RESEARCH ARTICLE

Mitochondrial damage and senescence phenotype of cellsderived from a novel frataxin G127V point mutation mouse modelof Friedreich’s ataxiaDaniel Fil1, Balu K. Chacko2,3,4, Robbie Conley1, Xiaosen Ouyang2,3,4,5, Jianhua Zhang2,3,4,5, Victor M. Darley-Usmar2,3,4, Aamir R. Zuberi6, Cathleen M. Lutz6, Marek Napierala1 and Jill S. Napierala1,*

ABSTRACTFriedreich’s ataxia (FRDA) is an autosomal recessiveneurodegenerative disease caused by reduced expression of themitochondrial protein frataxin (FXN). Most FRDA patients arehomozygous for large expansions of GAA repeat sequences inintron 1 of FXN, whereas a fraction of patients are compoundheterozygotes, with a missense or nonsense mutation in one FXNallele and expanded GAAs in the other. A prevalent missensemutation among FRDA patients changes a glycine at position 130 tovaline (G130V). Herein, we report generation of the first mousemodelharboring an Fxn point mutation. Changing the evolutionarilyconserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reducednumber of offspring. Like G130V in FRDA, the G127Vmutation resultsin a dramatic decrease of Fxn protein without affecting transcriptsynthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibitsignificantly reduced proliferation and increased cell senescence.These defects are evident in early passage cells and are exacerbatedat later passages. Furthermore, increased frequency of mitochondrialDNA lesions and fragmentation are accompanied by markedamplification of mitochondrial DNA in FxnG127V cells. Bioenergeticsanalyses demonstrate higher sensitivity and reduced cellularrespiration of FxnG127V cells upon alteration of fatty acid availability.Importantly, substitution of FxnWTwith FxnG127V is compatible with life,and cellular proliferation defects can be rescued by mitigation ofoxidative stress via hypoxia or induction of the NRF2 pathway. Wepropose FxnG127V cells as a simple and robust model for testingtherapeutic approaches for FRDA.

KEY WORDS: Friedreich’s ataxia, Senescence, Mitochondria,Frataxin, Point mutation, Oxidative stress

INTRODUCTIONFriedreich’s ataxia (also called FA or FRDA) is an autosomalrecessive disease with an estimated prevalence of 2-4 per 100,000individuals (Leone et al., 1990; Winter et al., 1981). Patientsdevelop progressive ataxia of all four limbs, associated withcerebellar dysarthria, absent reflexes in the lower limbs, sensory lossand abnormal pyramidal signs. Additionally, some patients developoptic atrophy, sensorineural hearing loss, diabetes mellitus, footdeformity, scoliosis and hypertrophic cardiomyopathy (Filla et al.,1996; Harding, 1981). Currently, there is no effective treatment.

FRDA is caused in the majority (96%) of cases byhyperexpansion of trinucleotide GAA repeats located in the firstintron of the frataxin (FXN) gene on both alleles. The remaining(4%) of the patients are compound heterozygotes for GAAexpansion on one FXN allele and a point mutation on the other(Cossee et al., 1999; Durr and Brice, 1996; Filla et al., 1996; Galeaet al., 2016). Although not affecting the coding sequence, GAArepeats impede transcription, leading to low mRNA and proteinlevels. Regarding point mutations on the protein coding sequence,≥30 pathogenic point mutations have been identified (Clark et al.,2019; Cossee et al., 1999; Galea et al., 2016), and only FXN R165Chas been identified in individuals in a homozygous state (Candayanet al., 2020). Many of the point mutations affect FXN mRNAexpression or the initiation of translation and result in more severeclinical presentations when compared with classic repeat expansionpatients (Bidichandani et al., 1997; Cavadini et al., 2000b; Galeaet al., 2016; Santos et al., 2010). Interestingly, a prevalent pointmutation in FRDA, a single nucleotide change (c.389G>T)resulting in a missense substitution at amino acid 130 of glycineto valine (G130V), is associated with a milder clinical presentationand slower disease progression than most FRDA cases arising fromhomozygous GAA repeat expansions (Bidichandani et al., 1997;Clark et al., 2017; McCabe et al., 2000; Puccio and Koenig, 2000).Symptoms common for homozygous repeat expansion FRDA,including dysarthria, loss of tendon reflexes and ataxia, are nottypically observed in G130V individuals, although they dodistinctively exhibit an early onset spastic gait and increasedprevalence of optic disk pallor and diabetes (Cossee et al., 1999;Galea et al., 2016). This suggests that the frataxin G130V(FXNG130V) protein is biologically active and contributes to aunique pathological mechanism.

Frataxin is a nuclear-encoded mitochondrial protein that is highlyconserved throughout evolution, with homologs in eukaryotesincluding mammals, invertebrates and yeast (Dhe-Paganon et al.,2000). Complete absence of wild-type Fxn (FxnWT) is incompatiblewith life, because Fxn null mice die early during embryogenesis(Cossee et al., 2000), and mouse embryonic fibroblasts (MEFs)lacking FxnWT fail to divide (Calmels et al., 2009). After translation

Handling Editor: Steven ClapcoteReceived 9 April 2020; Accepted 16 June 2020

1Department of Biochemistry and Molecular Genetics, University of Alabama atBirmingham, 1825 University Boulevard, Birmingham, AL 35294, USA.2Department of Pathology, University of Alabama at Birmingham, 901 19th StreetSouth, Birmingham, AL 35294, USA. 3Center for Free Radical Biology, University ofAlabama at Birmingham, Birmingham, AL 35294, USA. 4Mitochondrial MedicineLaboratory, University of Alabama at Birmingham, Birmingham, AL 35294, USA.5Department of Veteran Affairs Medical Center, Birmingham, AL 35294, USA. 6TheRare and Orphan Disease Center, JAX Center for Precision Genetics, 600 MainStreet, Bar Harbor, ME 04609, USA.

*Author for correspondence ( jsbutler@uab.edu)

J.S.N., 0000-0002-5141-0822

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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in the cytoplasm, frataxin is transported into the mitochondria usingan N-terminal targeting sequence. Upon entry, it undergoes a two-step proteolytic cleavage by mitochondria processing peptidase(MPP) to generate the mature protein (Koutnikova et al., 1998). Ithas been reported that the G130V mutation modulates interactionwith MPPβ and affects the maturation process (Cavadini et al.,2000a). In fact, the ratio of intermediate to mature frataxin protein isincreased in fibroblasts from FRDA G130V patients, suggesting animpairment in maturation processing (Clark et al., 2017). TheG130V mutation has been found to destabilize the protein structure,leading to low levels of mature frataxin in yeast (Cavadini et al.,2000a).Despite the availability of numerous disease models, no single

model perfectly mimics the human FRDA state. Several FRDAmouse models have been generated thus far, but all of them aimed torecapitulate a condition of low frataxin expression by engineeringlong tracts of the expanded GAAs (Miranda et al., 2002; Pook et al.,2001), conditional deletion of Fxn (Puccio et al., 2001) or transcriptdepletion (short hairpin RNA and small interfering RNA)(Chandran et al., 2017). Thus far, no animal models have beenreported that specifically analyze pathological consequences offrataxin point mutations.To determine the impact of the G130V mutation in vivo, we

generated mice harboring the homologous mutation in murinefrataxin (G127V). Our studies indicate that in the absence of FxnWT,

expression of the FxnG127V protein is sufficient for viability,although significant postnatal lethality was observed. Moreover,cells derived from homozygous mutant embryos (FxnG127V/G127V;MUT) demonstrate a significant increase in a senescent phenotype,significant mitochondrial damage and reduced proliferative capacitythat can be rescued after incubation in a hypoxic environment ortreatment with the nuclear factor erythroid 2 (NF-E2)-relatedfactor 2 (NRF2) activators dimethylfumarate (DMF) oromaveloxolone (RTA 408), conditions and compounds thatmitigate oxidative stress (La Rosa et al., 2020; Saidu et al., 2019;Wong et al., 1999). This new cellular model could be considered asa screening platform for testing the efficacy of small molecules andhuman FXN or FXN replacement therapies aimed to alleviate theeffects of frataxin deficiency.

