Causative role of oxidative stress in a Drosophila model of Friedreich ataxia

The FASEB Journal • Research Communication

Causative role of oxidative stress in a Drosophila modelof Friedreich ataxia

Jose V. Llorens,*,1 Juan A. Navarro,*,†,1 Maria J. Martınez-Sebastian,* Mary K. Baylies,‡

S. Schneuwly,† Jose A. Botella,† and Maria D. Molto*,2

*Departament de Genetica, Universitat de Valencia, Burjassot, Valencia, Spain; †Institute of Zoology,University of Regensburg, Regensburg, Germany; and ‡Developmental Biology Program, MemorialSloan Kettering Cancer Institute, New York, New York, USA

ABSTRACT Friedreich ataxia (FA), the most com-mon form of hereditary ataxia, is caused by a deficit inthe mitochondrial protein frataxin. While several hy-potheses have been suggested, frataxin function is notwell understood. Oxidative stress has been suggested toplay a role in the pathophysiology of FA, but this viewhas been recently questioned, and its link to frataxin isunclear. Here, we report the use of RNA interference(RNAi) to suppress the Drosophila frataxin gene (fh)expression. This model system parallels the situation inFA patients, namely a moderate systemic reduction offrataxin levels compatible with normal embryonic de-velopment. Under these conditions, fh-RNAi fliesshowed a shortened life span, reduced climbing abili-ties, and enhanced sensitivity to oxidative stress. Underhyperoxia, fh-RNAi flies also showed a dramatic reduc-tion of aconitase activity that seriously impairs themitochondrial respiration while the activities of succi-nate dehydrogenase, respiratory complex I and II, andindirectly complex III and IV are normal. Remarkably,frataxin overexpression also induced the oxidative-mediated inactivation of mitochondrial aconitase. Thiswork demonstrates, for the first time, the essentialfunction of frataxin in protecting aconitase from oxi-dative stress-dependent inactivation in a multicellularorganism. Moreover our data support an important roleof oxidative stress in the progression of FA and suggesta tissue-dependent sensitivity to frataxin imbalance.We propose that in FA, the oxidative mediatedinactivation of aconitase, which occurs normally dur-ing the aging process, is enhanced due to the lack offrataxin.—Llorens, J. V., Navarro, J. A., Martınez-Sebastian, M. J., Baylies, M. K., Schneuwly, S.,Botella, J. A., Molto, M. D. Causative role of oxida-tive stress in a Drosophila model of Friedreich ataxia.FASEB J. 21, 333–344 (2007)

Key Words: frataxin � aconitase � mitochondrial respiration� hyperoxia � RNAi

Friedreich ataxia (fa), the most common form ofhereditary ataxia, is a recessive neurodegenerative dis-ease affecting the central and peripheral nervous sys-tems (1, 2). Extraneural organs are also affected during

the course of the disease, as a significant proportion ofpatients develop cardiomyopathy (1, 3). This disablingcondition manifests usually in childhood or adoles-cence. Patients develop progressive ataxia of all fourlimbs, dysarthria, sensory loss, and pyramidal signs (1).Consequently, they have a diminished life quality, re-sulting in confinement to a wheel chair and a reducedlife expectancy mostly due to the hypertrophic cardio-myopathy.

FA is caused by reduction of the frataxin proteinmostly due to an abnormal GAA repeat expansion inthe first intron of the human FRDA gene (4), which, inturn, inhibits transcription (5). Frataxin shows highconservation throughout evolution, with orthologs inessentially all eukaryotes and some prokaryotes (6). Itspresence in Gram-negative bacteria supports the mito-chondrial localization found in eukaryotes (7, 8). Con-siderable effort has been made to explain the primarydysfunction in the FA pathogenesis. Frataxin has beenproposed to play several different roles: regulatingefflux of iron from mitochondria (9), storing iron inbioavailable and nontoxic form within mitochondria(10–12), regulating OXPHOS (13, 14), producingiron-sulfur (Fe-S) clusters (15); controlling heme groupsynthesis (16), and modulating mitochondrial aconi-tase activity (17).

Oxidative stress has also been suggested to have animportant role in the pathophysiology of FA (18). Highlevels of oxidative stress markers in FA patient samples,such as malondialdehyde in plasma (19), urinary-ex-creted oxidized DNA (20), and glutathione in blood(21), have been found. Studies carried out in differentmodels of FA, including yeast, cell culture, and mouse,have reported an alteration in intracellular oxidativestatus (22–25). In addition, overexpression of frataxinled to an up-regulation of some antioxidant pathways(26, 27). However, recent studies have questioned the

1These authors contributed equally to this work.2Correspondence: Departament de Genetica, Facultat de

Ciencies Biologiques, Universitat de Valencia, Carrer DoctorMoliner 50, 46100-Burjassot, Valencia, Spain. E-mail:[email protected]

doi: 10.1096/fj.05-5709com

3330892-6638/07/0021-0333 © FASEB

Downloaded from www.fasebj.org by (87.162.60.25) on February 24, 2018. The FASEB Journal Vol. 21, No. 2, pp. 333-344.

role of oxidative stress in FA. In those reports, theoverexpression of antioxidant enzymes as catalase,Cu,Zn-superoxide dismutase, and Mn-superoxide dis-mutase or MnTBAP treatment (a compound mimick-ing the Mn-superoxide dismutase action) failed torescue the loss of frataxin function phenotypes in bothmouse and fly (28, 29). Furthermore, Seznec et al. (28)did not detect increased level of oxidative stress mark-ers, suggesting that oxidative stress may not be a majorcontributor to the pathology.

