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Human Molecular Genetics Pages 425-430  

The Friedreich’s ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis
Introduction
Results
   Levels of frataxin mRNA levels are reduced in FRDA fibroblasts
   FRDA fibroblasts are more sensitive to H2O2 and iron than controls
   An iron chelator rescues FRDA fibroblasts preferentially from death
   Pyruvate and uridine supplementation preferentially boosts growth of FRDA fibroblasts
   Depletion of intracellular calcium ion rescues FRDA fibroblasts and controls from oxidant-induced death
   Apoptosis inhibitors preferentially rescue FRDA fibroblasts from death
Discussion
Materials And Methods
   Reagents
   Patients, DNA analysis and cell culture conditions
   Frataxin expression analysis
   Cell viability assay
   Measurement of mitochondrial iron
Acknowledgements
References

The Friedreich’s ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

The Friedreich’s ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

Alice Wong1, Joy Yang1, Patrizia Cavadini2,3, Cinzia Gellera3, Bo Lonnerdal4, Franco Taroni2,3,* and Gino Cortopassi1,*

1Department of Molecular Biosciences, 1311 Haring Hall and 4Department of Nutrition, University of California, Davis, CA 95616, USA and 2Laboratory of Cellular Pathology and 3Division of Biochemistry and Genetics, Istituto Nazionale Neurologico ‘Carlo Besta’, Milan, Italy

Received September 9, 1998; Revised and Accepted December 9, 1998

Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich’s ataxia (FRDA), an autosomal recessive neurodegenerative disease. Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency. These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients. FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls. The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40%. Treatment of cells with the intracellular Ca2+ chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death. Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine-induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts. These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.

INTRODUCTION

Friedreich’s ataxia (FRDA), the most common hereditary ataxia, is an autosomal recessive disease characterized by unsteady gait, muscle weakness of the legs, neuropathy and cardiomyopathy (1). Degeneration of the spinal cord, mainly the posterior columns and spinocerebellar tracts, occurs in FRDA patients. The cervical region is most severely damaged along with loss of large cells in the dorsal root ganglia, followed by loss of large myelinated axons in the peripheral nerve (2).

The genetic defect observed most frequently in FRDA patients is a GAA repeat expansion in the first intron of the gene encoding frataxin, a protein of unknown function (3). The GAA repeat expansion appears to inhibit both gene transcription and replication (4). While normal individuals possess 8-30 GAA repeats, FRDA patients have from 160 to >1200 copies of the GAA repeat (3,5). The genome of Saccharomyces cerevisiae contains a frataxin homolog, YFH1 (yeast frataxin homolog), knockout of which causes increased sensitivity to oxidative stress induced by hydrogen peroxide (H2O2), iron and copper, and an increased level of mitochondrial iron (6,7). Depletion of mtDNA and a decrease in respiration were observed in [Delta]YFH1 cells (7,8). In human cells, frataxin has been localized to mitochondria (9), and is highly expressed in heart, muscle, liver and pancreas (3,9). Interestingly, decreases in the activity of the mitochondrial iron-sulfur-containing proteins aconitase and respiratory chain complexes I, II and III have been observed in heart biopsies from a limited number of FRDA patients (10), which would suggest a pathogenic role for oxidant stress, as iron-sulfur proteins are exquisitely sensitive to the accumulation of reactive oxygen species.

Studies reported here address whether human FRDA fibroblasts may be a reasonable model system in which to study the cellular effects of GAA expansion (i.e. whether the mutation confers a predisposition to cell death), and also to test in this model system potential therapeutic agents that would be expected to inhibit death induced by oxidant stress on mechanistic grounds.

RESULTS

Levels of frataxin mRNA levels are reduced in FRDA fibroblasts

Frataxin mRNA levels were measured by RT-PCR and normalized to those of an unrelated mitochondrial protein, [beta]-MTP, the [beta]-subunit of mitochondrial trifunctional protein. The level of frataxin expression was 33.6% of the control value (see Materials and Methods).

FRDA fibroblasts are more sensitive to H2O2 and iron than controls

The sensitivity of five fibroblastoid FRDA cell lines and five control lines to H2O2 was examined. FRDA cells exhibited significantly greater sensitivity than controls to H2O2 at doses of 50 and 100 µM H2O2 (Fig. 1a). FRDA and control cells exhibited 81.7 ± 6.8 (95% CI) versus 93.2 ± 1.0% viability at 50 µM H2O2, and 51.6 ± 3.2% and 86.4 ± 6.2% at 100 µM H2O2, respectively.