RESULTSGeneration of frataxin G127V knock-in miceHuman and mouse mature frataxin proteins share 71.5% amino acidsequence identity (CLUSTALW; 73% by BLASTP), indicating thatthe outcome of the amino acid change is comparable between thetwo (Fig. 1A). To study the effects of the disease-causing pointmutation on the endogenous Fxn protein in vivo, we used CRISPR/Cas9 to introduce the (c.401G>T) mutation in exon 4 of murine Fxn(Fig. 1B). The resulting G127V substitution is equivalent to thehuman pathogenic G130Vmissense mutation. Additionally, a silent

Fig. 1. Generation of FxnG127V mice via CRISPR/Cas9-mediated knock-in strategy. (A) Alignment of human and mouse frataxin amino acid sequences, withthe position of glycine 130/127 in bold type and surrounded by a box. (B) Sequence of the edited region of the Fxn gene: scissors illustrate location of DNA break inexon 4 of Fxn; gray box indicates position of single guide RNA (sgRNA); arrow indicates location of missense mutation (red box) and silent mutation (green box).The result of the modification is G→V substitution and creation of an AatII restriction site. F and R illustrate locations of genotyping primers. The abbreviationssODN refers to the single-stranded oligodeoxynucleotide donor template. PAM, protospacer adjacent motif. (C) Representative agarose gel electrophoresisimage showing restriction fragment length polymorphism genotyping products for WT, HET and MUT cells. (D) Observed frequency of genotype distribution atweaning is plotted, with the number of animals per group indicated on each pie chart; n=276 biological replicates. (E) The observed frequency of the genotypedistribution of embryos at 20 dpc is plotted, with the number of animals per group indicated on each pie chart; n=67 biological replicates.

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mutation (c.399T>C) was introduced to serve as a Cas9 blockingmutation and to create a new restriction site (AatII/ZraI) forgenotyping (Fig. 1C).After CRISPR/Cas9 editing, the integrity of the Fxn coding

sequence and the incorporation of the G127V point mutation wereverified by sequencing complementary DNA generated fromcerebella of two mice heterozygous for the FxnG127V allele(FxnG127V/+). The results from both animals indicated thepresence of only the two engineered point mutations, with theremaining Fxn coding sequence being free of errors. Inter-crossingof FxnG127V/+ mice resulted in progeny of all possible genotypes:WT, mice homozygous for wild-type frataxin (Fxn+/+; n=77; 28%);HET, mice heterozygous for Fxn+ and FxnG127V alleles (FxnG127V/+;n=175; 63%); and MUT, mice homozygous for the FxnG127V allele(FxnG127V/G127V; n=24; 9%). MUT mice were viable but wereunder-represented postweaning (9% instead of the expected 25%;Fig. 1D). However, reduced representation of MUT animals was notdetected at the late embryonic stage, 20 days postcoitum (dpc)(Fig. 1E; Fig. S1), suggesting that MUT mice develop normally inutero but fail to thrive shortly after birth. Notably, late-stage MUT

embryos were significantly underweight compared with their WTand HET counterparts, suggesting a disadvantage for competitivesurvival after birth (Fig. S1). Furthermore, mating of HET to WT(C57BL/6J) did not result in a significant difference in mutant alleletransmission (P=0.074; Table S1).

Owing to the low number of MUT animals immediately availablefor study, we conducted molecular analyses on effects of the G127Vmutation on Fxn expression and function using MEFs, because theycould readily be isolated and cultured ex vivo. This homogeneouscellular model allows, for the first time, in-depth analyses of theendogenous FxnG127V protein in the absence of FxnWT.

Endogenous Fxn protein levels are markedly reduced by theG127V mutationTo determine how the mutation affected Fxn expression, we firstmeasured the levels of Fxn mRNA and Fxn protein in MEF celllines established from WT, HET and MUT embryos. Analyses byquantitative reverse transcription PCR (RT-qPCR) demonstratedthat the G127V mutation did not decrease Fxn mRNA expression(Fig. 2A). In addition, because glycine 127 is the second amino acid

Fig. 2. FxnG127VmRNA expression is not correlatedwith immunodetectable protein levels. (A) FrataxinmRNA expression levels measured by real-time RT-qPCR using RNA extracted from WT, HET and MUT MEFs. Fxn transcripts were normalized to Gapdh and plotted relative to WT samples. Data are shown forthree different primer pairs used for analyses: Ex2_Ex3 (spanning exons 2 and 3; upstream of the mutation); Ex3_Ex4 (spanning exons 3 and 4; encompassingthemutation site); and Ex4_Ex5 (spanning exons 4 and 5; downstream of the mutation). Bars show the mean±s.d. and represent data from two independent MEFlines per genotype (n=2 biological replicates), combined from two independent experiments; total measurements per bar=4. Student’s unpaired t-test (*P<0.05).(B) Frataxin protein expression levels in WT and MUT MEFs was determined by western blot. FxnG127V protein can be detected only with enhanced sensitivitywestern blotting techniques. To avoid oversaturation of the signal, 10 μg of WT lysate and 100 μg of MUT lysate were loaded per lane. The blot shown isrepresentative of three experiments performed with two independent MEF lines per genotype (n=2 biological replicates); total measurements=6. (C) Western blotanalysis of protein samples fractionated to soluble and insoluble fractions is shown, with Hprt and Ponceau S staining serving as loading controls. The blot shownis representative of two experiments performed with two independent MEF lines per genotype (n=2 biological replicates); total measurements=4. (D) Arepresentative western blot showing mitochondria-enriched fractions prepared from WT, HET and MUT MEFs. Fifty micrograms of each fraction was loaded perlane. Hprt serves as a positive control for the cytosolic fraction, whereas Nfs1 and Iscu are positive controls for the mitochondrial fraction. The blot shown isrepresentative of two experiments performed with two independent MEF lines per genotype (n=2 biological replicates). Quantitative values are plotted as themean±s.d. (total measurements per bar=4), with significant differences determined using ordinary one-way ANOVA (**P<0.01, ***P<0.001). IB, immunoblot.