To assess both the roles of frataxin and oxidativestress in FA, we induced posttranscriptional silencing ofthe Drosophila frataxin gene (fh) by transgenic double-stranded RNA interference (RNAi). We have generateda scenario where fh levels are reduced to 30% com-pared to the controls, thus overcoming the preadultlethality observed in other fly models (29). This allowedus to study the effects of Drosophila frataxin (FH)reduction and the contribution of oxidative insult inadult individuals. Furthermore, the effect of generaland tissue-specific overexpression of fh was also ana-lyzed.

In this work, we show that FH plays an essential rolein Drosophila melanogaster protecting against the delete-rious effects of oxidative stress. We have also confirmedthe prediction concerning the mitochondrial localiza-tion of FH (30). Both frataxin-deficient and frataxin-overexpressing adult flies showed reduced life span andclimbing ability. Moreover, both the reduction and theincrease of FH function seriously compromised aconi-tase activity in an unbalanced redox environment. FHdecrease also impaired mitochondrial respiration in anoxidative atmosphere. This work presents the firstevidence regarding the essential function of frataxin inprotecting aconitase from oxidative stress-dependentinactivation in a multicellular organism and supportsan important role of oxidative stress in the progressionof Friedreich ataxia.

MATERIALS AND METHODS

Drosophila stocks

The yw strain was used as a control and for the injection of theUAS-fhIR and UAS-fh constructs. The following driver lineswere obtained from the Bloomington Stock Centre: actin-GAL4, daG32-GAL4, 24B-GAL4, Ddc-GAL4, Dot-GAL4, neural-ized-GAL4, and the D42-GAL4 was kindly provided by G.Boulianne (University of Toronto). The crosses of the GAL4drivers and the fh responder lines were carried out at 25°C or29°C.

Generation of the fh constructs

FH-enhanced GFP construct

The coding sequence of fh was amplified from poly (A)-RNAisolated from Drosophila embryos. The gene-specific primersused were MECAD (5�-AAGTTGCGGCCGCCGCAACTGG-GATTTGTA-3�) and MEDAR (5�-ACTAATTCTAGAATTAAC-TACAGTAGGGCA-3�). The resulting polymerase chain reac-

tion (PCR) product was cloned into pCRscript SK(�) to yieldthe pCR-fh construct. The fh coding region was amplifiedfrom this construct using the fhpEGFPN3F (5 � -CTCGAGAAATGTTTGCCGGTCGTTTGAT-3 �) andfhpEGFPN3R (5�-GGATCCACTACAGTAGGGCAGGCGTAG-GAAG-3�) primers and subcloned in frame with the enhancedGFP (EGFP) protein into pEGFPN3 vector (BD BiosciencesClontech, Mountain View, CA).

UAS-fhIR construct

This construct was generated according to Piccin et al. (31).The pCRfh construct was used to obtain two copies of fh inopposite directions. Both copies were separated using afragment of the green fluorescent protein (GFP) as spacer.This fragment was amplified with the primers GFPRNAiF(5�-CCCAAGCTTCACGAATTCTTCAAGTCCGCC-3�) andGFPRNAiR (5�-CCGCTCGAGCTGGATCCGGACTTGTA-CAGC-3�). Finally, the construct containing the two fh se-quences in opposite orientation and separated by the GFPspacer was cloned into the pUAST vector (UAS-fhIR).

UAS-fh construct

The fh coding sequence was subcloned from the pCR-fhconstruct into the appropriate restriction sites in thepolylinker of the pUAST vector (UAS-fh).

Cellular transfections

Transient tranfections were carried out in CHO-K1 mamma-lian cells. The day before transfections, sterile cover slips wereplaced into 6-well dishes seeded with 1 � 106 cells. Transfec-tions were performed with FH-enhanced GFP fusion con-struct using 1 �g of DNA and 3 �l of FuGENE 6 transfectionreagent (Roche Diagnostics, Laval, Canada), according to themanufacturer’s protocol. A pEGFPN3 empty vector was alsotransfected into CHO-K1 as a negative control. Twenty-fourhours after transfections, the coverslips were rinsed with PBS,and cells were fixed with paraformaldehyde 4%. MitotrackerOrange CMTMRos (Invitrogen, Carlsbad, CA, USA) wasemployed as a control for the mitochondrial pattern. Slideswere observed under a Leica fluorescence microscope andimages were analyzed using Adobe Photoshop 7.0.

Generation of fly transformants

P-vector transformants were generated by standard embryo-injection methods, according to Rubin and Spradling (32). Atotal of 10 independent transforming lines were generated.The UAS-GAL4 system (33) was carried out to generate thefh-RNAi and the fh-overexpressed flies. Every experiment wasalways done simultaneously for control (yw x GAL4 driver)and for fh responder flies (UAS-fhIR x GAL4 driver andUAS-fh x GAL4 driver) at 25°C or 29°C.

Quantitative real-time PCR

Total RNA was isolated from 100 males or from age-matchedembryos using TriPure Isolation Reagent, according to themanufacturer’s instructions (Roche Diagnostics). cDNA wassynthesized with Expand Reverse transcriptase (Roche Diagnos-tics) and oligo-dT primers. Quantitative real-time PCR wasperformed with ABI PRISM 7500 sequence detection system(Applied Biosystems, Foster City, CA). TaqMan probes for RP-49(control) and fh containing 6-carboxyfluorescein (6-FAM) at the5� end, and the appropriate primers were synthesized by Applied

334 Vol. 21 February 2007 LLORENS ET AL.The FASEB Journal


Biosystems. Data analysis was performed in triplicate experi-ments. Statistical significance was evaluated using Student’s t testand P � 0.05 was considered significant.

