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Mitochondria-targeted antioxidants protect Friedreich Ataxia fibroblasts from endogenous oxidative stress more effectively than untargeted antioxidants¹

MATTHIAS L. JAUSLIN, THOMAS MEIER, ROBIN A. J. SMITH^* and MICHAEL P. MURPHY^,2

MyoContract Ltd., CH-4410 Liestal, Switzerland;
^* Chemistry Department, University of Otago, Dunedin, New Zealand; and
MRC-Dunn Human Nutrition Unit, Wellcome Trust, Cambridge CB2 2XY, UK

²Correspondence: MRC-Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK. E-mail: mpm{at}mrc-dunn.cam.ac.uk

SPECIFIC AIMS

Friedreich Ataxia (FRDA) is caused by defective expression of the mitochondrial protein frataxin, which leads to increased mitochondrial oxidative damage; therefore, antioxidants targeted to mitochondria should be particularly effective at slowing disease progression. To test this hypothesis, we compared the potency of mitochondria-targeted antioxidants derived from coenzymeQ₁₀ or vitamin E with untargeted analogs in preventing cell death due to endogenous oxidative stress in fibroblasts from FRDA patients.

PRINCIPAL FINDINGS

1. The mitochondria-targeted coenzyme Q derivative MitoQ prevents cell death in fibroblasts from FRDA patients more effectively than the untargeted analogs decylubiquinone or Idebenone
FRDA, the most common recessively inherited ataxia, is caused by decreased expression of the gene for the mitochondrial protein frataxin. While the function of frataxin is uncertain, its deficit in FRDA patients increases mitochondrial oxidative stress and iron accumulation, which are considered to be the major causes of the progressive pathophysiology of FRDA. Therefore, antioxidant therapies that decrease oxidative damage may help to delay the onset and slow the progression of FRDA. Supporting this, the antioxidants coenzyme Q and its short-chain analog Idebenone (Fig. 1 A) decrease oxidative damage and improve disease-specific parameters in FRDA patients. However, these antioxidants distribute throughout the cell and body, with only a small proportion accumulating in mitochondria, the principal site of damage in FRDA. We developed mitochondria-targeted antioxidants by covalently coupling antioxidant moieties to the triphenylphosphonium cation. These lipophilic cations easily permeate phospholipid bilayers by a non-carrier-mediated process and accumulate several hundred-fold within mitochondria due to the large membrane potential (150–170 mV, negative inside). One of these mitochondria-targeted antioxidants, MitoQ (Fig. 1A ), is a derivative of coenzyme Q and is more effective at protecting mitochondria from exogenous pro-oxidants than untargeted analogs. MitoQ is of particular interest for FRDA because its antioxidant moiety is the same as Idebenone, which has shown some efficacy in treating FRDA-related cardiomyopathy. We tested whether the mitochondria-targeted antioxidant MitoQ is more potent than its untargeted analogs Idebenone and decylubiquinone (Fig. 1A ) in a cell model of FRDA.

View larger version (20K): [in this window] [in a new window]   Figure 1. Prevention of cell death in FRDA fibroblasts by coenzyme Q derivatives. A) Structures of the untargeted coenzyme Q derivatives Idebenone and decylubiquinone and of the mitochondria-targeted derivative MitoQ. B) Dependence of protection against BSO-induced cell death on concentration. The effect of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 1 µM) in the presence of MitoQ is shown. Cells were grown in 25% (v/v) M199 with Earle's balanced salts (EBS) and 64% (v/v) minimal essential medium-EBS supplemented with 10% (v/v) fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 µg/mL insulin, 10 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, and 2 mM glutamine. Cells were plated in 96-well plates at 3000 cells/well and allowed to attach overnight. Fresh culture medium containing the antioxidant compounds was added. FCCP was added 30 min before the antioxidants. 24 h later 1 mM BSO was added and cell viability was assessed 48 h after that. The cells were stained with calcein-AM in PBS and its conversion to calcein by esterases within live, but not dead, cells was measured fluorometrically to yield a percentage cell viability. Data shown are from a typical experiment, repeated at least 3 times, and each point represents the mean ± SD of 4 determinations. The methyltriphenylphosphonium cation (TPMP) did not protect against cell death up to at least 50 µM (data not shown). C) Concentrations of antioxidants that prevent 50% of cell death (EC50) in the absence (filled bars) or presence (open bars) of 1 µM FCCP. Dose/response curves as in panel B were used to calculate the EC50 concentrations and data are means ± SD of at least 3 independent experiments. *P < 0.01 by Student’s unpaired t test.

