Activation of mTOR signalling in the RPE cells induces alterations in melanogenesis by suppressing autophagy
Authors: Sinha Debasish 
Published: December 2022

AbstractThe 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 catalyse the production of melanin. Defective melanosome formation (melanogenesis) causes hypopigmented RPE, leading to RPE cell loss and decline in retinal function. Recent studies demonstrated that autophagy plays a wide variety of roles in melanogenesis. The mammalian target of rapamycin (mTOR) is now recognized as the master regulator of autophagy. We speculated that activation of mTOR specifically in RPE cells by overexpressing mTOR Associated Protein, LST8 Homologue (mLST8), an important component of both mTOR complexes 1 and 2 (mTORC1 and mTORC2), would have deleterious effects on melanosome formation, resulting in oxidative stress. We hypothesize that both mTORC1 and mTORC2 complexes are involved in regulating melanogenesis in RPE cells. Herein, we speculate that modulating the expression of mLST8 in the RPE cells will provide novel evidence towards understanding the role of both mTOR complexes in melanosome biology. We have generated Bestrophin‐1 (Best1) promoter‐driven mLST8 constitutive knock‐in (KI) mice as a tool for this study. We performed transmission electron microscopy, haematoxylin–eosin (H&E) staining on RPE sections and electroretinography (ERG) on young and old wild type (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, Catalase (CAT) and Superoxide dismutase (SOD1) were also determined by western blot. 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 mTORC2 substrates, S6 kinase 1 (S6K1) and AKT Serine/Threonine Kinase 2 (AKT2), and oxidative stress markers, SOD1 and CAT, along with decreased expression of tyrosinase in the mLST8 KI RPE, relative to WT. Moreover, mLST8 KI RPE show abnormalities in melanosome number and membrane irregularities along with deregulation in melanogenesis and autophagy pathway mediators. Interestingly, treatment with Torin (mTOR inhibitor) rescued the alterations in phospho‐ S6K1 and AKT2, SOD1, CAT and tyrosinase in mLST8 KI RPE explants. Our results suggest that mLST8 overexpression activated both mTORC1 and mTORC2 thereby triggering defects in melanosome formation and leading to oxidative stress in RPE cells.