Figure 1. (a) (top) Sensitivity of FRDA fibroblasts to oxidative stress. Cells were exposed to increasing concentrations of H2O2 for 6 h, after which time viability was determined by the trypan blue exclusion assay. Means of 28 independent experiments are shown and bars represent two standard errors of the mean, which in normally distributed data are considered to represent 95% confidence intervals. Filled squares and circles represent the mean survival of five control and five FRDA fibroblast lines, respectively. Significance levels at each individual dose are represented by asterisks: *P < 0.05 and **P < 0.005. (b) (bottom) Hypersensitivity of FRDA fibroblasts to Fe3+. Five control and five FRDA fibroblast cell lines were exposed to increasing concentrations of iron. [square], controls; [circle], FRDA. Significance levels at each individual dose are represented by asterisks: **P < 0.005. Means of three independent experiments and error bars representing two standard errors of the mean are shown.

FRDA fibroblasts were hypersensitive to iron, an element known to induce oxidative stress, relative to controls (Fig. 1b). Control cells maintained nearly 100% viability at up to 5 mM FeCl3, while only 50% of the FRDA fibroblasts were viable at 1 mM FeCl3.

An iron chelator rescues FRDA fibroblasts preferentially from death

Deferoxamine mesylate is a specific iron chelator used in clinical practice (11). FRDA fibroblasts (Fig. 2b) treated with deferoxamine were rescued from H2O2-induced death to a greater extent than controls (Fig. 2a).ab


Figure 2. Protection of cells from H2O2-induced death by the iron chelator deferoxamine mesylate (DF). Cells were exposed to 250 µM DF for 18 h prior to the addition of H2O2. [square], cells; [circle], cells + 250 µM DF. The mean survival of (a) five control and (b) five FRDA fibroblasts is shown. Significance levels at each individual dose are represented by asterisks: **P < 0.005. Averages of 28 and three independent experiments are shown for cells with or without deferoxamine, respectively, and bars represent two standard errors of the mean.

One possible cause of increased sensitivity to oxidant stress in the human FRDA fibroblasts is an increased concentration of mitochondrial iron, as has been observed in the yeast knockout of the frataxin homolog (6,7). We did observe a small (40%) increase in mean concentration of mitochondrial iron in FRDA fibroblasts: 8.93 versus 6.36 nmol/mg in controls; however, this difference was not statistically significant (Fig. 3a). Other experiments with three FRDA and three control lymphoblast lines gave similar results, i.e. a 30% increase in mean mitochondrial iron content, but the difference in means was not significant at the 95% confidence level (data not shown).ab


Figure 3. (a) (top) Mitochondrial iron levels of FRDA and control fibroblasts. FRDA fibroblasts 53 and 1037, and control cell lines 269 and 305 were used. Cells were grown in media and mitochondria isolated. Iron was measured by the atomic absorption method (26). Averages of two independent experiments are shown, and error bars represent two standard errors of the mean. (b) (bottom) Growth of FRDA and control fibroblasts in pyruvate- and uridine-supplemented media. FRDA and control cell lines were grown in MEM + 10% FBS + 8 mM glutamine (-P, -U) or MEM + 10% FBS + 8 mM glutamine + 1 mM pyruvate + 50 mg/ml uridine (+P, +U) for 24 h, after which total cell number was determined. The mean percentage growth from five FRDA and five control fibroblasts is represented from six independent experiments. Growth in media containing pyruvate and uridine was significant, P < 0.005 for both FRDA and control fibroblasts, in comparison with media without pyruvate and uridine. Bars represent two standard errors of the mean.

Pyruvate and uridine supplementation preferentially boosts growth of FRDA fibroblasts

Supplementation of tissue culture medium with uridine and pyruvate rescues growth defects resulting from mitochondrial dysfunction, presumably because the main function of mitochondria in a high-glucose tissue culture environment is to provide pyrimidines for nucleic acid synthesis (12,13). If frataxin expansions cause mitochondrial dysfunction, then one would expect that supplementation of tissue culture medium with uridine and pyruvate would preferentially boost the growth rate of FRDA versus control fibroblasts. Supplementation with pyruvate and uridine strongly and significantly (P < 0.005) boosted the growth rate of FRDA fibroblasts, consistent with a mitochondrial biochemical defect (Fig. 3b).

