Mutations in the Mitf gene affect autophagy in mouse primary RPE cells.

Abstract

Purpose : Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types including melanocytes, retinal pigment epithelium and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The purpose of this study was to examine the role of Mitf in regulating autophagy in mouse primary RPE cells by studying mouse Mitf mutations.

Methods : Primary 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 characteristic morphology and the expression of RPE65 of these RPE cells were observed by antibody staining and microscopy. Expression of LC3 and MITF was analyzed with western blot analysis and confocal microscopy in primary RPE cells from C57BL/6J mice, untreated or treated with the mTOR inhibitor Torin1 for 3 hours or after incubation in starvation media for 1 hour. Torin1 and starvation are known activators of autophagy. The levels of LC3 and MITF were measured and compared by western blot in RPE cells from wild type and the Mitf mutant mice.

Results : Wild type RPE cells express MITF and also basal levels of LC3. The treatment with starvation media and Torin1 treatment increased the levels of LC3 in RPE cells. Furthermore, both starvation and Torin1treatment resulted in reduced MITF protein levels. Wild type treated RPE cells with starvation media showed phagosomes around the nuclei. 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.

Conclusions : This 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.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.