Western blot analysis

Total protein was extracted from age-matched embryos fol-lowing the method previously described (34). Protein levelswere quantified by Bradford assay. Fifty micrograms of totalprotein was applied to each lane. Samples were separated on5% stacking, 15% resolving Tris-glycine SDS-polyacrylamidegels. Resolved proteins were electroblotted to Hybond en-hanced chemiluminescence (ECL) nitrocellulose membrane(Amersham Biosciences) and probed with mouse anti-Frataxin monoclonal antibody (mAb) (MAB-10485, Immuno-logical Sciences) in combination with goat anti-mouse IgG-horseradish peroxidase conjugate (Amersham Biosciences,Little Chalfont, Buckinghamshire, UK). Detection was car-ried out using ECL Detection Reagents (Amersham Bio-sciences). The mouse anti-�-tubulin was used in combinationwith goat anti-mouse IgG-horseradish peroxidase conjugate(Amersham Biosciences) as a control. The protein MW wasestimated with a prestained protein molecular weight marker(Fermentas).

Immunocytochemistry staining

Whole mount embryos and stainings using the horseradishperoxidase technique were carried out as described previ-ously (35). The primary antibodies used were: mouse anti-myosin heavy chain (anti-MHC) 1:8, mouse mAb 22C10 1:50,mouse 1D4 antifasciclin II 1:20, mouse mAb EC11 antiperi-cardin 1:2, mouse mAb BP 102 anti-central nervous system(central nervous system (CNS)) axons 1:200 and rabbit Eve(Even-skipped protein) 1:1000.

Life span and climbing assays

Life span and climbing assays were performed as described inBotella et al. (36). Kaplan-Meier analysis of survival data withsemiparametric log rank test was performed using the Graph-Pad Prism 2.0 software. Climbing data were examined fordifferences with a one-way ANOVA test, and means werecompared using the SNK test. All statistical analyses werecarried out with the package Statistical Packages for the SocialSciences (SPSS) V.12.0.1 and values of P � 0.05 were consid-ered statistically significant.

Hyperoxia treatment

Hyperoxia treatment commenced 1 day posteclosion and wasperformed by exposing flies in a glass container with aconstant flux of 99.5% oxygen under a low positive pressureat 25°C. Flies were confined in groups of 20 and weretransferred every day to new vials containing regular food.

Mitochondria isolation

Mitochondria were isolated from 100 adult male flies after theprocedure described in Bioxytech Aconitase-340TM with somemodifications. Flies were homogenized in the appropriatebuffer, and the resulting mashes were centrifuged twice at 800g for 10 min at 4°C discarding the pellet every time. Super-natants were further centrifuged at 13000 g for 10 min at 4°C.Pellets were resuspended in 500 �l of ice-cold homogeniza-tion buffer at a concentration of 500 �g/ml. Mitochondrial

pellets were disrupted by sonication for 30 s four times on asetting of 2 with 1 min. interval between each sonication.

For measurements of complexes I-IV respiratory rates,mitochondria were softly isolated to prevent break. Flies wereplaced in 500 �l of cold isolation buffer (154 mM KCl, 1 mMEDTA, pH 7.4), gently pounded and homogenates werefiltered through 0.5 cm of cheesecloth placed in a new tube.Filtrate was centrifuged at 4°C for 8 min at 1500 g to removecellular debris. Supernatants were discarded, and the result-ing pellets were washed with 200 �l of isolation medium andfinally resuspended in 50 �l of isolation medium. All subse-quent assays were performed within 3 h, keeping the mito-chondria suspensions on ice.

Enzyme assays

Aconitase activity was determined using Bioxytech Aconitase-340TM Spectrophotometric Assay kit. Succinate dehydroge-nase was determined by the procedure described by Munujoset al. (37).

Mitochondrial respiratory assays

Rates of mitochondrial respiration (state 3, ADP-stimulatedstate and state 4, ADP-deleted state) were determined byoxygen consumption using a fiberoptic oxygen microsensorof Microx TX3 (Precision Sensing GmbH, Regensburg Uni-versity, Germany). Temperature was maintained at 28°C andthe reaction volume was 50 �l. Freshly isolated mitochondriawere added to the respiration buffer (10 mM KH2PO4, 5 mMMgCl2, 120 mM KCl, 1.25 mg/ml BSA, pH 7.4) allowing toequilibrate for 30 s.

Complex I and complex II contribution was measuredusing NADH (60 mM) and succinate (10 mM) plus rotenone(100 �M), respectively. Pyruvate plus malate (200 �M each)were used to analyze the oxygen rate consumption after theactivation of the Krebs cycle. Both substrates were added tothe chamber and allowed to equilibrate for 30 s, followed bythe addition of 5 mM ADP when required.

In the case of the respiration after addition of succinateplus rotenone (activation of complex II), only the state 3values have been reported because no difference was ob-served with state 4, probably due to the inability of succinateto cross the inner mitochondrial membrane as has beenpreviously observed by Ferguson et al. (38).

RESULTS

Drosophila frataxin protein localizes inthe mitochondria

We have previously predicted by computer analyses themitochondrial localization of the Drosophila frataxin-like protein (FH) (30). To confirm the subcellularlocalization of FH, colocalization experiments using afrataxin-enhanced green fluorescent protein (FH-en-hanced GFP) and a mitochondrial marker were per-formed. Transfection of a FH-enhanced GFP fusionconstruct into CHO-K1 cells revealed strong fluores-cence signal in the mitochondrial reticulum that colo-calized with the mitochondrion-selective probe mito-tracker Orange CMTMRos (Fig. 1). The subcellularlocalization of the Drosophila FH suggested an equiva-lent function to that of the human ortholog; therefore,

335A DROSOPHILA MODEL OF FRIEDREICH ATAXIA


a Drosophila strain showing a reduction of FH functionwould be of great value for modeling the progression ofFA disease. In a model recently published by Andersonet al. (29), the general silencing of fh expression toundetectable levels resulted in lethality during develop-ment and, therefore, precluding any study of the effectsof FH reduction in adult individuals.

