Activation of mTOR signaling in the RPE cells induces alterations in melanogenesis thereby triggering oxidative stress

Abstract

Purpose : The retinal pigmented epithelium (RPE) is a monolayer of post-mitotic, polarized and highly specialized pigmented cells at the back of the eye. An important and evolutionarily conserved role of the RPE is the absorption of stray light to increase visual acuity and reduce oxidative damage. This activity requires functional melanosomes, which contain enzymes that catalyze the production of melanin. Defective melanosome formation (melanogenesis) causes hypopigmented RPE, leading to RPE cell loss and decline in retinal function. It is known that mTOR plays a key role both in RPE function as well as melanogenesis. We speculated that activation of mTOR specifically in RPE cells by overexpressing mLST8, an important component of both mTOR complexes 1 and 2 (mTORC1 and 2), would have deleterious effects on melanosome formation, resulting in oxidative stress.

Methods : We have generated Best1 (mLST8) constitutive knock-in (KI) mice as a tool for this study. We performed transmission electron microscopy, hematoxylin-eosin (H&E) staining on RPE sections and electroretinography (ERG) on 3- and 9-month-old WT and mLST8 KI mice. Western blot analysis was performed on RPE explants from WT and mLST8 KI mice to evaluate autophagy flux. Expressions of mTORC1 and mTORC2 downstream components, melanosome marker, tyrosinase, and oxidative stress markers, CAT and SOD1 were also determined by western blot.

Results : Our results revealed the presence of abnormal melanosomes, as evident from their marked depigmentation and fragmentation, in the mLST8 KI RPE. H&E staining revealed noticeable hypopigmented patches in the RPE layer along with patchy loss of RPE. ERG analysis also showed a decline in retinal function in mLST8 KI mice, compared to age-matched controls. Western blot analysis indicated reduced autophagy flux, increased expressions (phosphorylation) of mTORC1 and 2 substrates, S6K and Akt2, and oxidative stress markers, SOD1 and CAT, along with decreased expression of tyrosinase in the mLST8 KI RPE, relative to WT. Interestingly, treatment with Torin (mTOR inhibitor) rescued the alterations in phospho- S6K and Akt2, SOD1, CAT and tyrosinase levels in mLST8 KI RPE explants.

Conclusions : Our results suggest that mLST8 overexpression activated both mTORC1 and 2 thereby triggering defects in melanosome formation and leading to oxidative stress in RPE cells.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.