Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Feb 4;12(2):381.
doi: 10.3390/antiox12020381.

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status

Inês Ramos Rego  1   2   3   4 , Daniela Silvério  1   2   3   4 , Maria Isabel Eufrásio  1   2   3   4 , Sandra Sofia Pinhanços  2   5 , Bruna Lopes da Costa  6   7 , José Teixeira  2   5 , Hugo Fernandes  2   5   8 , Yang Kong  7 , Yao Li  7 , Stephen H Tsang  6   7   9 , Paulo J Oliveira  2   5 , Rosa Fernandes  1   2   3   10   11 , Peter M J Quinn  7 , Paulo Fernando Santos  1   2   3   12 , António Francisco Ambrósio  1   2   3   10 , Celso Henrique Alves  1   2   3   10
Affiliations

TRAP1 Is Expressed in Human Retinal Pigment Epithelial Cells and Is Required to Maintain their Energetic Status

Inês Ramos Rego et al. Antioxidants (Basel). .

Abstract

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and blindness in elderly people worldwide. The damage to the retinal pigment epithelium (RPE) triggered by oxidative stress plays a central role in the onset and progression of AMD and results from the excessive accumulation of reactive oxygen species (ROS) produced mainly by mitochondria. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone that contributes to the maintenance of mitochondrial integrity by decreasing the production and accumulation of ROS. The present study aimed to evaluate the presence and the role of TRAP1 in the RPE. Here, we report that TRAP1 is expressed in human adult retinal pigment epithelial cells and is located mainly in the mitochondria. Exposure of RPE cells to hydrogen peroxide decreases the levels of TRAP1. Furthermore, TRAP1 silencing increases intracellular ROS production and decreases mitochondrial respiratory capacity without affecting cell proliferation. Together, these findings offer novel insights into TRAP1 functions in RPE cells, opening possibilities to develop new treatment options for AMD.

Keywords: age-related macular degeneration (AMD); mitochondria; oxidative stress; retinal pigment epithelium (RPE); tumor necrosis factor receptor-associated protein 1 (TRAP1).