We used fibroblasts from FRDA patients. We had shown that while exposure to the glutathione synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO) leads to a 70% decrease in the glutathione (GSH) content of FRDA and control cells, only the FRDA fibroblasts die within 24–48 h of this treatment. This differential susceptibility to GSH depletion is due to elevated endogenous oxidative stress in the FRDA cells, thus the cause of cell death is similar to that seen in FRDA patients. The BSO-induced death of FRDA fibroblasts was blocked by exogenous antioxidants such as Idebenone, making this an good model to test the potency of MitoQ relative to untargeted antioxidants. We next compared the ability to prevent cell death of the targeted antioxidant MitoQ with the untargeted coenzyme Q derivatives Idebenone and decylubiquinone (Fig. 1A ). Although neither Idebenone nor decylubiquinone prevented cell death, MitoQ was potent at far lower concentrations (Fig. 1B ). In Fig. 1C , the ability of the antioxidants at preventing cell death is expressed as the concentration required to rescue 50% of the FRDA cells (EC₅₀). These data show that MitoQ (EC₅₀ 0.51±0.50 nM) is 800-fold more potent than Idebenone (EC₅₀ 426±102 nM) and 50-fold more potent than decylubiquinone (EC₅₀ 26.5±19 nM). The lower concentration at which MitoQ afforded protection is likely to be due to its selective accumulation into mitochondria driven by the membrane potential. To see whether this was the case, we abolished the mitochondrial membrane potential with the uncoupler FCCP, preventing the selective uptake of MitoQ into mitochondria. The concentration of FCCP used (1 µM) was the highest tolerated by the FRDA cells in the absence of BSO (data not shown) and is known to abolish the mitochondrial membrane potential in fibroblasts. Figure 1B shows that FCCP decreased the potency of MitoQ 25-fold, making its EC₅₀ statistically indistinguishable from that of the untargeted antioxidant decylubiquinone (Fig. 1C ; P=0.192). FCCP did not affect the potency of Idebenone or decylubiquinone, as the EC₅₀ values for prevention of cell death in the absence (filled bars) and presence (open bars) of FCCP were similar (Fig. 1C ). The data in Fig. 1 indicate that MitoQ is more potent antioxidant than Idebenone or decylubiquinone because of its membrane potential-dependent accumulation by mitochondria within fibroblasts.

2. The mitochondria-targeted vitamin E derivative MitoVit E protects against cell death in fibroblasts from FRDA patients more effectively thanuntargeted analogs
We next considered whether MitoVit E (Fig. 2 A), a mitochondria-targeted version of vitamin E (-tocopherol), was more effective at preventing BSO-induced cell death in FRDA fibroblasts than untargeted analogs (Fig. 2A ). We compared MitoVit E with the water-soluble vitamin E derivative Trolox (Fig. 2A ), which lacks the hydrophobic side chain of vitamin E, and with native vitamin E (Fig. 2A ). MitoVit E did prevent cell death in FRDA cells (EC₅₀ 23.6 nM±14.0 nM) and was 350-fold more potent than Trolox (EC₅₀ 8.2±6.3 µM) and 20-fold more potent than vitamin E (EC₅₀ 416±76 nM) (Fig. 2B ). However, the EC₅₀ of MitoVit E did not increase upon dissipating the mitochondrial membrane potential with FCCP (Fig. 2B ). When compared with the coenzyme Q derivatives (Fig. 1C, B ), MitoVit E was of similar potency to decylubiquinone, 20-fold more potent than Idebenone, and 46-fold less potent than MitoQ. Therefore, targeting the active moiety of vitamin E to mitochondria increases its antioxidant efficacy in a way similar to the targeting of coenzyme Q derivatives, but MitoVit E is less potent than MitoQ.

