PurposeThe Mitf (microphthalmia‐associated transcription factor) gene that is essential for the normal development of the retinal pigment epithelium (RPE). Mutations in this gene can cause hypopigmentation, microphthalmia and blindness. The purpose of this work was to analyze the retinal function and morphology in mice with specific Mitf mutations.MethodsThe following Mitf mutations were used: Mitfmi‐enu122 (398), Mitfmi‐wh/+, Mitfmi‐wh/Mitfmi and wild type (C5BL/6J) mice as a control. Mice were anesthetized by an intraperitoneal injection of 40 mg/kg−1 Ketamine and 4 mg/kg−1 Xylazine. Flash electroretinography (ERG), from mice with pupils dilated, with a corneal electrode and a reference electrode placed in the mouth, was used to determine the role of the MITF protein in retinal function. Histological retinal sections were stained with hematoxylin and eosin.ResultsERG recordings revealed that only one of the four mutants had any retinal function. The wild type mice had significantly higher mean amplitudes of the photopic a‐waves and scotopic oscillatory potentials than the Mitfmi‐enu122 (398) animals (α = 0.05). Furthermore, Mitfmi‐enu122 (398) had significantly shorter implicit times for the photopic b‐waves and c‐waves. Histology revealed that the RPE layer in the Mitfmi‐enu122 (398) and shows localized thinning of the RPE and their retinas look normal. However, the Mitfmi‐wh/+ showed a profound RPE degeneration and this layer is missing from the Mitfmi‐wh/Mitfmi animals. Furthermore, Mitfmi‐wh/+ and Mitfmi‐wh/Mitfmi have an immense retinal degeneration, lacking the photoreceptor and outer plexiform layers.ConclusionsThis study demonstrates that the Mitf gene has an impact on retinal function in mice, and the morphology of the neuroretina and the RPE.

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.

Purpose: Mutations in the Mitf gene can lead to hypopigmentation, microphthalmia, retinal degeneration, deafness and blindness1. We have previously shown that RPE function and structure is affected in mice with various mutations in the Mitf gene, which is expressed specifically in the RPE, resulting in some cases in retinal degeneration2. The current study is the first of its kind to analyse visual and retinal function in Mitfmi/+ mice. Methods: Mitfmi/+ and C5BL/6 J (as control) mice were used in this study. All mice were 1‐ and 3‐months old. Electroretinography (ERG) was performed under both dark‐ and light‐adapted conditions, along with fundus photography from anaesthetised mice. The ERG dark‐adapted a‐, b‐ and c‐waves were analysed. Light‐adapted b‐wave amplitude was analysed. Fundus photography was performed to visualize and compare fundus images from wild type and Mitfmi/+ mice. Results: Light‐adapted a‐wave amplitude was significant lower in 3‐month‐old mutants (7.34 ± 1.34 μV, 6.72 ± 1.19 μV, 8.58 ± 1.03 μV; p < 0.05) compared to wild type mice at 3, 5 and 7 cd*sec/m2 respectively. Light‐adapted b‐wave amplitude was also significantly reduced in 3‐month‐old mutants (22.13 ± 0.95 μV, 35.09 ± 2.72 μV, 57.715 ± 5.17 μV, 66.16 ± 4.48 μV, 85.87 ± 4.27 μV; p < 0.05) compared to control animals at all luminance tested. Interestingly, ERG c‐wave amplitude was significantly lower in 1‐month‐old Mitfmi/+ (150.80 ± 11.91 μV; p < 0.05) compared to control mice at 10 cd*sec/m2 light intensity. Progressive hyper‐ and hypopigmentation areas were found in the fundi from the mutants. Conclusions: Retinal function was greatly affected in Mitfmi/+ mutant mice. Significantly lower c‐wave response and hypopigmentation provide evidence for severe RPE pigmentation and likely dysfunction as well in the mutants at 1‐month‐old. Furthermore, lower light‐adapted a‐ and b‐ wave responses indicates selective cone‐dystrophy in this heterozygous mutation in the Mitf gene. References 1 Steingrímsson E et al. Annu Rev Genet, 2004; 38:365–411. 2 García‐Llorca A et al. Sci Rep, 2019; 28; 9(1):15386.

Purpose Mice carrying different mutations in the microphthalmia‐associated transcription factor (Mitf) gene, affect the normal the retinal pigment epithelium (RPE) resulting in microphthalmia. The MitfK243R/K243R; Tyr::Cre mice have an induced conditional mutation, where Lysine 243 is mutated to Arginine, thus affecting DNA binding. The purpose of this study was to characterize eye development and function in these mutant mice. Our analysis shows retinal degeneration and slow progressive GA in the eyes of these mice. Methods C5BL/6J (wild type) and MitfK243R/K243R; Tyr::Cre mice of 1, 3 and 12 months old were used in this study. Fundus and optical coherence tomography (OCT) images were taken from wild type and mutant mice. Retinal function was assessed by electroretinography (ERG). RNA isolation from posterior eye cups and reverse‐transcription quantitative PCR (RT‐qPCR) were done to analyze Mitf mRNA expression. Results Bright field images indicate that eyes from B6‐MitfK243R; Tyr::Cre mice have alternating areas of severe hypo‐ and hyperpigmentation and vascular abnormalities. OCT images from 1 and 3 months old mutants demonstrated that some regions of the retina have the RPE, photoreceptor and neuronal layers intact, while neighboring areas show severe degeneration of these cell types. However, at one year of age the RPE, photoreceptor and outer retina layers are missing in the mutants. ERG recordings revealed that at 1 and 3 months old the mutants had significantly reduced (p < 0.05) photopic b‐wave, scotopic a‐ and b‐waves, while at 12 months old the mutant cone and rod ERG responses were flat or severely reduced compared to wild type. Gene expression analysis showed that Mitf expression was elevated in the mutants as compared to wild type, suggesting compensatory mechanisms. Conclusions We have found that mice with the MitfK243R conditional mutation driven by the Tyr::Cre driver may be the first mouse model of slow progressive retinal GA. This mutant shows similar characteristic as seen in human age‐related macular degeneration (AMD) and may therefore provide a model for that disease.