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of exon 4, we tested whether the nucleotide substitutions introducednear the 5′ end of this exon affected splicing of the Fxn transcript.RT-qPCR results using primer pairs designed upstream, spanningand downstream of the targeted site revealed that introduction of themutations did not decrease steady-state levels of the Fxn transcript(Fig. 2A). Moreover, RT-qPCR products spanning the Fxn exon 3-4junction were amplified from MUT mRNA, cloned and sequenced,and the results (5/5 clones) demonstrated no abnormally splicedtranscripts. Taken together, analyses of Fxn mRNA from WT, HETand MUTMEFs indicated that introduction of the G127V mutationdid not decrease production or negatively affect splicing of Fxntranscripts.Next, we turned our attention to how the G127V mutation

impacted Fxn protein levels, because we have previously reportedgreatly reduced total Fxn levels, especially of the mature (M)isoform, in fibroblasts derived from FRDA G130V patients (Clarket al., 2017). We first tested three distinct, commercially availableantibodies using lysates prepared from HEK-293T cells transientlyexpressing exogenous murine FxnWT or FxnG127V proteins toaddress the possibility of epitope masking by the point mutation(Fig. S2A). Albeit at much lower levels than FxnWT, the FxnG127V

protein was detected by both FXN polyclonal antibodies (first andsecond panels). However, as previously reported (Koutnikova et al.,1998), the FXN monoclonal antibody (clone 1G2) failed torecognize the mutant protein (third panel). Western blotting ofwhole cell lysates revealed that FxnG127V protein levels wereprofoundly decreased in MUT compared with HET or WTMEFs tosuch an extent that western blot techniques with enhancedsensitivity were necessary to detect the FxnG127V protein in wholecell lysates (Fig. 2B,C). Similar results were obtained with the otherFXN polyclonal antibody tested (Fig. S2B; GTX54036).In light of the low western blot signal from MUT samples, we

examined the possibility of FxnG127V protein aggregation. Asevident from analysis of insoluble fractions of whole cell lysates,neither FxnWT nor FxnG127V was found to aggregate (Fig. 2C).Finally, despite lower immunodetectable levels, the FxnG127V

protein was exclusively found in mitochondria-enriched fractions,in a similar manner to FxnWT protein (Fig. 2D). Western blotquantitation revealed that FxnG127V levels in MUT mitochondrialextracts were only 5% of those observed for FxnWT protein in WTmitochondrial extracts (Fig. 2D). These results indicated thatsteady-state levels of immunodetectable FxnG127V protein were

below the levels of FxnWT protein measured in human FRDA casescaused by homozygous repeat expansions (10-30% of levels inhealthy people) (Deutsch et al., 2010; Long et al., 2017; Nachbaueret al., 2011; Saccà et al., 2013) and other FRDAmouse models [e.g.knock-in/knockout (KIKO) mice (25-36% of WT) (Miranda et al.,2002; Perdomini et al., 2013)]. Nevertheless, this low amount ofFxnG127V protein was sufficient for MEF survival and mousedevelopment.

Cells expressing only FxnG127V exhibit reduced proliferationWe next turned our attention to overt cellular phenotypes observedwhile establishing and culturing independent batches of MUT, HETand WT MEF cell lines. To measure growth rates of linesrepresenting the three genotypes, equal numbers of cells wereseeded, and the cells were counted every 24 h over a 6-day period.We consistently noted slower proliferation of FxnG127V

homozygous MUT MEFs when compared with WT and HET celllines that were established in parallel (Fig. 3A,B). Calculatedpopulation doubling times (PDTs) of WT, HET and MUT MEFsfrom three independent experiments were 31.8, 30.7 and 62.3 h,respectively (Fig. 3C), which is a 2-fold slower growth rate of MUTMEFs when compared with WT or HET.

To test whether the diminished growth rate of MUT MEFs wasattributable to slower progression through the cell cycle, weanalyzed their distribution between G1, S and G2 phases usingflow cytometry. In synchronized cultures, a smaller percentage ofMUT cells were actively dividing compared withWTor HETMEFs(Fig. 4A,B). The WT and HET MEFs entered S phase ∼18 h afterserum supplementation, whereas MUT MEFs mostly remainedquiescent beyond 26 h (the last measurement taken). These resultsaligned with the calculated PDTs (Fig. 3C) and demonstrated aprofound proliferation defect in cells expressing a low level ofFxnG127V protein in the absence of FxnWT.

FxnG127V MUT MEFs exhibit increased senescenceIn addition to suppression of the cell cycle, we considered additionalprocesses that could negatively impact MUT cell numbers in culture(Fig. 3). Given that most of the MUT cell population appeared to bearrested in the G1 phase (Fig. 4B), we turned our attention first tocellular senescence. It was previously shown that acute and severedepletion of FXN (∼80% reduction) in immortalized cells resultedin inhibited proliferation, accompanied by increased G1 phase arrest

Fig. 3. FxnG127V MUT MEFs exhibit slow growth in culture. (A) Representative phase-contrast images of WT, HET and MUT MEFs at days 1 and 3 inculture after equal plating. Three independent growth curve experiments were performed using two independent MEF lines per genotype (n=2 biologicalreplicates). Scale bars: 500 μm. (B) Growth analysis of WT, HET and MUT MEFs over 6 days in culture. Cells (at passage 3) were seeded at equal densities induplicate wells at day 0, then counted every 24 h. The experiment was repeated three times using two independent MEF lines per genotype (n=2 biologicalreplicates), and a representative curve is shown as themean±s.d. (C)WT, HETandMUTMEF population doubling times as calculated from the growth curves (B).Bars show the mean±s.d. calculated from three independent growth curve experiments; total measurements per bar=3. Significant differences were determinedusing ordinary one-way ANOVA (*P<0.05).

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and cellular senescence (Bolinches-Amoros et al., 2014). To testwhether MUT MEFs, expressing low levels of FxnG127V, wereprone to a similar fate, we examined induction of senescence bydetection of senescence-associated β-galactosidase (SA-β-gal)activity, a known characteristic of senescent cells (Dimri et al.,1995). As expected, WT and HETMEF cultures were nearly devoidof senescent cells, with only 2.4±1.6% and 1.7±0.5% of the culturesstaining positive for SA-β-gal activity, respectively (Fig. 5A,B). Incontrast, cells positive for SA-β-gal activity represented 10.3±1.1%in MUT MEF cultures, indicating a ∼4.3-fold increase insenescence induction in MUT MEFs compared with WT(Fig. 5B). Moreover, we found that MUT MEFs senesced at asignificantly faster rate than WT cells when compared at eachpassage (Fig. S3).In addition to senescence, acute frataxin depletion has been

shown to induce cell death via apoptosis (Loría and Diáz-Nido,2015; Mincheva-Tasheva et al., 2014; Palomo et al., 2011). Todetermine whether MUTMEFs were predisposed to apoptosis, wequantified the percentage of viable, apoptotic and dead cells inactively dividing cultures using flow cytometry. WT and HETMEF populations were composed mostly of viable cells (89±4%and 92±0.7%, respectively), whereas the percentage of viable cellsin MUT MEF populations was significantly lower (75±0.8%;P<0.0001) (Fig. 5C,D). By contrast, the percentage of dead cells[propidium iodide (PI) and annexin V positive] was significantlyhigher for MUT populations (12.8±1.7%) compared with WT (4.5±0.8%; P<0.001) or HET (3.3±0.5%; P<0.001). Finally, although

the proportion of apoptotic cells was higher in MUT than in WTand HET MEF populations, the difference did not reach statisticalsignificance (MUT, 7.7±1.1%; WT, 5.5±2.7%; and HET, 3.5±0.4%). Taken together, these data demonstrated that in theabsence of FxnWT, expression of FxnG127V was sufficient forsurvival, but that murine cells expressing only the mutant proteinhad reduced proliferative capacity and augmented cell death andsenescence.