View larger version (17K): [in this window] [in a new window]   Figure 2. Prevention of cell death in FRDA fibroblasts by vitamin E derivatives. A) Structures of native vitamin E (-tocopherol) and a water-soluble derivative, Trolox. Neither is targeted to mitochondria, in contrast to the mitochondria-targeted derivative MitoVit E. B) The concentrations of antioxidants that prevent 50% of cell death (EC50) in the absence (filled bars) or presence (open bars) of 1 µM FCCP. Experimental details are as described in legend to Fig. 1 . Data are means ± SD of at least 3 independent experiments. **P < 0.001 by Student’s unpaired t test.

CONCLUSIONS AND SIGNIFICANCE

We have shown that the mitochondria-targeted antioxidants MitoQ and MitoVit E prevent cell death in BSO-treated FRDA fibroblasts. BSO causes GSH depletion, which leads to the death of FRDA cells, but not controls, consistent with the cell death arising from endogenous mitochondrial oxidative stress. Thus, the antioxidants block endogenous, FRDA disease-specific oxidative stress. This is the first demonstration that mitochondria-targeted antioxidants are able to reduce cell death arising from endogenous oxidative stress.

We wanted to see whether mitochondria-targeted antioxidants were more potent at preventing cell death than those that distributed throughout the cell. MitoQ was 800-fold more potent than the untargeted antioxidant Idebenone. That this increased potency was due to the mitochondrial accumulation of MitoQ driven by the membrane potential is suggested by the 25-fold decrease in its effectiveness when the mitochondrial membrane potential was abolished. Under these conditions, the protection against cell death by MitoQ was indistinguishable from that by the untargeted antioxidant decylubiquinone. The targeting of the antioxidant moiety of vitamin E to mitochondria also increased its antioxidant potency, MitoVit E being 350-fold more potent than Trolox and 20-fold more potent than vitamin E.

These data indicate that mitochondria-targeted antioxidants may be more effective as therapies than untargeted antioxidants. Figure 3 shows a schematic of the uptake of MitoQ into cells driven by the plasma membrane potential, followed by its accumulation into mitochondria driven by the mitochondrial membrane potential. This selective accumulation leads to high local concentrations of antioxidants within the mitochondria, thereby protecting the organelle against the mitochondrial oxidative damage that is the principal cause of FRDA pathophysiology. Furthermore, these compounds have been shown to accumulate within the heart and central nervous system after feeding to mice, suggesting that this procedure may be an efficient way of increasing the antioxidant content of mitochondria in those tissues most affected in FRDA. In many ways FRDA is a paradigm for other degenerative diseases that involve mitochondrial oxidative stress, such as Parkinson disease and Huntington disease. Therefore, the finding of efficacy in a FRDA cell model suggests that this approach may be worth extending to other diseases. However, there are still many uncertainties to be resolved. The targeted version of vitamin E was 350-fold more potent than Trolox and 20-fold more potent than vitamin E; however, its potency was unaffected by dissipating the mitochondrial membrane potential. This contrasts with the dramatic effect of uncoupling on the potency of MitoQ. The reasons for this are not known, but factors that may contribute include the greater hydrophobicity of MitoQ relative to MitoVit E (octanol/phosphate-buffered saline partition coefficients of 160 and 7.4 respectively) and the ability of the respiratory chain to recycle MitoQ, but not MitoVit E. These differences may also underlie the fact that MitoQ is 46-fold more effective as an antioxidant than MitoVit E in this system. Further exploration of these findings will lead to the development of new targeted antioxidants and to a deeper understanding of mitochondrial reactive oxygen species production and oxidative damage.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the selective uptake of MitoQ into the cytoplasm driven by the plasma membrane potential (p) and its subsequent accumulation by mitochondria driven by the mitochondrial membrane potential (m). Within mitochondria, this several hundred-fold accumulation of MitoQ relative to its concentration in the external fluid will protect the organelle from oxidative damage far more effectively than untargeted antioxidants.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0240fje; doi: 10.1096/fj.03-0240fje

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