Autophagy is affected by Mitf in mouse primary RPE cells

Abstract

PurposeMicrophthalmia‐associated transcription factor (MITF) regulates the differentiation and development of the retinal pigment epithelium (RPE). Mice that lack functional MITF do not develop the RPE and have microphthalmia. Recent studies have involved MITF in autophagy regulation in other cell types. The purpose of this study was to examine if the Mitf gene plays a fundamental role in regulating autophagy in primary RPE cells using various mutations in the Mitf gene.MethodsPrimary RPE cells from wild type and MITF mutant mice Mitf mi‐enu122(398), Mitf Mi‐Wh/+ and Mitf Mi‐Wh/Mitf mi‐mi) were isolated by enzymatic dissociation. The levels of LC3 and MITF were measured and compared by western blot in the primary RPE cultures from wild type and mutant mice. Basal autophagy was also analysed with western blots and confocal imaging using same markers in primary RPE cells from C5BL/6J mice. Untreated cells were compared to cells treated with the mTOR inhibitor Torin1, cells incubated in starvation media and cells treated with the autophagy inhibitor, bafilomycin A1 (Baf A1).ResultsThe treatment with starvation media and Torin1 increased the levels of LC3 in RPE cells. Furthermore, both starvation and Torin1treatment resulted in reduced MITF protein levels. Cotreatment of Torin1 or starvation with Baf A1 restored the protein levels of MITF and LC3. Only the LC3II protein was detected in RPE cells from MITF mutant whereas wild type RPE cells showed both LC3I and II, suggesting that the degradation pathway of LC3 is stalled in the RPE from Mitf mutant mice.ConclusionsThis study suggests that autophagy is affected in Mitf mutant mice. This is consistent with in vitro data showing that MITF regulates expression of genes involved in autophagy.