PubMed Disclaimer

Conflict of interest statement

Stephen H. Tsang receives research support from Abeona Therapeutics, Inc and Emendo. He is also the founder of Rejuvitas and is on the advisory board for Nanoscope Therapeutics. Peter M.J. Quinn receives research support from Rejuvitas, Inc. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
TRAP1 is expressed in RPE cells. TRAP1 mRNA (A) and protein (B) are expressed in human ARPE-19 cells. TRAP1 is located mainly in the mitochondria of ARPE-19 cells where it co-localizes with MitoTracker green (C) and the mitochondrial protein SAM50 (D). TRAP1 mRNA (E) is also expressed in iPSC-derived RPE cells (iRPE, two different lines), in RPE from human donors and in RPE spheroids (E). TRAP1 protein is also present in protein extracts from RPE spheroids (two different lines) (F) and co-localizes with the mitochondrial marker TOM20-GFP (G). NTC: Non-template control. Scale bars: 10 µm.
Figure 2
Figure 2
TRAP1 levels decrease upon challenge with hydrogen peroxide. ARPE-19 cells were treated with different doses of H2O2 (0; 31.25; 62.5; 125; 125; 250; 500; 1000 and 2000 μM) for 24 h (A). The dose of 500 μM was selected for the following experiments. ARPE-19 cells were treated, or not, with 500 μM H2O2 for 24 h (B–E). Scale bars in (B,C): 20 µm. Exposure to 500 μM H2O2 for 24 h resulted in lower levels of TRAP1. NS: Non-stimulated (black bars); ST: stimulated with H2O2 (grey bars). The values are presented as a percentage of the control (non-treated cells). Cell metabolism was assessed using the resazurin assay. Four independent experiments were performed (n = 4). For the graph (A), statistical significance was calculated by using the Mann–Whitney U test. For the graph (E), statistical significance was calculated by using an unpaired t-test. Statistically significant values: * p < 0.05, *** p < 0.001. In the graphs, all values are expressed as mean ± standard error of the mean (SEM).
Figure 3
Figure 3
TRAP1 silencing decreases ARPE-19 cellular metabolism. Seventy-two hours after TRAP1 silencing using specific siRNAs (siTRAP1), the levels of TRAP1 were reduced by approximately 50% (A,C–E). Some cells still expressed high levels of TRAP1 (C, arrowheads).The scramble (control) RNA (siCTL) did not affect TRAP1 levels (A,B–E) compared with the incubation with only the transfection reagent (lipofectamine RNAiMAX) (A,D,E). Scale bars in (A–C): 20 µm. TRAP1 siRNA-mediated silencing decreased the cell metabolic activity as measured by the resazurin assay (F). TRAP1 silencing did not further decrease cell metabolic activity upon treatment of cells with 500 µM H2O2 for 24 h (G). The total number of cells (H), cell mass measured by SRB assay (I) and the number of proliferating Ki67-positive cells (H) did not change upon TRAP1 silencing. Black bars: cells incubated with lipofectamine RNAiMAX reagent only. Grey bars: cells transfected with lipofectamine RNAiMAX reagent and siCTL. White bars: cells transfected with lipofectamine RNAiMAX reagent and siTRAP1. Number of independent experiments: (E) n = 5; (F) n = 8; (G) n = 4; (H) n = 5; (I) n = 6; (J) n = 4. Statistical significance was calculated by using the Mann–Whitney U test. Statistically significant values: * p < 0.05, ** p < 0.01, *** p < 0.001. In the graphs, all values are expressed as mean ± standard error of the mean (SEM).
Figure 4
Figure 4
TRAP1 silencing in ARPE-19 cells increase the levels of superoxide anion. Representative confocal microscopy images of fluorescent detection of ROS by dihydroethidium (DHE) in cells transfected with siCTL (A,D) and siTRAP1 (B,E) for 24 h (A,B) and 72 h (D,E). Scale bars: 20 µm. Mean fluorescence intensity (MFI) of at least 4 images per condition/experiment was measured using Image J v1.53C and divided by the number of the DAPI-positive nuclei (MFI/cell) (C,F). Four independent experiments were performed (n = 4). Statistical significance was calculated by using the Mann––Whitney U test. Statistically significant values: * p < 0.05, ** p < 0.01. In the graphs, all values are expressed as mean ± standard error of the mean (SEM).
Figure 5
Figure 5
TRAP1 silencing increases mitochondrial elongation without affecting mitochondrial ultrastructure. Transmission electron microscopy (TEM) images of mitochondria from ARPE-19 cells transfected with siCTL (A) or siTRAP1 (B). Representative mitochondria are presented. Scale bars in A and B: 100 nm. Analysis of confocal microscopy images obtained using Image-iT™ TMRM Reagent (TMRM) for mitochondrial labelling in ARPE-19 cells transfected with siCTL (C) or siTRAP1 (D). Scale bars in C and D: 50 µm. The mitochondrial interconnectivity was similar between the two experimental groups (E). siTRAP1 cells showed increased mitochondrial elongation (F). Statistical significance was calculated by using an unpaired t-test. Statistically significant values: *** p < 0.001. In the graphs, all values are expressed as mean ± standard error of the mean (SEM).
Figure 6
Figure 6
TRAP1 silencing drives a quiescent metabolic state. Representative images of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements using a Seahorse XFe96 Extracellular Flux Analyzer (A–F). siTRAP1 cells presented a decreased basal and maximal OCR compared with the siCTL cells (B,C). A shift towards a more quiescent phenotype was observed when the average mitochondrial basal OCR was plotted against the average basal ECAR (D). A decrease in mitochondrial OCR was paralleled by a slight decrease in ECAR (D). siTRAP1 silencing significantly reduced the ATP production-linked (E) and proton leak-related (F) OCR. Data are the mean ± SEM of 3 independent experiments and show the effects of mitochondrial inhibitors (a) oligomycin (2 µM), (b) FCCP (1 µM) and (c) antimycin A (1 µM) plus rotenone (1 µM) injected as indicated. The results are expressed as pmol O2/min/cell mass for OCR and mpH/min/cell mass for ECAR. Statistical significance was calculated by using an unpaired t-test. Statistically significant values: ** p < 0.01. In the graphs, values are expressed as mean ± standard error of the mean (SEM).
Figure 7
Figure 7
Graphical representation of the highlights of our study. TRAP1 is present in retinal pigment epithelial cells and is located mainly in the mitochondria. TRAP1 silencing increases reactive oxygen species (ROS) production and decreases mitochondrial respiratory capacity.
See this image and copyright information in PMC

References

    1. Deng Y., Qiao L., Du M., Qu C., Wan L., Li J., Huang L. Age-related macular degeneration: Epidemiology, genetics, pathophysiology, diagnosis, and targeted therapy. Genes Dis. 2021;9:62–79. doi: 10.1016/j.gendis.2021.02.009. - DOI - PMC - PubMed
    1. Algvere P.V., Kvanta A., Seregard S. Drusen maculopathy: A risk factor for visual deterioration. Acta Ophthalmol. 2016;94:427–433. doi: 10.1111/aos.13011. - DOI - PubMed
    1. Mitchell P., Liew G., Gopinath B., Wong T.Y. Age-related macular degeneration. Lancet. 2018;392:1147–1159. doi: 10.1016/S0140-6736(18)31550-2. - DOI - PubMed
    1. Fields M.A., Del Priore L.V., Adelman R.A., Rizzolo L.J. Interactions of the choroid, Bruch’s membrane, retinal pigment epithelium, and neurosensory retina collaborate to form the outer blood-retinal-barrier. Prog. Retin. Eye Res. 2020;76:100803. doi: 10.1016/j.preteyeres.2019.100803. - DOI - PubMed
    1. Alves C.H., Fernandes R., Santiago A.R., Ambrósio A.F. Microglia Contribution to the Regulation of the Retinal and Choroidal Vasculature in Age-Related Macular Degeneration. Cells. 2020;9:1217. doi: 10.3390/cells9051217. - DOI - PMC - PubMed

LinkOut - more resources