Generation of RNAi frataxin flies

To bypass the preadult lethality obtained by the almostcomplete depletion of FH, we reduced fh expression toa level that allowed study of adult individuals. In thisway, we generated a model that would more closelyparallel the situation in FA patients.

Several Drosophila transgenic lines carrying the UAS-fhIR construct were generated. The presence of thisconstruct was verified by Southern blot and after exclu-sion of position-dependent effects, a line carrying theUAS-fhIR construct on the second chromosome wasselected for further analysis.

To test the efficiency of our model and reveal itspotential, knockdown of FH was performed throughoutthe organism, as well as in specific tissues because mostpatients are affected by progressive polyneuropathy,myopathy and hypertrophic cardiomyopathy (39). FHknockdowns were generated using induction of RNAiby means of the GAL4/UAS system at 29°C. Table 1shows the GAL4 lines used and the results obtained.The gene silencing performed using the ubiquitousdrivers, actin-GAL4 and da-GAL4, resulted in lethalityshowing, in agreement with Anderson et al. (29), thatthe function of FH is essential during development.The lethality observed with 24B and Dot drivers, dem-onstrated that FH plays a key role in the developmentof the early mesoderm (precursor tissue of muscles anddorsal vessel) and heart. Silencing of fh using differentnervous system drivers resulted in viable progeny with-out any gross morphological abnormalities. The off-spring from these crosses were examined both for theirlife span and climbing ability. A statistically significantreduction of the life span (Fig. 2A) and a 15% declinein climbing capability was found only in the case ofneuralized-GAL4, which is expressed in all sensory or-gans and their precursors. The general silencing of fhin motoneurons (D42-GAL4), brain (c698a-GAL4), anddopaminergic neurons (Ddc-GAL4) did not result inany distinguishable phenotype (Fig. 2B-D). The resultsabove were in agreement with the clinical aspectsdescribed for FA patients, where sensory neurons seemto be the most affected component of the nervoussystem and validated the use of our RNAi model tofurther study FA pathology in flies.

Moderate reduction of fh expression shortens lifespan and impairs climbing ability

Next, we determined experimental conditions thatcould allow us to parallel more closely the situation inFA patients. Two parameters were crucial to achievethis: first, reduction of fh expression ubiquitously andsecond, presence of a given fh level able to circumvent

Figure 1. Drosophila frataxin protein (FH) localizes in themitochondria. A) CHO-KI cells transfected with a FH-en-hanced GFP construct showed a mitochondrial fluorescentpattern. B) Staining with the orange CMTMRos mitochon-drial marker; C) Overlap of both signals indicated localizationof fly frataxin to the mitochondria.

TABLE 1. Effects of general and selective RNAi and overexpression of fh on Drosophila viability at 29 °C

Expression pattern GAL4 drivers RNAi Overexpression

Ubiquitous actin Lethal at mature pupae Lethal at early pupaeda Lethal at mature pupae Lethal at 3rd instar larvae

Muscle system and heart 24B Lethal at mature pupae Lethal at early pupaeDot Lethal at mature pupae Lethal from early pupae to adults

Nervous system Ddc Viable ViableD42 Viable ViableC698a Viable Viableneuralized Viable Viable



developmental defects leading to lethality. We achievedthe optimal level of fh silencing using the ubiquitousdriver actin-GAL4 at 25°C. Under these conditions, thefh mRNA level detected using real-time PCR showed thepresence of only 30% of fh messenger when comparedto control flies. This amount of fh mRNA is similar tothe expression levels reported in FA patients (7, 40).

Survival analysis revealed a reduction of mean andmaximum life span of 60% and 32%, respectively infh-RNAi adult flies when compared to controls (Fig.3A). Moreover, we also found a reduction in climbingability of 45% in 5-days-old fh-RNAi individuals (Fig.3B). These results suggested that the function of FH isnot only essential for development but also required forthe maintenance of vital functions of the adult individ-ual. Alternatively, the reduction in survivorship and thedecay in climbing performance may also be influencedby, in our hands, undetectable developmental defects.

fh mediates protection against oxidative stress

In Drosophila, reductions of life span and decline inclimbing capabilities have been often related to in-creased sensitivity to oxidative damage (36, 41). To testwhether the phenotypes caused by reducing fh expres-sion might be a consequence of an enhanced suscepti-bility to oxidative stress, we exposed fh-RNAi and con-trol flies to a high oxidative atmosphere (99.5% O2).Hyperoxia has been found to be a relevant strategy to

recreate the effect of unbalanced cellular oxidativestatus in Drosophila (42). Under these conditions, con-trol flies showed an average maximum life span of 9days, whereas fh-RNAi flies showed a 44% reduction inthis value (Fig. 3C). This result indicated that FHfunction might be involved in a protection mechanismagainst oxidative stress and suggested that oxidativedamage plays a role in the phenotypes induced infh-RNAi flies during the aging process. The associationfound between lack of FH function and oxidative stressis in agreement with findings reported in other models(22–27) yet contradicts recent suggestions (28, 29) thatthere is no role for oxidative stress in FA pathology.

Figure 3. General reduction of fh expression impairs life spanand climbing ability and increases sensibility against oxidativeinsult. Kapplan-Meier analysis and one-way ANOVA with posthoc Student-Newman-Keuls test (both with P � 0.05) wereperformed for life span and climbing data, respectively.Control genotypes (open squares/bar): yw, actin-GAL4/�and RNAi genotypes (gray squares/bar): �/UAS-fhIR; actin-GAL4/�. A) Life span under normoxia. Decrease of fhexpression reduced the mean and maximum life span. B)Walking ability of 5-day-old adult individuals. Loss of fhfunction diminished performance of flies in negative geotaxisexperiments. C) Life span under hyperoxia (99.5% O2).Reduction of fh expression enhanced susceptibility to oxida-tive injures.