Mitochondrial integrity in FxnG127V MUT MEFs decreasesover timeConsidering that consequences of frataxin deficiency are acutelyobserved in mitochondria (Stepanova and Magrané, 2020), wesought to evaluate effects of FxnG127V expression on functions ofthis organelle. Increased mitochondrial fragmentation has beenreported for some neuronal subtypes derived from FRDA mousemodels (Lin et al., 2017b; Mollá et al., 2017), and fragmentation inFRDA patient cells becomes apparent after an external oxidativeinsult (Lefevre et al., 2012). To assess mitochondrial morphologyand network integrity in WT and MUT MEFs, cells were labeledwith MitoTracker Deep Red and imaged. As shown in Fig. 6A,mitochondrial networks were not qualitatively different between thecell lines at early passages, but fragmented networks were observedmore frequently in MUT MEFs at late passages compared withthose in WT cells. Measurement of the length of mitochondria withIMARIS Filament Tracer software revealed shorter filament lengthsin late passage MUT cells (Fig. 6B).

Fig. 4. FxnG127V MUT MEFs proliferate more slowly after cell cycle synchronization. (A) A schematic representation of the cell cycle synchronizationprotocol is shown, whereby quiescence is induced by serum deprivation for 72 h, followed by serum restoration and progression into the cell cycle. (B) Flowcytometry analysis of cell cycle distribution of WT, HET and MUT MEFs (at passage 4) monitored from 16 to 26 h after serum restoration. Each timepoint is an average of two replicates.

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Frataxin deficiency is also linked with mitochondrial DNA(mtDNA) damage (Haugen et al., 2010; Karthikeyan et al., 2003).Indeed, our previous study in FRDA fibroblasts found that low FXNlevels led to increased mtDNA damage and decreased mtDNArepair capacity (Bhalla et al., 2016). To determine whether theintegrity of mtDNA was compromised in cells expressing onlyFxnG127V, we analyzed mtDNA isolated from WT and MUT MEFsusing a quantitative PCR assay based on the principle that DNAlesions can slow down or block the progression of DNA polymerase(Bhalla et al., 2016; Ponti et al., 1991; Redmann et al., 2018). Thistype of assay detects various forms of DNA damage, from single- ordouble-strand DNA breaks and gaps to specific chemical lesions(Lehle et al., 2014). Quantitation of long (10,085 bp) and short(117 bp) mtDNA fragments amplified from WT and MUT MEFsrevealed an increase in lesion frequency in cells expressing onlyFxnG127V (Fig. 6C), consistent with a higher level of mtDNAdamage.Finally, we calculated the mtDNA copy number by comparing

the amount of mtDNA with an intergenic region of nuclear DNA[fragment of the hexokinase 2 (Hk2) gene]. These results revealedthat the mtDNA content was not changed in early passage MUTMEFs but that it increased significantly in later passage cellscompared with WT MEFs (Fig. 6D). Taken together, these effectson mtDNA integrity and the number of mitochondria suggested thatFxnG127V MUT MEFs accumulated defective mtDNA over timewhich might then contribute to the emergence of the senescencephenotype.

Mitochondria of FxnG127VMUTMEFsexhibit altered fattyacidutilizationFXNWT knockdown (KD) and overexpression both negativelyimpact mitochondrial energy production (Bolinches-Amoros et al.,2014; Vannocci et al., 2018). However, the effect of FXN pointmutations on cellular bioenergetics has not been studied. To assessthe effect of FxnG127V expression on bioenergetics, we measuredcellular oxygen consumption rate (OCR) directly in living cells.Initially, we performed mitochondrial stress tests for WT and MUTMEFs in basal conditions (unchallenged; Fig. 7A,B) (Dranka et al.,2011; Hill et al., 2012). Interestingly, basal and maximal respirationrates were not different betweenWT and MUTMEFs at early or latepassages in these conditions. However, an increase in proton leakwas detected in the MUT cells (compared with WT) at both earlyand late passages (Fig. 7B), which is consistent with a decrease inbioenergetic efficiency, possibly related to mitochondrialmembrane damage.

Next, we assessed the status of fatty acid oxidation in FxnG127V

MUT MEFs. Depletion of endogenous fatty acids followed bypalmitate supplementation revealed similar profiles for earlypassage WT and MUT MEFs, suggesting that both had thecapacity to utilize exogenous fatty acids as an energy source in thesetest conditions (Fig. 7C,D). To determine the dependence of thecells on oxidation of endogenous fatty acids, we next treated theMEFs with etomoxir, which, at low concentrations (10 µM), blocks∼80% of mitochondrial import of fatty acids via irreversibleinhibition of carnitine palmitoyltransferase 1 (CPT1) (McGarry

Fig. 5. FxnG127V MUT MEFs are prone to senescence and cell death. (A) Representative phase-contrast images of WT, HET and MUT MEFs stained fordetection of SA-β-galactosidase (blue cells) taken at ×200 total magnification. Scale bars: 200 μm. (B) Quantification of senescent cells inWT, HETandMUTMEFcultures is plotted as a percentage of total cells counted. Each bar represents the mean±s.d. of six independent fields, in which ≥125 cells were counted per field;n=2 biological replicates, total measurements per bar=6. A significant difference between the MUT group and the WT and HET groups was determined byordinary one-way ANOVA (****P<0.0001). (C) Representative scatter plots are shown for flow cytometry analyses after annexin V and propidium iodide (PI)staining of WT, HET and MUT MEFs. The histograms illustrate the number of cells stained with PI (y-axis) and/or annexin V (x-axis), and the populations aredivided into quadrants (Q1-Q4). Ten thousand events were collected for each measurement, and measurements were repeated in two independent experimentswith two technical replicates per experiment; n=2 biological replicates, total measurements=4. (D) The percentages of live (Q4), apoptotic (Q3) and dead (Q2)cells within WT, HET and MUT MEF cultures were averaged from two independent annexin V/PI flow cytometry experiments; n=2 biological replicates; totalmeasurements per bar=4. Significant differences were determined between MUT and WT, HET groups by two-way ANOVA using Tukey’s method formultiple comparisons (***P<0.001, ****P<0.0001). Cells used for all staining experiments were early passage (passage 3 or 4).

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et al., 1991; Yao et al., 2018). After etomoxir treatment, weobserved a significant reduction of maximal respiration in both WTand MUT MEFs, with a greater inhibition shown by MUT cells forboth ATP-linked and maximal respiration, in addition to theirreserve capacity (Fig. 7E,F). In a similar manner, we analyzed fattyacid utilization of late passage WT and MUT MEFs and observedsignificant decreases in OCR for both types of cells after bothtreatments, with exacerbated effects observed for MUT MEFs(Fig. 7G-J).

