The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

<p>Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD.</p

MOESM1 of Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

Additional file 1: Fig S1. Confocal images of non-wounded (A) and wounded (B) Regea08/017 hESC-RPE cultures in which COLI is shown in red and nuclei in blue. As seen from the ortho-sections on the right and above, the COLI is concentrated close to the substrata. This is indicated also with white arrows. The background labeling, which would be visible is other areas of the cell, is very low. Scale bars are 10 ¾m

Functional testing of ATP-dependent efflux transporter proteins and viability of cultured cells. A

<p>) Calcein retention in ARPE-19, undifferentiated hESC, fusiform, and cobblestone hESC-RPE, and hFF cells in the presence or absence ( =  control) of efflux protein inhibitors. Retention is expressed as a percentage of fluorescence relative to control (control = 100%). The studies were repeated at least three times for ARPE-19 and fusiform hESC-RPE, and once each for undifferentiated hESC, cobblestone hESC-RPE, and hFF. Data are expressed as mean±SD, *p<0.05, **p<0.01, ***p<0.001. B-E) Representative images of viable (green fluorescence) and dead (red fluorescence) ARPE19 (B,C) and fusiform hESC RPE cells (D,E). The scale bar 100 µM.</p

MOESM3 of Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

Additional file 3: Video S2. The video corresponds Fig. 7C, D. Calcium wave in hESC-RPE monolayer (28d + 7d) followed for 300 s after mechanical stimulation. Prior the stimulation hESC-RPEs were loaded with fluorescent Ca2+ sensitive dye Fluo-4 AM that reflects [Ca2+]i concentration in the cytoplasm. The site of mechanical stimulation is marked with white an arrow. Mechanical stimulation of a single cell in a hESC-RPE monolayer resulted in a [Ca2+]i increase, seen as an increase in fluorescent signal, in the stimulated cell that propagates in a wave-like manner to neighbouring cells

MOESM2 of Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

Additional file 2: Video S1. Calcium wave in hESC-RPE monolayer (9d + 7d) followed for 300 s after mechanical stimulation. The video corresponds Fig. 7A, B. Prior the stimulation hESC-RPEs (9d + 7d) were loaded with fluorescent Ca2+ sensitive dye Fluo-4 AM that reflects [Ca2+]i concentration in the cytoplasm. The site of mechanical stimulation is marked with white an arrow. Mechanical stimulation of a single cell in a hESC-RPE monolayer resulted in a [Ca2+]i increase, seen as an increase in fluorescent signal, in the stimulated cell that propagates in a wave-like manner to neighbouring cells

Localization of ATP-dependent efflux transporter proteins. A-L

<p>) Confocal micrographs after indirect immunofluorescence labeling with efflux pump proteins MRP-1, -4, or -5 (green), and eye-specific proteins MITF and cellular retinaldehyde-binding protein (CRALBP, both red), the polarization marker Na⁺/K⁺ ATPase (red), and the nuclear label 4′,6′-diamidino-2-phenylidole (blue). In figures <b>M-P</b>) the brightfield micrographs show the same ARPE-19 cells and fusiform, early cobblestone, and cobblestone hESC-RPE as shown in the confocal images. Scale bars, 10 µm.</p

Expression of ATP-dependent efflux transporter genes.

<p>Relative expression of MRP1, MRP3, MRP4, MRP5, P-gp, and MRP6 genes. D407 used as reference sample for all genes except MRP-6, for which HEK-293 was used instead. Values that are significantly different from those of the reference sample are marked with an asterisk (*). For better visualization, fold-change is represented on a logarithmic scale. Standard deviations of fold-change from three separate experiments are presented as error bars.</p

Reverse-transcriptase–PCR primer sequences and used annealing temperatures.

<p>Reverse-transcriptase–PCR primer sequences and used annealing temperatures.</p

Morphology and gene expression of hESC on different maturation stages.

<p>Brightfield micrographs of cell cultures showing the representative morphology of <b>A</b>) undifferentiated hESC (Regea08/017), <b>B</b>) fusiform hESC-RPE, <b>C</b>) epithelioid hESC-RPE, <b>D</b>) cobblestone hESC-RPE. Scale bars, 100 µm, <b>E</b>) Gene expression of <b>1</b>: D407, <b>3</b>: ARPE-19, <b>5</b>: undifferentiated hESC, <b>7</b>: fusiform hESC-RPE, <b>9</b>: epithelioid hESC-RPE, <b>11</b>: cobblestone hESC-RPE, <b>13</b>: hFF. –RT- negative controls (i.e., samples not treated with reverse transcriptase) are placed adjacent to each sample in the same order: <b>2</b>: D407, <b>4</b>: ARPE-19, <b>6</b>: undifferentiated hESC, <b>8</b>: fusiform hESC-RPE, <b>10</b>: epithelioid hESC-RPE, <b>12</b>: cobblestone hESC-RPE, <b>14</b>: hFF. <b>F</b>) Culture periods of the studied samples in all analyses. Cells were selected based on their morphology rather than the culture period.</p

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

<div><p>Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates <i>SQSTM1/p62</i> mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.</p></div