Abstract
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
Highlights
The retinal pigment epithelial (RPE) cells, which are situated between the photoreceptor cells and choroid in the back of the eye, are vitally important for vision by maintaining the viability of photoreceptor cells [1]
Our aim was to assess the functionality of mature human embryonic stem cell (hESC)-RPE cells; it was important to verify that the used hESC-RPE cells have been successfully differentiated and matured from pluripotent stem cells
This was done by assessing gene expression with Reverse Transcription-Polymerase Chain Reaction (RT-PCR), protein expression and localization with immunofluorescence labelling, confocal microscopy and evaluating cell morphology and pigmentation with brightfield phase contrast microscopy and Transmission Electron Microscopy (TEM)
Summary
The retinal pigment epithelial (RPE) cells, which are situated between the photoreceptor cells and choroid in the back of the eye, are vitally important for vision by maintaining the viability of photoreceptor cells [1]. Central characteristics of RPE cell degeneration associated with aging include subtle changes in pigmentation, as well as accumulation of intracellular lipofuscin, a highly cross-linked aggregate of oxidized proteins, and extracellular drusen deposits. Autophagy is essential for maintaining healthy cell homeostasis: the autophagic process begins with the formation of isolation membranes known as phagophores [6], which become elongated and form mature double membrane autophagosomes. These autophagosomes engulf portions of cytoplasm containing oligomeric protein complexes and organelles, fuse with lysosomes, and their content is degraded by lysosomal enzymes. A decline in autophagy is usually accompanied by the accumulation of aggregates expressing autophagy markers p62 ( known as sequestosome-1, SQSTM1) and LC3 (microtubule-associated protein 1A/1B-light chain 3, known as MAP1LC3A), normally degraded by lysosomal enzymes in autophagic processes [3,5,15]
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