Mitigation of oxidative stress increases viability of FxnG127V

MUT MEFsIt was recently demonstrated that hypoxic conditions rescue frataxinnull organisms from lethality (Ast et al., 2019). Indeed, we observeda significant increase in proliferation of MUT MEFs, rescued torates similar to those observed for WT and HET MEFs, when thecells were cultured in a hypoxic environment (Fig. 8A). Thecalculated PDTs for WT, HET and MUT cells grown in hypoxicconditions were 29.4, 28.4 and 31.1 h, respectively, with nosignificant differences determined between the genotypes (Fig. 8B).We also used XTT assays to test the effects of several compoundsshown to improve biochemical and cellular phenotypes of FRDAcell line models, including idebenone (IDB) and omaveloxolone(RTA 408; Omav), which are molecules that have been or arecurrently included in clinical trials for FRDA (Zhang et al., 2019),and dimethyl fumarate (Tecfidera; DMF), which is a moleculeapproved for treatment of multiple sclerosis (http://www.fda.gov)(Gold et al., 2012; Saidu et al., 2019). Viability of MUT MEFs wasimproved after 24 h of treatment with Omav (Fig. 8C) and DMF

(Fig. 8D) to levels that no longer differed significantly fromuntreated WT cells, whereas idebenone treatment had no effect(Fig. 8E). Taken together, these results implicated oxidative stress asa driver of reduced survival and proliferation of FxnG127V MUTMEFs and suggested that restoring the redox balance via NRF2activation significantly improved the viability of these cells.

DISCUSSIONHerein, we report generation of mice carrying a G127V mutation inthe endogenous Fxn locus, analogous to the pathogenic G130Vmutation observed in individuals diagnosed with FRDA(Bidichandani et al., 1997). Previous studies of FXNG130V inMEF cultures demonstrate that transgenic expression of the mutanthuman protein can rescue the lethal phenotype of Fxn knockout(Calmels et al., 2009). The viability of our homozygous FxnG127V

mice also demonstrates that the FxnG127V mutant protein retains alevel of function that, on its own, sustains life in a whole organism.Individuals carrying homozygous FXN G130V mutations have notbeen identified. The possibility exists that homozygous FXNG130V mutations could result in embryonic or perinatal lethality,an idea supported, in part, by our results demonstrating reducedrepresentation of FxnG127V MUT mice after birth. Othercontributory factors could be under- or misdiagnosis owing tounderutilization of high-throughput sequencing methods to supportmolecular diagnoses and atypical clinical presentations.

The normal Mendelian genotype distribution of mouse embryosat 20 dpc and reduced representation ofMUT neonates is intriguing.MUT mice seem to develop normally in utero, but the majority failto thrive shortly after birth. It is plausible that deficiencies resulting

Fig. 6. Increasedmitochondrial damage in FxnG127V MUTMEFs. (A) Representative confocal images of WT andMUT passage (p) 4 and 6MEFs stained withMitoTracker DeepRed FM are shown (z-stack maximum intensity projections). Cells were imaged using an oil immersion ×63 objective. Three independentexperiments were conducted (staining and imaging) using two independent MEF lines per genotype (n=2 biological replicates). (B) Fields from ten images pergroup (A) were analyzed, and mitochondrial network filament lengths were calculated from an average of 415 measurements per field using IMARIS FilamentTracer software. Each bar represents the mean±s.d. of the averaged measurements per field; total averaged measurements per bar=6-9. The significantdifference between MUT andWT filament lengths was determined by Student’s unpaired t-test (*P<0.05). (C) Relative quantitation of mitochondrial DNA lesionsin WT and MUT MEFs calculated from the ratio of long PCR (10 kb) product normalized to the short PCR (117 bp) product is plotted as the mean±s.d; n=2biological replicates, total measurements per bar=4. Significant differences were determined by Student’s unpaired t-tests (*P<0.05, **P<0.01). (D) Relativequantitation of mitochondrial DNA copy number normalized to genomic DNA is plotted as themean±s.d; n=2 biological replicates, total measurements per bar=4.Significant differences were determined by Student’s unpaired t-tests (ns, not significant; *P<0.05, **P<0.01).

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from low levels of FxnG127V protein hinder adequate metabolicadaptation at birth, when the neonate must make a transition from acontinuous transplacental supply of glucose to a variable fat-basedfood supply via a series of coordinated metabolic and hormonalchanges coupled with a dramatic increase in oxygen availability(Ward Platt and Deshpande, 2005). Extremely low levels of Fxn(WT or G127V) could potentially exaggerate iron sulfur cluster(ISC) deficiency, affect multiple ISC-dependent processes,including respiration (Brzóska et al., 2006), DNA replication andrepair (Fuss et al., 2015) or ribosome maturation (Kispal et al.,2005), and curtail the survival of MUT mice in the crucial periodafter birth. Comprehensive studies will be necessary to assessdevelopmental milestones and to determine the precise mechanismand timing of premature lethality in MUT mice. Owing to reducedrepresentation of postnatal mutant animals, we focused our initialanalyses on MEFs isolated from FxnG127V MUT mice, and fullmolecular and behavioral phenotyping of FxnG127V mice will bedescribed elsewhere.

Molecular analysis of MUT MEFs revealed that the G127Vmutation does not reduce Fxn mRNA levels but has a profoundnegative impact on immunodetectable Fxn protein levels. TheG130V mutation was shown to reduce the solubility of purifiedrecombinant human FXN proteins in vitro (Correia et al., 2008) orwhen exogenously expressed at high levels (Clark et al., 2017).However, our data indicate that the FxnG127V protein expressed fromthe endogenous locus is detectable exclusively in soluble fractions.Moreover, endogenous FxnG127V is localized to mitochondria, as isexpected and observed for the FxnWT protein.

Depending on the FRDA model, different effects on the integrity(e.g. ultrastructure), number and function of mitochondria havebeen reported. For instance, reduced mitochondrial biogenesis and,subsequently, the number of mitochondria was reported in FRDApatient-derived cells, in FXNWT KD control cells and in brain andskeletal muscle from the KIKO mouse model (Jasoliya et al., 2017;Lin et al., 2017a,b), whereas accumulation of damagedmitochondria was observed in FRDA induced pluripotent stem

Fig. 7. Bioenergetic characteristics of FxnWT and FxnG127V MUT MEFs. (A-J) Shown are OCRs and mitochondrial function indices recorded duringmitochondrial stress tests conducted on the following: (A,B) early (p4) and late (p8) passage WT and MUT MEFs; (C,D) early (p4) passage WT and MUT MEFswith or without palmitate-BSA (25 µg/ml) treatment; (E,F) early (p4) passage WT and MUT MEFs with or without etomoxir (10 µM) treatment; (G,H) late passage(p8) WT and MUT MEFs with or without palmitate-BSA (25 µg/ml) treatment; or (I,J) late passage (p8) WT and MUT MEFs with or without etomoxir (10 µM)treatment. Data for unchallenged WT and MUT MEFs (A,B) are replotted on all graphs for side-by-side comparison of all treatments/conditions. Comparisonswere madewith Student’s unpaired t-tests betweenWT and MUTMEFs for each condition. Three independent experiments were performed for panels A-J, eachwith at least four technical replicates per sample. Representative plots are shown for each as the mean±s.e.m.; total measurements per bar=12 (unchallenged),4 (etomoxir) and 4 (palmitate). AntiA, antimycin A; Eto, etomoxir; FCCP, carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone; Oligo, oligomycin; Palm,palmitate. *P<0.05, **P<0.01, ***P<0.0.001, ****P<0.0001.