Figure 2. Selective alteration of fh expression in sensoryorgans and in motorneurons shortens life span. Kapplan-Meier analysis of survival data with semiparametric log ranktest (P�0.05) was performed for each group of data. Controlgenotypes (open squares): neuralized-GAL4/� (A), D42-GAL4/� (B), wc698a-GAL4/yw (C), Ddc-GAL4/� (D). RNAigenotypes (gray squares): �/UAS-fhIR neuralized-GAL4/�(A), �/UAS-fhIR; D42-GAL4/� (B), wc698a-GAL4/yw; �/UAS-fhIR (C), �/UAS-fhIR; Ddc-GAL4/� (D). Overexpres-sion genotypes (black squares): �/UAS-fh; neuralized-GAL4/� (A), �/UAS-fh; D42-GAL4/� (B), wc698a-GAL4/yw; �/UAS-fh (C), �/UAS-fh; Ddc-GAL4/� (D).



fh protects against oxidative stress-inducedaconitase inactivation

Since the mitochondrial enzyme aconitase has beenidentified as a specific target of oxidative stress in avariety of organisms (43–45), and its activity is found tobe seriously affected in FA tissues, we next assessed thefunctional integrity of this enzyme in our Drosophilamodel. As shown in Fig. 4A, aconitase activity in controlflies was reduced, as expected, during aging (46).Surprisingly, this reduction was similar in fh-RNAi flies,indicating that a decrease in the FH function did notappear to have an effect on the overall aconitase activitymeasured during the aging process (see Discussion).Interestingly, when aconitase activity was measuredafter one day of hyperoxia treatment, a dramatic reduc-tion in the enzymatic activity was observed in fliesdefective for FH (Fig. 4B). As reported in Das et al. (44),aconitase activity in wild-type (WT) flies decreasesunder hyperoxia, demonstrating the vulnerability ofthis enzyme to high levels of reactive oxygen species(ROS). As this reduction was enhanced 30% in fh-RNAiflies, the result shows the importance of FH function inprotecting aconitase against its oxidative stress-inducedinactivation. This result is in agreement with the pro-posed function for frataxin acting as an aconitasechaperone (17).

In contrast, the activity of succinate dehydrogenase(SDH), a mitochondrial Fe-S cluster containing enzyme

also found affected in FA patients, showed no change inaging (Fig. 4C) or after one day under hyperoxiaconditions in controls and fh-RNAi flies (Fig. 4D). Ourresults were in agreement with other reports that showthat SDH is not especially sensitive to ROS during thenormal aging process (47). Moreover, this fact couldexplain why in hyperoxic conditions, the activity of thisenzyme does not decrease due to a deficit of FH.

Taken together, these results suggested that a declinein aconitase activity by ROS inactivation is one of theprimary events responsible for the physiological defectsobserved in fh-RNAi flies.

Frataxin deficiency seriously compromisesmitochondrial respiration

A decrease in aconitase activity would be predicted toimpair the citric acid cycle, diminishing the supply ofreducing equivalents for electron transport and, there-fore, decreasing ATP production. Moreover, inhibitionof aconitase activity has been shown to decrease oxygenconsumption in cultured cells (48). To test whether thereduction of FH function would affect mitochondrialrespiration, we measured oxygen consumption rates ofisolated mitochondria from control and fh-RNAi flies innormoxia and hyperoxia conditions. No difference wasfound in state 3 (active respiration state) or state 4(basal respiration state) between 1-day fh-RNAi andcontrol flies under normoxic conditions (Fig. 5A). A37% reduction in oxygen consumption in state 3 wasreadily observed in controls when maintained 1 dayunder hyperoxia. Strikingly, a 73% decrease was found(state 3) in fh-RNAi flies after oxidative stress treatment,a two fold reduction when compared to the hyperoxicvalues in control flies (Fig. 5A). This result indicatedthat on oxidative insult, the function of FH is necessarybut not sufficient to warrant the supply of energy forthe cells.

There can be several causes for the decreased respi-ration observed in FH-deficient flies. These includedefects in respiratory complexes and/or a diminutionof reducing equivalents necessary for electron transportfrom the citric acid cycle due to the aconitase defi-ciency described above. To distinguish between thesetwo possibilities, oxygen consumption induced by stim-ulation of mitochondrial respiratory complex I and IIwas analyzed.

On addition of NADH (complex I), no differencewas observed in oxygen consumption rate betweencontrol and fh-RNAi flies under normoxic or hyperoxictreatment, although hyperoxia was able to equallydecrease this rate by 38% in both cases (Fig. 5B). Theanalysis of respiration using succinate plus rotenone(complex II) showed no difference between controlsand fh-RNAi flies under normoxia or hyperoxia treat-ment (Fig. 5C). This was in agreement with the above-mentioned lack of effect on SDH activity observed infh-RNAi flies. Altogether, these results indicated thatcomplexes I and II (and indirectly III and IV) do notseem to be affected by the lack of FH under our

Figure 4. Loss of fh function induces oxidative stress-mediatedaconitase inactivation. Aconitase and succinate dehydroge-nase (SDH) activities were measured from extracts of adultsmaintained under normoxia (N) or hyperoxia (H) condi-tions. The data represent the mean � sem of three indepen-dent determinations. Control genotypes (open bar): yw,actin-GAL4/�, RNAi genotypes (gray bar): �/UAS-fhIR; ac-tin-GAL4/� and overexpression genotypes (striped bar):�/UAS-fh; actin-GAL4/�. A) Aconitase activity decreased inan age-dependent manner without showing any differencesbetween control and fh-RNAi flies. B) One-day hyperoxictreatment induced enhanced aconitase inactivation in fh-RNAi and in fh-overexpressed flies. C) SDH activity showed nochange during aging in both, control, and fh-RNAi flies. D)SDH activity remained intact in control and fh-RNAi flies after1-day hyperoxic exposure.



experimental conditions. Thus, in our frataxin model,the oxidative inactivation of aconitase, and not theinstability of the respiratory chain complexes, appearsto be the initial cause of the observed reduction inrespiration rate.

