulatory function of αA-crystallin in the inflammatory response associated with metabolic stresses.
Methods :
Primary Müller glial cells (MGCs) isolated from Ko-aA-crystallin mice were transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non phosphorylatable mutants (T148A) of αA-crystallin. The cells were then exposed to multiple metabolic stress including serum starvation (SS) or high glucose with TNF-alpha (HG+T) before being evaluated for the expression of inflammatory markers by qPCR and protein expression of NFkB1 & NLRP3 components by western blot.
Results :
Elevated levels of inflammatory markers in SS were diminished in MGCs overexpressing WT (IL-6-97%; IL-1β- 88%; MCP-1-89%; IL-18-72%) and further in T148D (IL-6-87%; IL-1β- 86%; MCP-1-28%; IL-18-64%) as compared to EV. The HG+T induced increase in IL-6, IL-1β, MCP-1 & IL-18 were dampened by WT (55%, 36%, 85% & 93% respectively) and even more significantly by T148D (IL-6- 92%; IL-1β-54%; MCP-1-98%; IL-18-55%) overexpression, whereas T148A was ineffective in either stress. Further, overexpression of WT or the T148D, also lead to a significant reduction of Nlrp3 (85%), Asc (61%) and caspase-1 (68%) expression in serum deprived MGCs and nearly abolished the NF-kB induction (αA-WT-97%; αA-T148D-95%) in HG+T stress. The protein levels of NLRP3 components and NF-kB were consistent with the transcriptomic data in a metabolic stress specific manner.
Conclusions :
The data gathered in this study demonstrate the central regulatory role of αA-crystallin and its modulation by phosphorylation on T148 in retinal MGC. This study demonstrates that αA-crystallin can dampen sustained expression of pro-inflammatory cytokines through modulation of multiple key inflammatory pathways, therefore, suggesting its potential as therapeutic target for modulation of chronic neuroinflammation.
This is a 2021 ARVO Annual Meeting abstract.