Abstract
Purpose :
Increasing evidence suggests an association between autophagy, and age-related degenerative disorders. AHR is a transcription factor mainly known for its role in toxin metabolism. Recently however, it has also been shown to regulate inflammation and autophagy. Aged Ahr-/- mice exhibit several phenotypic features of dry AMD including increased expression of inflammatory markers in the RPE/choroid, as shown by RNAseq and morphological flatmount analyses. Herein, we further investigated the role of AHR expression and function in regulating inflammation and autophagy in RPE cells.
Methods :
In vivo, aged Ahr-/- and Ahr+/+ mice were used to (1) study inflammation in the sub-retinal space by staining RPE-choroid flatmounts for F4/80 (macrophages) and phalloidin (RPE cell borders); (2) generate a cytokine profile of the posterior retina; and (3) measure levels of autophagy proteins (LC3B, SQSTM1/p62, Beclin1, Atg5, Atg7) in the posterior retina. In vitro, human and mouse primary RPE cultures were used to study the effect of AHR knockdown and modulation of AHR activity (Agonists: n=4, Antagonists: n=2) on autophagy. Finally, we tested the therapeutic potential of modulating AHR activity (chronic/acute), on autophagy and inflammation in RPE cells exposed to oxidative insults.
Results :
Ahr-/- mice displayed a significant upregulation of F4/80+ sub-retinal cells in aged mice as compared to Ahr+/+ mice (3.5-fold in 12-14 month old, 3-fold in 18-22 month old, n=4, p<0.04). RPE-choroid protein lysates exhibited significant upregulation of LC3B in 12-14 month old (2.5-fold) as well as 18-22 month old (2-fold) in Ahr-/- mice as compared to Ahr+/+ mice (n=4, p<0.05). Interestingly, RPE/choroid samples from a 12-14 month old cohort, showed significant downregulation of p62/SQSTM1 protein levels (2-fold, n=4, p<0.05). In vitro results, consistent with the in vivo findings, showed an induction of autophagosome formation in RPE cells as measured by LC3B II/ LC3B I ratio, with AHR knockdown (4.5-fold, n=3, p<0.05).
Conclusions :
The absence of Ahr is associated with an increase in inflammation and autophagosome formation in the RPE-choroid milieu. These finding along with our previous reports of RPE cell dystrophy and accumulation of extracellular sub-RPE deposits in aged Ahr-/- mice, support the premise that targeting the AHR signaling pathway may improve RPE cellular turnover and health.
This is a 2021 ARVO Annual Meeting abstract.