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cell-differentiated cardiomyocytes and cardiac Fxn conditionalknockout (cKO) mice (Hick et al., 2013; Perdomini et al., 2014;Vyas et al., 2012). Our results demonstrate a significantly increasedfrequency of mtDNA lesions in FxnG127VMUTMEFs, even at earlypassages, whereas an increased number of mitochondria iscorrelated with time in culture (i.e. with increasing passage).Elevated mitochondrial biogenesis could be a compensatorymechanism initiated by FxnG127V MUT cells to overcome theunderlying load of damaged mitochondria. Indeed, this type ofmechanism was previously proposed to explain similar resultsobtained using FRDA fibroblasts harboring homozygous repeatexpansions (García-Giménez et al., 2011). In addition to totalcontent, analyses of the dynamic state of mitochondria alsoprovided insight into the networks formed over time in FxnG127V

MUT MEFs. Although the mitochondrial networks formed in earlypassageMUT cells appear indistinguishable from those inWT cells,filament lengths in later passage MUT MEFs are significantlyshorter than those of WT cells at matched passage. Increasedfragmentation could serve as an independent indicator of excessivemitochondrial damage in late passage FxnG127V MUT MEFs.Elevated mitochondrial content is one feature of senescent cells

and is often accompanied by increased production of reactiveoxygen species, reduced membrane potential and reducedrespiratory coupling (Korolchuk et al., 2017), additionalphenotypes that were revealed by our bioenergetic studies of early

and late passage MUTMEFs. Extracellular flux analyses have so farbeen reported only recently in FRDA cellular models, namely insensory neurons differentiated from induced pluripotent stem cellsderived from patients harboring homozygous repeat expansions (i.e.chronically low FXNWT expression) (Igoillo-Esteve et al., 2020)and an inducible FXN overexpression HEK293 cell line (i.e. short-term high FXNWT expression) (Vannocci et al., 2018). Basal andmaximal respiration and ATP production were significantly reducedin FXNWT-deficient sensory neurons (Igoillo-Esteve et al., 2020),and no beneficial effects were observed for these parameters uponFXNWT overexpression (Vannocci et al., 2018). Expression of onlyFxnG127V protein was sufficient to maintain basal respiration atlevels observed for WT MEFs at early and late passages.

Impaired glucose utilization and increased β-oxidation in FRDApatients was revealed by metabolic labeling of freshly isolatedplatelets (Worth et al., 2015), and lipid metabolism defects have beenreported in various FRDA cellular and animal models (Tamarit et al.,2016). Our study on FxnG127V MUT MEFs also reveals alteredendogenous fatty acid utilization, and our results could indicate thatMUT MEFs depend on oxidation of endogenous FAs for energyproduction more than WT MEFs do, or that their available fatty acidpool is lower. The increased dependence of MUT mitochondria onfatty acid oxidation could also be a compensatory mechanism forimpairments in glycolysis or other metabolic pathways and might berelated to increased susceptibility to oxidative stress.

Fig. 8. Viability of FxnG127V MUT MEFs can be rescued by mitigating oxidative stress. (A) Growth analysis of WT, HET and MUT MEFs grown in hypoxicconditions over 6 days in culture. Cells were seeded at equal densities in duplicate wells at day 0, then counted every 24 h. The experiment was repeated twiceusing two independent MEF lines per genotype (n=2 biological replicates), and a representative curve is plotted as the mean±s.d. (B) WT, HET and MUT MEFpopulation doubling times as calculated from the growth curves shown in A. Bars represent themean±s.d.; total measurements per bar=4. (C-E) XTTassays wereperformed after 24 h treatments of WT and MUT MEFs with respective compounds. Absorbances were recorded (specific A465 nm and background A660 nm), anddata for each treatment are expressed relative to untreated WT cells. Each bar represents the mean±s.d. of at least three independent experiments (black dots)performed using two independent MEF lines per genotype (n=2 biological replicates); total measurements per bar=5 (Omav), 4 (DMF) and 3 (IDB). Significantdifferences were determined by ordinary one-way ANOVA comparing each treatment with untreated WT (*P<0.05, **P<0.01, ****P<0.0001).

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Some growth phenotypes typified by FXNWT-deficient cellularmodels, such as reduced proliferation and viability and increasedsensitivity to oxidative stress (Carletti et al., 2014; Cotticelli et al.,2019; La Rosa et al., 2019; Loría and Diáz-Nido, 2015; Palomoet al., 2011), are observed in FxnG127V MUTMEFs. However, mostof these studies report significantly increased apoptosis upondepletion of FxnWT, whereas FxnG127V MUT MEFs insteadundergo growth arrest and senescence, suggesting a residualfunction of the mutant protein that sustains cell viability. Theability of certain antioxidant agents to restore proliferation of MUTMEFs supports this hypothesis.Activation of the NRF2 pathway as a therapeutic target in FRDA

has gained attention because positive results of a phase 2 study forOmav treatment of FRDA patients were recently reported(NCT02255435) (La Rosa et al., 2020; Lynch et al., 2019).Increased NRF2 levels (mRNA and protein), in addition to theinduction of NRF2 target gene expression, are observed after 24 h oftreatment of FRDA fibroblasts with DMF, idebenone or Omav(Petrillo et al., 2019). Moreover, treatment of fibroblasts from FRDApatients with DMF was shown to increase FXN mRNA levels(Jasoliya et al., 2019), but treatment with Omav or idebenone had noeffect on FXN gene expression (Petrillo et al., 2019). Finally, Omavtreatment resolved several mitochondrial defects attributed to lowFXNWT/FxnWT levels in FRDA fibroblasts andwas protective againstoxidative stress-induced cell death (Abeti et al., 2018). Likewise, ourstudy indicates that activation of NRF2 signaling in cells expressingonly the FxnG127V protein is protective against cell death, at least viaOmav- and DMF-mediated actions. Omav and DMF both work toincrease NRF2 levels and transcriptional activity by regulating itsrepressor, Kelch-like ECH-associated protein 1 (KEAP1), therebypreventing its degradation and augmenting its nuclear translocation(Probst et al., 2015; Takaya et al., 2012). We can speculate that thetypical NRF2 antioxidant transcriptional response is hampered, butwe cannot rule out the involvement and regulation of cell cycleprotein expression, and therefore additional studies will be necessaryto determine the mechanism(s) of action of DMF and Omav inenhancing the viability of FxnG127V MUT MEFs.Our results demonstrate that cells expressing only the FxnG127V

mutant protein exhibit many shared pathophysiologicalcharacteristics of cells expressing low levels of FxnWT protein,especially reduced growth potential and sensitivity to oxidative stress.However, some phenotypes, especially those pertaining to lipidhandling, appear to be attributed uniquely to expression of theFxnG127V mutant protein. These results support a hypothesis ofFxnG127V/FXNG130V proteins assuming alternative functions ratherthan loss of function. Changes in Fxn/FXN function induced by theG127V/G130V mutation, whether it be differential association withISC assembly proteins or by eliciting new interactions, could help toshed light on uniquemolecular events underlying the atypical clinicalpresentation of FRDA G130V patients. It will be crucial to definefunctional relationships of the FxnG127V protein in neurons involvedin prominent FRDA G130V symptoms, such as spastic paraparesis.Examining the effects of FxnG127V expression on disease-relevanttissues, such as those of the central nervous system and the heart, inthe mouse model, and accounting for temporal aspects ofdevelopment and aging processes are of immediate interest.

MATERIALS AND METHODSAnimalsMice were housed in the animal facility at the University of Alabama atBirmingham under 12 h/12 h light/dark conditions and fed ad libitum. Allexperimental procedures were conducted in accordance with the Guide for

the Care and Use of Laboratory Animals published by the US NationalInstitutes of Health (NIH publication no. 85-23, revised 1996) and wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Alabama at Birmingham.