Overexpression of fh induces similar defects to itsreduction

To study the consequences of increasing fh expression,several Drosophila transgenic lines carrying the UAS-fh

construct were also generated. After exclusion of posi-tion-dependent expression of the transgene, a linecarrying that construct on the second chromosome wasselected for further analysis.

Overexpression analyses were performed at 29°Cusing the same GAL4 lines (ubiquitous pattern, ner-vous system, muscles and heart) as in the fh-RNAiexperiments. The results obtained are summarized inTable 1. An early and ubiquitous overexpression of fhwith the drivers actin and da, led to the death of allindividuals at early pupae stage and as third instarlarvae, respectively. Full lethality was also observed with24B and Dot drivers, similar to that seen in the fh-RNAiexperiments. Although fh overexpression with Dot-GAL4 began during embryogenesis (49), 95% of indi-viduals died in pupal stages with the remaining 5%dying during eclosion from puparium. The postembry-onic lethality can be explained because Drosophila cancomplete embryonic development despite an affectedheart, but the presence of a functional organ is re-quired for the rest of the life cycle (50). Taking theRNAi and overexpression data together, these resultssuggested that the same process is being disrupted byeither a FH excess or deficit and support a criticalfunction of FH in the early development of muscles andheart. In addition, overexpression of fh using neuralspecific GAL4 drivers was compatible with normalpreadult development. Thus, life span and climbingexperiments were conducted to monitor age-relatedchanges. Overexpression using neuralized (sensory or-gans) and D42 (motoneurons) GAL4 drivers resulted ina reduction of 64% and 49% of mean life span values(Fig. 2A, B) and a 85% and 60% decline in climbingability respectively, effects stronger than those dect-ected in the fh-RNAi flies. No phenotype was observedeither with c698a (brain) or with Ddc (dopaminergiccells) GAL4 drivers (Fig. 2C, D). These results rein-forced the important role of FH activity in the periph-eral nervous system (PNS) of Drosophila.

Ubiquitous overexpression of fh impairs development

To quantify the level of fh overexpression, fh-mRNA wasdetected in da-GAL4�UAS-fh embryos using real-timePCR. A 9-fold increase was found in these embryoswhen compared to controls. This induction of expres-sion correlated with the results obtained in Westernblot analysis (Fig. 6A).

Immunohistochemistry was carried out to determinethe underlying defects associated with the lethal phe-notypes obtained from da-driven overexpression of fh.Anti-MHC staining revealed muscles clearly disruptedwhen compared to controls (Fig. 6B, C). In almost allembryos, we unequivocally observed three specific par-tially formed muscles: dorsal acute 3, dorsal oblique 3,and dorsal oblique 4. To investigate whether the ner-vous system was also affected, two different neuralmarkers (1D4 antifasciclin II and 22C10) were used.ID4 labels the 6 longitudinal axons of the CNS and theintersegmental (ISN) and segmental (SN) nerves; and

Figure 5. Oxidative stress compromises mitochondrial respi-ration in fh-RNAi flies. Oxygen consumption rates weremeasured from isolated mitochondria of adult flies main-tained under conditions of normoxia (N) or hyperoxia (H).The data represent the mean � sem of 3 independentdeterminations. Control genotypes (open bar): yw; actin-GAL4/� and RNAi genotypes (gray bar): �/UAS-fhIR; actin-GAL4/�. A) Enhanced reduction in respiration rate inhyperoxia-treated fh-RNAi flies. B) Oxygen consumption ratesafter the stimulation of complex I did not show differencesbetween control and fh-RNAi flies in normoxic or hyperoxictreatment. (C) No difference was detected in the analysis ofrespiration stimulating complex II.



22C10 labels the PNS. We detected aberrant axonaltracks in 70% of da-GAL4�UAS-fh embryos when usingthe 1D4 staining (Fig. 6D, E). 22C10 staining showed anincrease in the number of sensory ventral neurons (Fig.6F, G) and axonal pathfinding defects (Fig. 6H) in 10%of the embryos. In contrast, no abnormalities weredetected in CNS with the Eve and BP 102 stainings(data not shown).

We also analyzed the possible defects caused by fhoverexpression in the heart, which is a key affectedtissue in FA. For this purpose, Dot-GAL4�UAS-fh em-bryos were stained with ECII antibody (Ab), whichlabels the extracellular matrix (ECM) surrounding thepericardial and cardial cells of the heart tube. Thisstaining revealed the lack of some pericardial cellsalong the tubular structure of the developing heart(Fig. 6I, J).

Taken altogether, these results indicated that over-expression of FH seriously affects the development ofembryonic muscles, peripheral nervous system, and theheart. We find that the key tissues affected in FApatients are unexpectedly sensitive to increase offrataxin function in Drosophila.

Frataxin overexpression inhibits mitochondrialaconitase activity under hyperoxia

To check whether oxidative stress might be a factorinvolved in the overexpression phenotypes, we mea-

sured the activity of mitochondrial aconitase in actin-GAL4�UAS-fh adult flies at 25°C (at this temperature,overexpression of fh using the ubiquitous driver actin-GAL4 provided adult individuals). When total aconi-tase activity was determined during aging, no differ-ences were detected between controls andoverexpressed flies (data not shown). However, whenflies were exposed to a high oxidative atmosphere(99.5% O2), fh-overexpressed flies showed a 40% reduc-tion in aconitase activity compared to control flies (Fig.4B). This result reinforced the relationship between FHand aconitase in Drosophila and the role of the oxidativestress in the phenotypes induced by fh misexpression.

