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FASEB J. 2010 Oct;24(10):3733-43. doi: 10.1096/fj.09-152728. Epub 2010 May 21.
Reactive oxygen species, oxidative stress, and cell death correlate with level of CoQ10 deficiency.
Quinzii CM1,
López LC,
Gilkerson RW,
Dorado B,
Coku J,
Naini AB,
Lagier-Tourenne C,
Schuelke M,
Salviati L,
Carrozzo R,
Santorelli F,
Rahman S,
Tazir M,
Koenig M,
DiMauro S,
Hirano M.
- 1
- Department of Neurology, Columbia University Medical Center, 630 W. 168th St., P&S 4-423, New York, NY 10032, USA.
Abstract
Coenzyme Q(10) (CoQ(10)) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ(10) deficiency is unknown. We determined previously that severe CoQ(10) deficiency in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations produces divergent alterations of bioenergetics and oxidative stress. Here, to better understand the pathogenesis of CoQ(10) deficiency, we have characterized the effects of varying severities of CoQ(10) deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10) biosynthesis. Levels of CoQ(10) seem to correlate with ROS production; 10-15% and >60% residual CoQ(10) are not associated with significant ROS production, whereas 30-50% residual CoQ(10) is accompanied by increased ROS production and cell death. Our results confirm that varying degrees of CoQ(10) deficiency cause variable defects of ATP synthesis and oxidative stress. These findings may lead to more rational therapeutic strategies for CoQ(10) deficiency.
Figure 1.
CoQ10 biosynthesis pathway. CoQ10 is composed of a benzoquinone and a decaprenyl side chain. ADCK3 (CABC1) is a kinase that modulates CoQ10 synthesis possibly through phosphorylation of COQ3 (). The function of COQ9 is unknown.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 2.
Cell growth rates over 24, 48, and 72 h in galactose medium supplemented with regular FBS (A) or galactose medium supplemented with dialyzed FBS (B). Values are means of ≥3 measurements. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 3.
Adenine nucleotide levels in CoQ10-deficient skin fibroblasts and controls after 48 h in galactose medium supplemented with regular FBS (A, B) or dialyzed FBS (C, D). Values are means of ≥3 measurements. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 4.
Assessment of mitochondrial membrane potential with TMRE in live cells cultured for 48 h in galactose medium with regular FBS (A) and dialyzed FBS (B). The y axis of the TMRE graph represents the relative percentage fluorescence intensity of each cell sample compared with control cells. Values are means of ≥3 measurements. *P < 0.05 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 5.
Quantitation of MitoSOX staining by flow cytometry and oxidation of lipids and proteins in CoQ10-deficient skin fibroblasts and controls after 48 h in galactose medium with regular FBS (A–C) or dialyzed FBS (D–F). The y axis of the MitoSOX graphs represents the fluorescence intensities of MitoSOX cell samples relative to unstained fibroblasts (units listed on left y axis). Diamonds indicate cellular CoQ10 levels (units listed on right y axis). Values are means of ≥3 measurements. *P < 0.05, ***P < 0.001 vs. controls. MDA, malondialdehyde; 4HE, 4-hydroxyalkenals.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 6.
Trypan blue staining of CoQ10-deficient skin fibroblasts and controls after 1 wk in galactose medium with regular FBS. Bars represent means of ≥3 values. ***P < 0.001 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 7.
Vybrant apoptosis assay was used to assess apoptosis (PO-PRO1; A) and necrosis (7-AAD; B) in COQ2 mutant fibroblasts after 1 wk in galactose medium supplemented with regular FBS. Bars represent means of ≥3 measurements. Red fluorescence 7-AAD labels necrotic cells. PO-PRO-1 dye, a violet-fluorescent nucleic acid stain, is permeant to apoptotic cells, but not live cells. *P < 0.05, ***P < 0.001 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 8.
Morphology of nuclei by DAPI staining in COQ2 mutant fibroblasts after 1 wk in galactose medium supplemented with regular FBS. Normal nuclei are seen in the left upper and lower panels (control); fragmented nuclei are seen in P6 (center) and in P2 (right). Original view: top panels, ×60; bottom panels, ×200. Figure shows an experiment representative of ≥3 different experiments. Scale bar = 20 μm.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 9.
Assessment of mitochondrial membrane potential by TMRE in COQ2 mutant fibroblasts after 1 wk in galactose medium supplemented with regular FBS. The y axis of the TMRE graph represents the fluorescence intensity of the samples (red trace, patients; black trace, controls) relative to the background intensity (gray trace, unstained fibroblasts). Values are means of ≥3 measurements.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 10.
Mitochondria morphology by MitoTracker Red in COQ2 mutant cells after 1 wk in galactose medium supplemented with regular FBS. Top panels: normal reticular mitochondria (control). Bottom panels: abnormal blebbed and fragmented mitochondria (P2). Original detail view: ×100 (right panels). Figure shows an experiment representative of ≥3 different experiments.
FASEB J. 2010 Oct;24(10):3733-3743.
Figure 11.
Quantitative real-time PCR showing mtDNA depletion in P6 fibroblasts after 1 wk in galactose medium supplemented with regular FBS. Bars represent means ± sd of ≥3 experiments. *P < 0.05 vs. controls.
FASEB J. 2010 Oct;24(10):3733-3743.
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