Generation of Fxn G127V miceThe G127V knock-in mutation (GGC→GTC codon change) was introducedusing CRISPR/Cas9 endonuclease-mediated genome editing viahomologous-directed repair. Sequences for the guide RNA andoligonucleotide donor used are provided in Table S2. A silent D126D(GAT→GAC) was co-introduced with the G127V mutation as a PAMblocker. C57BL/6J zygotes with well-recognized pronuclei weremicroinjected with guide RNA, the mutation-bearing oligonucleotidedonor and Cas9 nuclease. After injection, the embryos were transferred topseudopregnant females. Two founders in 31 progeny were identified thatcontained the desired D126D, G127V mutations. Both were mated toC57BL/6J, and a single founder demonstrated transmission of the targetedD126D, G127V mutation through the germline. This mouse strain wasdesignated JR 30822 (C57BL/6J-Fxnem8Lutzy/J). The population ofJR 30822 mice was expanded with an additional backcross to C57BL/6Jmice before intercrossing to score for the effect on homozygosity. Thecolony is maintained by backcrossing of heterozygous mice to wild type(C57BL/6J or wild-type littermates).

GenotypingMouse genomic DNA was isolated from ∼3 mm tail biopsies withQuickExtract™ DNA Extraction Solution (Lucigen # QE09050) and usedas a template for PCRs. A 300 bp fragment encompassing the Fxn pointmutation was amplified with JumpStart™ Taq DNA Polymerase (Sigma-Aldrich, #D9307) using Fxn G127V restriction fragment lengthpolymorphism primers found in Table S2. After amplification, the PCRproduct was digested overnight at 37°C with 0.5 μl of the restriction enzymeAatII (NEB, #R0117) added directly to the PCR tube. Subsequently, thereaction products were resolved by 1% agarose gel electrophoresis andvisualized with ethidium bromide.

Derivation and culture of MEFsMEFs were derived according to (Jozefczuk et al., 2012). Briefly, pregnantfemale mice at 13 or 14 dpc were sacrificed and embryos extracted into Petridishes. After removal of heads and red organs, the remaining tissues wereminced, trypsinized with 0.05% trypsin/EDTA and incubated for 15 min at37°C. Disrupted tissues were centrifuged at 300 g for 5 min, the supernatantwas removed, and pelleted cells were suspended in Dulbecco’s modifiedEagle’s medium (DMEM) high-glucose medium with pyruvate (LifeTechnologies, 11995) supplemented with 10% fetal bovine serum (HyClone,SH30396.03), 1% GlutaMAX (Life Technologies, 35051), and 1% penicillin-streptomycin (Life Technologies, 15140). Cells from each embryo were platedseparately on gelatin-coated 100 mm dishes and propagated in DMEM (asabove) supplemented with 10% fetal bovine serum and 1% GlutaMAX.Experiments denoted as ‘early passage’ were performed with cells betweenpassage numbers 3 and 5, whereas ‘late passage’ denotes cells of passages 6-9.MEF cell lines derived from at least two different embryos per genotype wereused for each experiment, and considered as independent biological replicates.

Treatments and XTT assaysWTandMUTMEFswere seeded at 20,000 cells per well in 96-well plates 24 hbefore treatments. Compoundswere added and cells incubated for an additional24 h, followed by incubation with XTT [(sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium)] reagent for 3 h.The XTT assays were performed as recommended by the manufacturer(ATCC). Commercially available compounds were obtained as follows: Omav(RTA 408; Cayman Chemical), DMF (Tocris) and idebenone (Sigma-Aldrich).

Quantitation of mRNARNAwas isolated fromMEFs using the RNeasy Mini Kit (Qiagen, #74104).Genomic DNA contamination was removed with the TURBO DNA-free™

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Kit (Invitrogen, #AM1907). The level of specific mRNAwas quantitated byPower SYBR™ Green RNA-to-CT™ 1-Step Kit (Applied Biosystems,#4389986). Transcript (mRNA) levels relative to that of the housekeepinggene glyceraldehyde phosphate dehydrogenase (Gapdh) were calculatedusing the ΔΔCt method. Primer sequences can be found in Table S2.

Western blotFor preparation of total protein lysates, MEFs were homogenized in NP-40lysis buffer containing 1% NP-40, 20 mM Tris-HCl pH 7.4, 150 mMNaCl,5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 10 mM sodium fluoride,1 mM sodium orthovanadate and 5 mM sodium pyrophosphate, withprotease inhibitor cocktail (Millipore, #539134). The lysates were rotatedfor 30 min at 4°C, followed by centrifugation at 15,800 g for 20 min. Thesupernatant was removed as the soluble fraction. To reduce carryover, thepellets were washed with lysis buffer and resuspended in urea-sodiumdodecyl sulfate buffer (NP-40 lysis buffer with 8 M urea/3% sodiumdodecyl sulfate) followed by sonication, with three 20 s pulses at 20%amplitude on a Fisher Scientific™ Model 120 Sonic Dismembrator. Thelysates were then centrifuged again at 15,800 g for 20 min at 4°C and thesupernatant was collected as the insoluble fraction. Protein concentration ofsoluble fractions were estimated by Bradford assay using Protein Assay DyeReagent (Bio-Rad, #5000006). Samples were heated with NuPAGE™ LDSSample Buffer (4×) (Invitrogen, #NP0008) and NuPAGE® Reducing Agent(10×) (Invitrogen, #NP0004) for 10 min at 70°C. Protein samples wereresolved on a 4-12% NuPAGE gel and transferred to nitrocellulosemembranes with an iBlot™ 2 Gel Transfer Device (Invitrogen, #IB21001).Membranes were blocked with 5% ECL™ Advance Blocking Reagent (GEHealthcare, #RPN418) followed by incubation with primary and secondary(anti-rabbit horseradish peroxidase linked, GE Healthcare, #LNA934V)antibodies. Antibody information is provided in Table S3. For enhancedsensitivity, membranes were incubated with SuperSignal™ Western BlotEnhancer (Thermo Fisher Scientific, #46640) for 10 min at roomtemperature (RT) before blocking, and high-intensity horseradishperoxidase substrate SuperSignal™ West Femto (Thermo FisherScientific, #34094) was used to produce a signal. All images werecaptured on a ChemiDoc MP Imaging System and analyzed usingImageLab v.6.0.1 software (Bio-Rad).

Mitochondrially enriched protein lysates were prepared as described (Linet al., 2017b), with modifications. Cells were resuspended in hypotonicbuffer [225 mM mannitol, 75 mM sucrose, 5 mM Hepes, 1 mM EGTA,0.1 mM EDTA (pH 8), 0.1% bovine serum albumin (BSA), 1% proteaseinhibitor cocktail (PIC; Sigma-Aldrich, P8340)], kept on ice for 10 min,then homogenized. The suspensions were centrifuged at 1500 g for 4 min at4°C, after which supernatants were transferred to fresh tubes, and thesuspensions were centrifuged again at 20,000 g for 15 min at 4°C.Supernatants were collected as ‘cytoplasmic’ fractions, and pellets werewashed with buffer [50 mM Tris (pH 7.5), 0.25 M sucrose, 0.2 mM EDTA(pH 8), 0.1%BSA and 1% PIC], then resuspended in lysis buffer [0.1%NP-40, 0.25 M NaCl, 5 mM EDTA, 50 mM Hepes (pH 7.5) and 1% PIC] andkept on ice for 10 min for protein extraction. Lysates were centrifuged at20,000 g for 10 min at 4°C, and clarified supernatants were transferred tofresh tubes as ‘mitochondrially enriched’ fractions.