DISCUSSION

Friedreich ataxia is the most frequent form of heredi-tary ataxia in the Caucasian population. Currently,model organisms are being used to understand thepathological mechanism of FA, as well as to test poten-tial therapies. The primary and secondary structure ofDrosophila FH is well conserved between flies and hu-mans and the Drosophila FH is predicted to have similarchemical properties as other frataxin-like proteins (30).In this work, we have confirmed the mitochondriallocalization of FH, suggesting that the molecular pro-cesses relevant to the disease might also be shared in a

Figure 6. Defects promoted in Drosophila embryos by fh overexpression driven by the da-GAL4 and Dot-GAL4 lines. A) Detectionof Drosophila frataxin protein. The two bands correspond to unprocessed precursor of 21 kDa and the mature form of 15kDa (30). The stronger signal of the mature form can be explained because the Anti-Frataxin MAB-10485 recognizes mainly theprocessed form on Western blot analysis of human tissues (7). Anti-�-tubulin was used as a loading control. Overexpressiongenotype: �/ UAS-fh; da/� (lane1) and control genotype: �/UAS-fh (lane 2). B–H) Muscular and nervous defects inda-GAL4�UAS-fh embryos. In these panels, anterior is toward the left, and all are lateral views. Anti-MHC staining revealedstrong abnormalities in stage 16 embryos (C) compared to control embryos of the same developmental stage (B). Incompleteformation of the muscles dorsal acute 3 (DA3) dorsal oblique 3 and dorsal oblique 4 (DO3/4) was observed. The staining with1D4 showed defects in the pathfinding of motoraxons in stage 14–17 embryos (E) compared to control ones (D). 22C10 stainingdetected an excess of some sensory ventral neurons (G) and axonal pathfinding problems of sensory nerves (H) in stage 16embryos with respect to stage 16 control embryos (F). Sensory neurons are divided in 4 clusters: dorsal (dc), lateral (lc), ventral’ (v’c)and ventral (vc). I, J) Heart defects in Dot-GAL4�UAS-fh embryos. In these panels, anterior is toward the left and both are dorsal views.Staining with EC11 showed the loss of pericardial cells (Pc) in 17-stage embryos (J) with respect to age-matched controls (I).



Drosophila model. Therefore, we report a new RNAimodel of FA in Drosophila, in which we developedexperimental conditions producing a moderate sys-temic reduction of frataxin level, which, in both hu-mans and flies, is compatible with normal embryonicdevelopment but results in clinical symptoms associatedwith FA. We also report the effects of an fh overexpres-sion system, which provides additional insight tofrataxin function.

In agreement with the essential role for frataxinduring development proposed in previous animal mod-els (29, 51), we have found that the silencing of fh usinggeneral and some tissue specific GAL4 lines at 29°C alsoresulted in lethality. Furthermore, we found a distinctsensitivity of different tissues to the reduction of FH.Silencing of fh in muscle and heart was lethal, whereasreduction of fh expression in neural tissues did notresult in preadult phenotypes. This shows that FHfunction in muscles and heart is critical during embry-onic development, whereas FH activity in the nervoussystem appears necessary during adulthood and isrestricted to the PNS.

In this work, we have created a scenario that results ina three-fold reduction of fh mRNA levels by combiningour RNAi transgenic flies with the actin-GAL4 driver at25°C. This reduction was compatible with a normalembryonic development but resulted in shortened lifespan and reduced climbing ability in adulthood. Theseresults strikingly parallel the situation in FA patients,whose motor abilities are mainly impaired from pubertyonward and show a decreased life expectancy (52).

The link between oxidative stress and FA is contro-versial. Variation in levels of oxidative stress markers inFA patients have been used to monitor the develop-ment of the pathology (19–21), and a relationshipbetween oxidative stress and progression of the FAdisease has been already proposed (18, 53). Recently,two reports have dismissed the role of oxidative damagein the progression of the disease (28, 29). To provideinsight into this controversy, we investigated the role ofthe oxidative insult in the development of the pathol-ogy using our model system. One day actin-GAL4/UAS-fhIR individuals were exposed to hyperoxia. The fh-RNAi flies showed a hypersensitive response tohyperoxia, thereby supporting a causative role of oxi-dative stress in FA. This result also suggested that theshortened life span and locomotor defects observedduring aging in fh-RNAi flies is due to the deleteriouseffects of ROS during their lifetime.

Since mitochondrial aconitase is the most affectedenzyme in FA and it has been shown to be, in contrastto other tricarboxylic acid (TCA) cycle enzymes, selec-tively inactivated by oxidative stress (44–46), we moni-tored its activity in fh-RNAi flies. Interestingly, theexposure to hyperoxia led to an extreme reduction ofaconitase activity in the fh-RNAi flies. This result indi-cated a role for FH in protecting aconitase activityagainst ROS-mediated inactivation, supporting frataxinfunction as an aconitase chaperone (17). Oxidativestress induced the release of the Fe-� aconitase Fe-S

cluster leading to its inactivation (54). Thus, withoutthe protection of frataxin, aconitase might not bereactivated at normal rates when oxidative stressreaches high levels, as happens in hyperoxia or duringthe aging process, resulting in its irreversible inactiva-tion.

Surprisingly, during aging, no difference betweencontrol and fh-RNAi flies was detected when totalaconitase activity was determined, though the activityshowed as expected an age-dependent reduction. Wehave shown in this work that the reduction of FHfunction distinctively affects diverse tissues. In addition,the different sensitivity of some tissues to oxidativestress might account for a dissimilar rate of aconitaseinactivation; therefore, the lack of a distinct decrease oftotal aconitase activity in fh-RNAi flies could be due tothe masking effect of intact aconitase when whole flyextracts were used.

