<i>In vitro</i> phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Olchawa, Magdalena; Krzysztyńska-Kuleta, Olga; Duda, Mariusz; Pawlak, Anna; Pabisz, Paweł; Czuba-Pelech, Barbara; Sarna, Tadeusz

doi:10.1080/10715762.2019.1603377

Cited by 15 publications

(23 citation statements)

References 74 publications

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“…Such compounds also target activities of the proteolytic pathway. The accumulation of these molecules further exacerbates the susceptibility of RPE cells to blue light-induced damage [ 22 , 23 ]. Constituents of long-chain polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), present at high quantities in POS, also contribute to oxidative stress in RPE cells.…”

Section: Introductionmentioning

confidence: 99%

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

Keeling

Culling

Johnston

et al. 2020

IJMS

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Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.

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Section: Introductionmentioning

confidence: 99%

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

Keeling

Culling

Johnston

et al. 2020

IJMS

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“…Our recent study provided evidence that sub‐lethal photic stress mediated by POS‐rhodopsin photobleaching products induced oxidation of cellular proteins (Olchawa et al., 2019). In an independent study, we have also demonstrated that photic stress mediated in ARPE‐19 cells by phagocytized lipofuscin granules led to oxidation of the cellular proteins and damage of the cell cytoskeleton, which plays an important role in nanomechanical properties of the cell (Wiktor et al, 2018).…”

Section: Resultsmentioning

confidence: 99%

“…The viability of cells subjected to photic treatment was analyzed by MTT assay as described previously (Olchawa et al., 2019). In brief, immediately or 24 hr after treatment, thiazolyl blue tetrazolium bromide in complete medium was added to the culture wells (final concentration of 0.5 mg/ml) followed by incubation for 0.5 hr at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…To monitor oxidation of proteins in ARPE‐19 cells subjected to irradiation with blue light, coumarin boronic acid (CBA) was used as the sensitive fluorogenic probe according to the previously described procedure (Michalski et al., 2014; Olchawa et al., 2019). In brief, control and blue light irradiated cells immediately after irradiation were incubated with catalase (580 U/ml) for 2 min.…”

Section: Methodsmentioning

confidence: 99%

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The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study

Olchawa

Szewczyk

Krzysztyńska-Kuleta

et al. 2020

Pigment Cell Melanoma Res

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Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α‐tocopherol, was analyzed by electron paramagnetic resonance oximetry, time‐resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE‐19 cells exposed to blue light. Phagocytosis of FITC‐labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells—their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

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“…Propidium iodide staining. Survival of HEK293 cells incubated with different concentrations of inhibitors (U73122 or BIM-46187) was also confirmed immediately and 24 h after treatment by quantifying nuclear staining with the membrane impermeant fluorescent dye propidium iodide (PI), according to previously described protocol (35). In brief, propidium iodide (final concentration 100 lM) was added to control cells and cultures incubated with selected inhibitors.…”

Section: Methodsmentioning

confidence: 99%

Melanopsin Signaling Pathway in HEK293 Cell Line with Stable Expression of Human Melanopsin: Possible Participation of Phospholipase C beta 4 and Diacylglycerol

Krzysztyńska-Kuleta

Olchawa

Sarna

2021

Photochem & Photobiology

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Melanopsin, a member of the G protein-coupled receptors family, is involved in non-image-forming functions including circadian rhythm, sleep regulation and pupil response. In spite of significant research efforts, the signaling cascade involving melanopsin photoactivation remains poorly characterized. Here, we analyzed the effects of photoactivation of melanopsin on phospholipase C (PLC) and diacylglycerol. As an in vitro model, HEK293 cells with stable expression of human melanopsin were used. Although both the PLC b1 and PLC b4 subtypes were activated by the cell exposure to blue light, only PLC b4 appeared to play a significant role in the studied melanopsin signaling pathway. We have demonstrated, for the first time, that cells expressing human melanopsin and enriched with 11-cis-retinal exhibited significantly increased diacylglycerol level. To determine the role of phospholipase C and involvement of diacylglycerols, two approaches were employed: inhibition of the G protein and phospholipase C (using the BIM-46187 and U73122 inhibitors, respectively), and gene silencing using siRNA of PLC b1 and PLC b4 . While silencing the PLC b4 gene and using U73122 inhibited the diacylglycerol and calcium ion responses, the FOS gene expression level was only partially reduced. These results may facilitate a better understanding of the role of phospholipase C and diacylglycerols in the melanopsin signaling pathway.

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In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Cited by 15 publications

References 74 publications

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study

Melanopsin Signaling Pathway in HEK293 Cell Line with Stable Expression of Human Melanopsin: Possible Participation of Phospholipase C beta 4 and Diacylglycerol

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