Depletion of intracellular calcium ion rescues FRDA fibroblasts and controls from oxidant-induced death

Although the complete mechanism by which oxidant stress kills cells is not completely understood, oxidant stress often causes a rise in intracellular Ca2+, which may be required for cell death (14,15). Treatment of cells with an intracellular Ca2+ chelator, BAPTA-AM, provided significant rescue of both control and FRDA fibroblasts from oxidant-induced death (Fig. 4), and depletion of Ca2+ from cell media also provided significant rescue (data not shown).a


Figure 4. Protection of FRDA and control fibroblasts from death induced by oxidant stress by the intracellular Ca2+ chelator BAPTA-AM. Survival of (a) (top) five control cell lines and (b) (bottom) five FRDA fibroblasts. [square], cells; [circle], cells + 10 µM BAPTA-AM. Significance levels at individual doses are represented by asterisks: **P < 0.005. Averages from three and 28 independent experiments are shown for cells with or without BAPTA-AM, respectively, with bars representing 95% CI.

We previously have shown that cyclosporin A (CsA) can protect cells with mitochondrial mutations from oxidant-induced death (14), presumably by blocking the mitochondrial permeability transition (MPT). To investigate this possibility of protection in FRDA fibroblasts, cells were pre-treated with 1 µM CsA for 30 min before addition of H2O2, and no protection was observed in FRDA nor control fibroblasts (data not shown).

Apoptosis inhibitors preferentially rescue FRDA fibroblasts from death

Recently, mitochondrial disruption has been shown to trigger the apoptotic cascade directly (16-20). If mitochondrial stress in mutant FRDA fibroblasts resulted in activation of the apoptotic cascade, then one might predict that inhibitors of apoptosis should rescue FRDA fibroblasts preferentially from death. The apoptosis inhibitor z-VAD.fmk conferred significant rescue on FRDA fibroblasts, but not controls (Fig. 5a and b). Other inhibitors of apoptosis, YVAD-CHO and DEVD-CHO, provided similar extents of rescue (data not shown). For example, at 250 µM H2O2, the addition of z-VAD.fmk increased the viability of FRDA fibroblasts from 21.0 ± 3.4 to 66.6 ± 8.8%, whereas the viability of control cells at this dose was not significantly different. z-VAD.fmk did not provide significant protection of control cells from death, consistent with the view that caspase activity is not required for death of control cells. In fact, at the 100 µM dose of H2O2, z-VAD.fmk appeared to accelerate death slightly, an unexpected result.abc

   a
   b

Figure 5. (a and b) Protection of fibroblasts from H2O2-induced death by z-VAD.fmk. Cells were treated with H2O2 and z-VAD.fmk for 6 h and viability was assessed by the trypan blue exclusion assay. [square], cells; [circle], cells + 50 µM z-VAD.fmk. Averages of three or 28 independent experiments are shown for cells treated with or without z-VAD.fmk, respectively. Significance levels at individual doses are represented by asterisks: *P < 0.05, **P < 0.005. (c) Induction of caspase 3 activity by staurosporine in FRDA fibroblasts versus controls. Specific activities for caspase 3 were 1.50 ± 0.24 (2× SEM) and 0.96 ± 0.19 pmol of p-nitroaniline/min/mg. Averages from 6-8 independent experiments are shown, with bars representing two standard errors of the mean.

If intrinsic mitochondrial disruption was occurring in FRDA fibroblasts, then one might expect more activation of the central death enzyme of apoptosis, caspase 3. Death was induced with the apoptotic agent staurosporine. A significantly higher activity of caspase 3 activity was observed in FRDA versus control fibroblasts (Fig. 5c).

