SIRT5/Akt2 signaling axis regulates TFEB activity and autophagy in the RPE cells: Implications in dry age-related macular degeneration

Abstract

Purpose : Lysosomes are critical for several homeostatic functions in the RPE cells, most importantly, phagocytosis and autophagy. Decline in lysosomal function and autophagy has been linked with AMD. Inflammation has emerged as a potential cause of AMD and has been known to regulate lysosomal function and autophagy. But how inflammation regulates these processes remains elusive. We and others have shown that the Akt2-dependent inflammatory pathway is activated in the retina as well as the macular RPE of human dry AMD donors. Akt2 is also known to regulate lysosomal function in several cell types. This study was undertaken to assess if Akt2 overexpression in RPE cells leads to deregulation of lysosome function and autophagy.

Methods : We have generated Best1 (Akt2) constitutive knock-in (KI) mice as tools to be used in this study. The mouse was characterized at increasing age for a dry AMD-like phenotype using transmission electron microscopy, fundus autofluorescence, electroretinography and hematoxylin-eosin staining. RPE cells from WT and Akt2 KI mice were used to perform western blots for p-Ezrin, Akt2, p-Akt2, p-TFEB, and lysosomal markers like Cathepsin D, Lamp1, Cathepsin L, and Sirt5. Autophagy flux was measured in WT, Akt2 KI, and PGC1α KO RPE explant cultures.

Results : We found a noticeable age-dependent decline in retinal function, accumulation of basal laminar deposits, pigmentary changes in the Akt2 KI mouse model. We also observed increased expression of p-Akt2 and downregulation of TFEB-dependent lysosomal markers like Lamp1, Cathepsin D and L along with decrease in p-Ezrin and Sirt5 in Akt2 KI RPE cells, relative to WT. To know the underlying signaling associated with these phenotypic changes we performed human-high throughput protein-protein interaction and further confirmed by co-immunoprecipitation studies that Akt2 and Sirt5 are binding partners. In vitro and in vivo analysis further showed that Sirt5 modulates Akt2 kinase activity and subsequent TFEB phosphorylation, whereas Akt2 activation inhibits Sirt5 expression through PGC1α signaling in the RPE cells.

Conclusions : It can be concluded from our results that overexpression of AKT2 in RPE cells results in an AMD-like phenotype with age. Akt2 activity in RPE cells is regulated by Sirt5, and the mutual cooperation between the two proteins modulate TFEB activity and autophagy.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.