Growth curves and population doubling time of MEFsEarly passage WT, HET and MUT MEFs were seeded in duplicate in six-well plates at 1×105 cells per well and cultured in either a cell cultureincubator with ‘normoxic’ gas composition (air supplemented with 5%CO2) or ‘hypoxic’ gas composition (90% N2, 5% CO2 and 5% O2). Cellswere detached with trypsin and counted daily after seeding (days 1-6). ThePDT was calculated during the logarithmic phase of the growth using theformula: PDT=T×ln2/ln(A/A0), where T corresponds to the time duration ofculture (in hours), A corresponds to the number of cells in the well at the timeof measurement and A0 is the initial number of cells.

Measurement of cell death and cell cycleMEFs were plated at 1×105 WT or 2×105 MUT cells per 100 mm dish andcultured until ∼80% confluent. Cells were detached with accutase andwashed with PBS. For cell death analysis, MEFs were processed with the

FITC Annexin V Apoptosis Detection Kit I (BD BioSciences, #556547)according to the manufacturer’s instructions. For cell cycle analysis, MEFswere fixed with cold 70% ethanol overnight, then stained with PBSsupplemented with Triton X-100 (final concentration 0.1%), RNase A (finalconcentration 200 µg/ml) and PI (final concentration 20 µg/ml) for 30 minat RT. Stained MEFs were counted with an LSRII Analyzer, BD Divav.8.0.1 (BD BioSciences) and data analyzed with the FlowJo softwarepackage using a Watson pragmatic model.

Senescence-associated β-galactosidase stainingCell staining was performed with a Senescence β-galactosidase Staining Kit(Cell Signaling Technology, #9860), following the manufacturer’sinstructions. At least ten images were taken per group at ×200 totalmagnification, and the fraction of blue-stained cells counted by a blindedinvestigator. To avoid staining attributable to cell confluence rather than toproliferative senescence (Severino et al., 2000), the assay was performed insubconfluent cultures displaying comparable cell densities.

Staining of mitochondriaMEFs were plated on glass coverslips, cultured until ∼60-80% confluentand stained with OptiMEM supplemented with 100 nM (finalconcentration) MitoTracker™ Deep Red FM (Thermo Fisher Scientific,#M22426) for 30 min at 37°C followed by 30 min incubation in OptiMEMwithout the dye. Stained MEFs were fixed with 3.7% formaldehyde for15 min at 37°C, mounted on microscope slides with ProLong™ GoldAntifade Mountant with 4′,6-diamidino-2-phenylindole (Invitrogen,#P36935) and imaged with a Nikon A1R confocal microscope using ×63objective magnification (University of Alabama at Birmingham HighResolution Imaging Facility). Maximum-intensity projections of confocalimage z-stacks were rendered as three-dimensional reconstructions usingImaris analytical software (Bitplane AG). Imaris Filament Tracer tool withsubjective thresholding was used to detect mitochondrial filament lengthsautomatically.

Mitochodrial DNA damage and copy number in MEFsMitochondrial DNA damage was determined by quantitative PCR (qPCR)as described by Furda et al. (2014), with some modifications. Briefly, totalDNA was isolated with the QIAamp DNA Mini Kit (Qiagen, #51304)according to the manufacturer’s instructions. Long mtDNA fragments wereamplified with high-fidelity AccuPrime™ Taq DNA polymerase (ThermoFisher Scientific, #12346086) and quantified with the Quant-iT™PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific, #P7589). Ashort mtDNA fragment was quantified with Power SYBR™ Green PCR(Thermo Fisher Scientific, #4367659) relative to genomic DNA.Quantitation of the average lesion frequency in mtDNA was performedassuming a random distribution of lesions in accordance with the Poissonequation (Westbrook et al., 2010). The mtDNA copy number was estimatedby determination of the mtDNA to nuclear DNA ratio with a qPCR assay asdescribed by Quiros et al. (2017). Primer sequences can be found inTable S2.

Measurement of mitochondrial respirationThe mitochondrial function of MEFs was determined using a SeahorseExtracellular Flux Analyzer (XF96 Analyzer; Agilent Technologies, SantaClara, CA, USA), which measures the OCR in live cells. Cells were plated at40,000 cells per well in a XF96 assay plate and allowed to attach for 4 h inculturemedium before the assay, washed usingXF-DMEMassaymedium andallowed to equilibrate in a non-CO2 incubator for 1 h at 37°C. Mitochondrialstress tests were performed by sequentially injecting oligomycin (Oligo, 1 µg/ml), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1.5 µM)and antimycin A (Anti A, 10 µM) (Chacko et al., 2013). To record fatty acid-mediated OCR measurements, BSA-palmitate substrate (25 µg/ml) wasincubated with MEFs for 30 min before the assay in XF-DMEM medium(Ravi et al., 2015). Etomoxir (10 µM) was added to determine fatty acid-sensitive OCR (Ravi et al., 2015). The datawere normalized to the total proteincontent in each well, measured using the Lowry HS protein assay (Bio-Rad)and expressed as the mean.

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Statistical analysesStatistical analyses were conducted using GraphPad Prism v.6. Statisticalsignificance was determined by performing Student’s unpaired two-tailedt-tests, multiple t-tests (with Holm–Sidak correction for multiplecomparisons), ordinary one-way ANOVA or two-way ANOVA (withTukey correction for multiple comparisons). Significant differences wereconsidered as P<0.05.

AcknowledgementsThe authors wish to acknowledge the technical support of the University of Alabamaat Birmingham High Resolution Imaging Facility. We also thank Ms Yu-Yun Chen forassistance with antibody validation experiments.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: A.R.Z., C.M.L., M.N., J.S.N.; Methodology: A.R.Z., C.M.L., M.N.,J.S.N.; Formal analysis: D.F., B.K.C., J.Z., V.M.D.-U., A.R.Z., C.M.L., M.N., J.S.N.;Investigation: D.F., B.K.C., R.C., X.O., A.R.Z., J.S.N.; Writing - original draft: D.F.,J.S.N.; Writing - review & editing: D.F., B.K.C., J.Z., V.M.D.-U., A.R.Z., C.M.L., M.N.,J.S.N.; Supervision: J.S.N.; Project administration: J.S.N.; Funding acquisition:M.N., J.S.N.

FundingThis work was supported by the Office of Extramural Research, National Institutes ofHealth, National Institute of Neurological Disorders and Stroke (R03 NS099953 toJ.S.N., R21 NS101145 and R01 NS081366 to M.N.) and Friedreich’s AtaxiaResearch Alliance (to J.S.N.). The Nathan Shock Center (P30 AG050886) grantsupported the bioenergetics measurements. The Jackson Laboratory’s GeneticEngineering Technologies Scientific Service partially supported the development ofthe Fxn mutant mouse model described in this publication. Mouse strain JR083822is available from the Jackson Laboratory mutant mouse collection (www.jax.org).

Supplementary informationSupplementary information available online athttps://dmm.biologists.org/lookup/doi/10.1242/dmm.045229.supplemental

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