DISCUSSION

The results presented above demonstrate that the GAA expansion in frataxin confers H2O2 sensitivity and iron hypersensitivity on human FRDA fibroblasts. The kinetics of FRDA fibroblast death were dependent on the concentration of H2O2 and iron, consistent with an oxidative stress hypothesis for FRDA. Iron is a crucial reagent in the Fenton reaction, as it can react with mitochondrially produced superoxide anion (·O2-) to produce toxic free radicals such as the hydroxyl radical (·OH-). Thus, consistent with the knockout yeast model [Delta]YFH1, the results presented here support a pathogenic role for oxidative stress in FRDA. The iron chelator deferoxamine specifically rescued FRDA and, to a lesser extent, control cells from death, consistent with a death mechanism dependent on Fenton chemistry. Although mean iron levels were slightly higher in FRDA mitochondria than in controls, the difference was not statistically significant in the two fibroblast (Fig. 3a) and three lymphoblast (data not shown) lines examined. The small difference in iron concentration may be the consequence of residual frataxin expression in FRDA fibroblasts, in contrast to the situation in knockout yeast with zero expression of YFH1. An alternative explanation is that in comparison with controls, FRDA fibroblasts either may experience intrinsically higher endogenous levels of oxidant stress or contain an oxidant-sensitive target, which would explain the sensitivity of FRDA fibroblasts to oxidants in the absence of a large difference in mitochondrial iron accumulation.

Increases in concentration of the intracellular Ca2+ ion are known to follow oxidant stress in many cell types (15). Chelation of intracellular Ca2+ rescued both FRDA and control fibroblasts from death, demonstrating a requirement for Ca2+ for oxidant-induced death, but not necessarily any difference in this Ca2+-dependent step in FRDA versus control fibroblasts. In contrast to our earlier results in osteosarcoma cells bearing pathogenic mitochondrial DNA mutations in which CsA provided preferential protection from death induced by oxidant stress, presumably by inhibiting the MPT (14), we observed no protection by CsA of FRDA or control fibroblasts (data not shown). The implication is that oxidative stress is not killing FRDA fibroblasts by induction of the MPT.

Presumably a common endpoint of multiple toxic stimuli to cells is activation of the apoptotic machinery, which recently has been shown to be regulated at least partially at the mitochondrial level. If the FRDA mutation conferred excess activation of the apoptotic pathway, one would predict a greater sensitivity to agents which induce cell death (i.e. Fig. 1), and also a greater degree of protection from cell death by apoptosis inhibitors. The staurosporine-induced caspase 3 activity was significantly higher in FRDA fibroblasts than in controls, and three known biochemical inhibitors of the apoptosis machinery provided significant protection of FRDA but not of control fibroblasts from death. These findings are consistent with the idea that the apoptotic pathway is activated preferentially in FRDA fibroblasts.

Altogether, our data suggest that FRDA may be the latest addition to the increasing list of neurodegenerative disorders caused by oxidative stress (21,22). The results indicate that expansions of GAA in the frataxin gene result in increased susceptibility of cells to oxidant-induced death, and thus the cells potentially could be used as a model to study the mechanism of death acceleration, which may relate to the FRDA disease process. In addition, the results demonstrate rescue from death by chelators of iron and calcium and by apoptosis inhibitors in the model system, which may suggest that antioxidant, chelation and anti-apoptotic therapy potentially could be considered for this devastating neurodegenerative disease.

MATERIALS AND METHODS

Reagents

H2O2 was purchased from Fisher (Pittsburgh, PA), 1,2-bis(2-aminophenoxy)-ethane-N,N,N[prime],N[prime]-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM) and deferoxamine mesylate were purchased from Sigma (St Louis, MO), z-VAD.fmk was purchased from Enzyme Systems (Livermore, CA), YVAD-CHO was purchased from Calbiochem (Cambridge, MA) and DEVD-CHO was purchased from Biomol (Plymouth Meeting, PA).

Patients, DNA analysis and cell culture conditions

All five FRDA fibroblast cell lines used in this study were from patients diagnosed with classical Friedreich’s ataxia. Mean passage numbers of FRDA and control fibroblasts were 15 and 13, respectively. Analysis of the GAA repeat expansion was performed by PCR as described (23) and the following allele sizes were determined: FRDA 13 (743/1046); FRDA 53 (658/1243); FRDA 209 (613/910); FRDA 1035 (365/742); FRDA 1037 (753/820); control 66LS (10/10); control 269 (8/11); control 305 (8/10); and controls GM00023 and GM00024 (NIGMS Human Genetic Mutant Cell Repository) (not determined). Expression of frataxin mRNA was measured by RT-PCR as described below. Mean expression of FRDA fibroblasts was 33.6% of the control mean. Fibroblasts were grown in minimal essential medium (MEM; Life Technologies, Gaithersburg, MD), supplemented with 8 mM glutamine, 10% fetal bovine serum (FBS), 50 mg/ml uridine, 1 mM sodium pyruvate, 10 µg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 50 ng/ml basic growth facor (bFGF), and gentamicin.