Succinate dehydrogenase activity did not show signif-icant alterations during aging or hyperoxia in controlor silenced flies. Altogether, these results suggestedthat, at least in our model, the decay in aconitaseactivity is the primary event in the development of thepathology, occurring before the reduction of SDHactivity reported in patient tissues and in other animalmodels (23, 55, 56).

Strikingly, overexpression of FH also enhanced theoxidative-mediated inactivation of mitochondrial acon-itase. It suggests that an excess of frataxin function isinducing defects in Drosophila likely in an aconitase-related manner as occurs when frataxin expression isdiminished. The inactivation of this enzyme wouldprobably compromise the TCA cycle and the energeticsupply from oxidative phosphorylation leading to thelethality observed. This may also explain why tissuessensitive to FH reduction are sensitive to FH increase.

Aconitase synthesis requires an intact [4Fe-4S] clus-ter for its function and may be affected in an environ-ment with limited iron availability. Overexpression ofmitochondrial ferritin, a protein related to iron metab-olism in the cell, has been reported to inhibit theactivity of the mitochondrial aconitase (57). Frataxinhas been shown to form in vitro iron-binding multimers(10), whose structure would be similar to ferritin mac-romolecules. Therefore, overexpression might induce anew in vivo function for frataxin, being partially respon-sible of the Fe nonavailability in the mitochondriaconducting to aconitase dysfunction. Alternatively,physical interactions between frataxin and other pro-teins such as SDH, aconitase, ferrochelatase, or Isu1have been reported in different organisms (14, 17, 58,59). Since those genes are also present in Drosophila(60) and our results support a direct interaction withone of them, all of these interactions may also exist inthe fly. An excess of frataxin might saturate that set ofinteractions leading to dysfunction of the TCA cycle,the respiratory chain and/or the FeS cluster assemblymachinery, provoking an energetic catastrophe. Thisinterpretation suggests that overexpression might beacting as a dominant negative mutation.



TCA cycle plays a pivotal role in mitochondria bioen-ergetics, and because of its biochemical design, adecrease in the activity of one enzyme will potentiallycompromises the entire respiratory process. The de-cline in aconitase activity could well explain the reduc-tion in ATP production observed in FA patients afterexercise (61, 62). To test that possibility, the oxygenconsumption rate of isolated mitochondria in ourmodel was measured. Our results showed that hyper-oxia led to a remarkable reduction in oxygen consump-tion rates in mitochondrial extracts in our fh-RNAi flies.The fact that this reduction was not detected by activat-ing specifically complex I or II and only found onaddition of pyruvate plus malate indicates that, infh-RNAi flies, the production of electron supply fromthe TCA cycle is defective.

The lack of respiration defects found by specificallystimulating complex II is also in agreement with theintact SDH activity determined under the same condi-tions and suggests mitochondrial aconitase as the pri-mary target of FH deficiency in the silenced flies.

According to our results, we suggest that in Fried-reich ataxia, the regular oxidative mediated inactiva-tion of aconitase occurring normally during the agingprocess is enhanced due to the lack of frataxin. Whenmitochondrial aconitase function is seriously compro-mised, a failure of the TCA cycle leads to an energeticcatastrophe (63). This could explain why in FA pa-tients, tissues with a high energetic demand such asskeletal muscle and heart are seriously affected (39, 61,64). Aconitase inactivation may initiate a harmful feedback loop by increasing oxidative stress in two ways: 1)an impaired electron transport chain would induce theaccumulation of substrates such as NADH that byautooxidation might generate a “reductive stress” con-dition increasing the production of ROS (43) andsecond, leading to an increase in citrate concentration,the immediate substrate of aconitase. Such accumula-tion has been already observed in mammals and corre-lates with the drop of aconitase activity during aging(65). Citrate is a known iron chelator and its toxicity isiron dependent in a yeast frataxin-deficient strain (66).Moreover, citrate Fe3� complexes have been shown toincrease oxidant damage in vitro (67). Interestingly,cardiac tissue shows the highest concentration of citrate(68), and in FA patients, it is where iron deposits havebeen found (39). This burst in oxidative damage mightaccelerate aconitase inactivation and affects other mac-romolecules, such as the Fe-S enzymes of the respira-tory chain.

We have shown in a multicellular model organismthat frataxin is primarily involved in protecting aconi-tase against oxidative-mediated inactivation and pro-pose that oxidative stress plays a central role in theprogression of Friedreich ataxia.

The authors thank Dr Jonathan B. Clark for his carefulreading of the manuscript and Dr. Dan Kiehart, Dr. ManfredFrasch and Dr. Francesc Palau for the generous gifts of theanti-MHC, anti-Eve and anti-Frataxin, respectively. The hy-bridoma antibodies 22C10, EC11, 1D4, and BP 102, which

were developed by Benzer, S., Gratecos, D. and Goodman, C.respectively, were obtained from the Developmental StudiesHybridoma Bank developed under the auspicies of theNICHD and maintained by the University of Iowa, Depart-ment of Biological Sciences, IA 52242. We also thank Dr.Gabrielle Boulianne and the Bloomington Stock Center forfly stocks. This work was supported, in part, by grants fromGeneralitat Valenciana (GV04B-089), and Fondo Investiga-ciones Sanitarias (ISCIII2003-G03/056–2; PI052024). J.V.L. isa recipient of a fellowship from Ministerio de Educacion yCiencia.

We thank J. C. Adell for his advice on the statistical analysesand the Servicio Central de Soporte a la Investigacion Exper-imental de la Universitat de Valencia for access to DNAanalysis resources and databases.

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Received for publication January 23, 2006.Accepted for publication September 14, 2006.



Causative role of oxidative stress in a Drosophila model of Friedreich ataxia

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