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. 2021 Nov 13;10(11):1806.
doi: 10.3390/antiox10111806.

The Protective Effect of Ubiquinone against the Amyloid Peptide in Endothelial Cells Is Isoprenoid Chain Length-Dependent

Javier Frontiñán-Rubio  1   2 , Yoana Rabanal-Ruiz  1   2 , Mario Durán-Prado  1   2 , Francisco Javier Alcain  1   2
Affiliations

The Protective Effect of Ubiquinone against the Amyloid Peptide in Endothelial Cells Is Isoprenoid Chain Length-Dependent

Javier Frontiñán-Rubio et al. Antioxidants (Basel). .

Abstract

Vascular brain pathology constitutes a common feature in neurodegenerative diseases that could underlie their development. Indeed, vascular dysfunction acts synergistically with neurodegenerative changes to exacerbate the cognitive impairment found in Alzheimer's disease. Different injuries such as hypertension, high glucose, atherosclerosis associated with oxidized low-density lipoprotein or inflammation induce NADPH oxidase activation, overproduction of reactive oxygen species, and apoptosis in endothelial cells. Since it has been shown that pretreatment of cultured endothelial cells with the lipophilic antioxidant coenzyme Q10 (CoQ10) displays a protective effect against the deleterious injuries caused by different agents, this study explores the cytoprotective role of different CoQs homologues against Aβ25-35-induced damage and demonstrates that only pretreatment with CoQ10 protects endothelial brain cells from Aβ25-35-induced damage. Herein, we show that CoQ10 constitutes the most effective ubiquinone in preventing NADPH oxidase activity and reducing both reactive oxygen species generation and the increase in free cytosolic Ca2+ induced by Aβ25-35, ultimately preventing apoptosis and necrosis. The specific cytoprotective effect of CoQ with a side chain of 10 isoprenoid units could be explained by the fact that CoQ10 is the only ubiquinone that significantly reduces the entry of Aβ25-35 into the mitochondria.

Keywords: Alzheimer´s disease; NADPH oxidase; amyloid beta; coenzyme Q; endothelial cells; ubiquinol.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Analysis of apoptosis and necrosis in bEnd.3 cells exposed to Aβ25–35 and different coenzyme Q homologues (CoQs). Cells were pretreated with CoQ2, CoQ6, CoQ9, or CoQ10 for 24 h and then treated with Aβ25–35 for 24 h. (A) Apoptosis was determined by morphological criteria in cells stained with Hoescht 33258 (white arrows point to apoptotic nuclei). (B) Necrosis was determined by quantifying necrotic cells (EtBr positive cells—red) versus viable cells (Calcein-AM positive cells—green). Results show the percentage of apoptotic or necrotic cells versus total cells. Data are presented as the mean ± SEM. Student’s t-test was used to compare the differences between groups treated with the same ubiquinone and Dunnett’s multiple comparison test to compare each Aβ25–35 (plus or minus ubiquinone) treatment with the control. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, (n = 3).
Figure 2
Figure 2
Characterization of NADPH oxidase levels. bEnd.3 cells were pretreated with CoQ2, CoQ6, CoQ9, or CoQ10 for 24 h and then treated with Aβ25–35 for 24 h. NADPH oxidase was assessed using a NOX1 ELISA kit. Results are expressed as pg/105 cells compared with the standard. Data are presented as the mean ± SEM. Student’s t-test was used to compare the differences between groups treated with the same ubiquinone and Dunnett’s multiple comparison test to compare each Aβ25–35 (plus or minus ubiquinone) treatment with the control. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 3).
Figure 3
Figure 3
Determination of ROS levels, free cytosolic Ca2+, and mitochondrial status in cells exposed to Aβ25–35 and different CoQs. bEnd.3 cells were pretreated for 24 h with CoQ2, CoQ6, CoQ9, or CoQ10 and then treated with Aβ25–35 for 24 h. (A) Levels of O2− were assessed by a MitoSOX red fluorescent probe. (B) H2O2 levels were quantified using a H2DCF-DA green fluorescent probe. (C) Free cytosolic calcium levels were quantified using a Fluo-4 green fluorescent probe. (D) Mitochondrial status was assessed using a Mitotracker CMXRos probe, which is dependent on mitochondrial membrane potential. Results show the RFUs normalized vs. control cells. Data are presented as the means ± SEM. Student’s t-test was used to compare the differences between groups treated with the same ubiquinone and Dunnett’s multiple comparison test to compare each Aβ25–35 (plus or minus ubiquinone) treatment with the control. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (n = 3) (scale bar = 20 µM).
Figure 4
Figure 4
Evaluation of the energetic cellular phenotype in cells exposed to Aβ25–35 and different CoQs. bEnd.3 cells were pretreated for 24 h with CoQ2, CoQ6, CoQ9, or CoQ10 and then treated with Aβ25–35 for 24 h. (A) OCR levels are expressed as oxygen consumption (picomoles) per minute. (B) ECAR levels are expressed as changes in pH per minute. Results were normalized against the total number of cells per well. Data are presented as the mean ± SEM. Student’s t-test was used to compare the differences between groups treated with the same ubiquinone and Dunnett’s multiple comparison test to compare each Aβ25–35 (plus or minus ubiquinone) treatment with the control. ns: not significant, * p < 0.05, ** p < 0.01 (n = 3).
Figure 5
Figure 5
Real-time characterization of Aβ25–35 internalization and mitochondrial dynamics. bEnd.3 cells were pretreated with CoQ2, CoQ6, CoQ9, or CoQ10 for 24 h and were then treated with Aβ25–35 HyLiteTM 488. (A) Levels of fluorescent Aβ25–35 were measured every two hours and normalized vs. 0 h of exposure. Images show internalized Aβ25–35 after 10 h of exposure in individual cells (white line represents the cell outline) (scale bar = 20 µM). (B) Mitochondrial status was quantified as the intensity of Mitotracker CMXRos compared to 0 h of exposure to Aβ25–35. Data are presented as the mean ± SEM. Dunnett’s multiple comparison test compared each Aβ25–35 plus ubiquinone treatment with Aβ25–35. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, (>30 cells/condition).
Figure 6
Figure 6
Aβ25–35 internalization and mitochondrial colocalization in bEnd.3 cells exposed to Aβ25–35 and different CoQs. bEnd.3 cells were pretreated with CoQ2, CoQ6, CoQ9, or CoQ10 for 24 h and then treated with Aβ25–35 HyLiteTM 488 for 24 h. (A) Internalized fluorescent Aβ25–35 is expressed as RFUs. (B) Mitochondrial-fluorescent Aβ25–35 was measured as the percentage of fluorescent Aβ25–35 present into mitochondria compared to total internalized Aβ25–35. (C) Images show cells stained with Mitotracker CMXros (red) and Aβ25–35 (green) and their colocalization. Detailed images show the colocalized areas in white (scale = 20 µM/scale augmented = 5 µM). Data are presented as the mean ± SEM. Dunnett’s multiple comparison test compared each Aβ25–35 plus ubiquinone treatment with Aβ25–35. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, (n = 3).
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