Specific changes of zinc and metallothioneins during the in vitro maturation and ageing of a retinal pigment epithelium cellular model

Abstract

AbstractPurposeDuring ageing, the retinal pigment epithelium (RPE) undergoes several morphological and physiological alterations such as loss of melanin, formation of extracellular deposits and metal dyshomeostasis. We examined the age‐related changes in the transcriptome and metallome (metal content in cellular cytosol, membrane and culture media) of the ageing RPE to give an insight into the origin of risk factors linked with neurodegenerative eye diseases like AMD.MethodsWe established a primary human RPE cells culture model to study the maturation (10, 21 and 59 days in culture) and ageing (91 and 133 days in culture) of cells in vitro. We analyzed the transcriptome by RNA sequencing (RNAseq) and determined the metallome by inductively coupled plasma mass spectrometry (ICP‐MS). Localization of specific proteins was carried out by immunocytochemistry.ResultsRPE differentiation markers increased significantly from 10 to 59 days in culture, followed by a decrease from 91 days onwards, as determined by RNAseq and immunocytochemistry assays. After 59 days in culture, there was an alteration in the expression of genes associated with essential biological processes, some of them being phosphorylation, oxidation‐reduction and ion transport. Metal content (i.e., Na, P, Mg, Ca, Cu and Zn) in the cellular cytosol and membranes showed mixed tendencies with time. Interestingly, cytosolic zinc levels decreased from 10 to 59 days (maturing RPE) but increased at 91 and 133 days (ageing RPE). This biphasic change was mirrored by the gene expression changes of specific isoforms of the zinc‐binding metallothioneins including MT1G and MT2A.ConclusionsGene expression and metal content showed time‐dependent changes in the in vitro RPE cell culture model. We observed a specific association between zinc levels and metallothionein expressions during maturation and ageing that, if confirmed by further evidences, may shed light for the metal dyshomeostasis observed during AMD.