Frataxin expression analysis

RT-PCR analysis of frataxin expression was performed in the five FRDA fibroblast cell lines as well as in five control cell lines. Poly(A)+ mRNA was prepared from 4.5 × 106 cells using the FastTrack kit (Invitrogen, Milan, Italy) following the manufacturer’s instructions. cDNA was synthesized from 1 µg of poly(A)+ mRNA using the Expand reverse transcriptase (Boehringer, Monza, Italy) and the ‘lock-docking’ protocol as previously described (24). A frataxin RT-PCR fragment of 480 bp was amplified using the sense oligonucleotide primer FA1/2-S (5[prime]-ACGCCCCGCCGCGCAAGTTCGAAC-3[prime]) and the antisense primer FA5A-AS (5[prime]-AGCATCTTTTCCGGAATAGGCCAA-3[prime]). As an internal control, a similar size (529 bp) fragment was amplified from the mRNA of 3-ketoacyl-CoA thiolase [beta]-MTP (GenBank accession no. D16481) using the sense primer IMA-S (5[prime]-ATCATGGCGGAGGAAAAGGCTCTG-3[prime]) and the antisense primer FINE-AS (5[prime]-AAATGGCATTGCCTAGTGTGAGTGTTG-3[prime]). Optimal conditions for semi-quantitative analysis of PCR products were selected, which included sampling during the log-phase of PCR and determination of the optimal amount of DNA loaded on the 2% agarose gel. Densitometric scanning of the gel was performed using a digital camera and the software Bio-Profil (Vilber-Lourmat, Paris, France). The mean of two independent experiments was used. Frataxin mRNA levels were expressed as a percentage of mRNA levels of the control transcript. The frataxin mRNA level in five FRDA cells was 0.037 ± 0.018 (95% CI) and of control cells (66LS, 269 and 305) was 0.110 ± 0.028 (95% CI).

Cell viability assay

Cell viability was determined as described (14). Briefly, 1 × 105 cells were grown in MEM supplemented with 8 mM glutamine and 10% FBS 24 h prior to use. Cells were given fresh media (MEM, 8 mM glutamine and 10% FBS) before the addition of H2O2. The following reagents, with their final concentrations, were added to the cells before H2O2: 10 µM BAPTA-AM [in dimethyl sulfoxide (DMSO)], 50 µM z-VAD.fmk (in DMSO), 100 µM YVAD-CHO (in water) and 100 µM DEVD-CHO (in water). Deferoxamine mesylate (250 µM final concentration, in water) was added to cells 18 h prior to H2O2. Six hours later, cells were removed by trypsin-EDTA and resuspended in phosphate-buffered saline. Viabilty was determined by the trypan blue exclusion assay. In the case of BAPTA and z-VAD.fmk, vehicle controls with DMSO were performed. Student’s t-tests were carried out to determine the significance values for both control and FRDA fibroblasts at individual doses.

Measurement of mitochondrial iron

Mitochondria were isolated from fibroblasts using the method of Rickwood et al. (25). Mitochondrial iron was measured by atomic absorption spectroscopy as described (26).

ACKNOWLEDGEMENTS

We thank the patients who have generously participated in this study; Dr Franca Mazzucchelli for her clinical help; and Dr Barbara Garavaglia and Ms Simona Allievi for their help in establishing and culturing the fibroblast cell lines. This work was supported by NIH grant AG 11967, a pilot grant from the Alzheimer’s Disease Research Center and the Center for Environmental Health Sciences to G.A.C., and by Telethon-Italia grant E.514 to F.T.

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*To whom correspondence should be addressed. Gino Cortopassi-Tel: +1 530 754 9665; Fax: +1 530 754 9342; Email: gacortopassi@ucdavis.edu; Franco Taroni-Tel: +39 02 239 4284; Fax: +39 02 266 4236; Email: taroni@tin.it

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