Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia

Abstract

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. Fxn^G127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in Fxn^G127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of Fxn^G127V cells upon alteration of fatty acid availability. Importantly, substitution of Fxn^WT with Fxn^G127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose Fxn^G127V cells as a simple and robust model for testing therapeutic approaches for FRDA.

Keywords: Frataxin; Friedreich's ataxia; Mitochondria; Oxidative stress; Point mutation; Senescence.

Fig. 1.

Generation of Fxn^G127V mice via CRISPR/Cas9-mediated knock-in strategy. (A) Alignment of human and mouse frataxin amino acid sequences, with the 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 in exon 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 abbreviation ssODN refers to the single-stranded oligodeoxynucleotide donor template. PAM, protospacer adjacent motif. (C) Representative agarose gel electrophoresis image showing restriction fragment length polymorphism genotyping products for WT, HET and MUT cells. (D) Observed frequency of genotype distribution at weaning is plotted, with the number of animals per group indicated on each pie chart; n=276 biological replicates. (E) The observed frequency of the genotype distribution of embryos at 20 dpc is plotted, with the number of animals per group indicated on each pie chart; n=67 biological replicates.

Fig. 2.

Fxn^G127V mRNA expression is not correlated with immunodetectable protein levels. (A) Frataxin mRNA 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 for three different primer pairs used for analyses: Ex2_Ex3 (spanning exons 2 and 3; upstream of the mutation); Ex3_Ex4 (spanning exons 3 and 4; encompassing the mutation 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 MEF lines 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. Fxn^G127V protein can be detected only with enhanced sensitivity western 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 is representative of three experiments performed with two independent MEF lines per genotype (n=2 biological replicates); total measurements=6. (C) Western blot analysis of protein samples fractionated to soluble and insoluble fractions is shown, with Hprt and Ponceau S staining serving as loading controls. The blot shown is representative of two experiments performed with two independent MEF lines per genotype (n=2 biological replicates); total measurements=4. (D) A representative western blot showing mitochondria-enriched fractions prepared from WT, HET and MUT MEFs. Fifty micrograms of each fraction was loaded per lane. Hprt serves as a positive control for the cytosolic fraction, whereas Nfs1 and Iscu are positive controls for the mitochondrial fraction. The blot shown is representative of two experiments performed with two independent MEF lines per genotype (n=2 biological replicates). Quantitative values are plotted as the mean±s.d. (total measurements per bar=4), with significant differences determined using ordinary one-way ANOVA (**P<0.01, ***P<0.001). IB, immunoblot.

Fig. 3.

Fxn^G127V MUT MEFs exhibit slow growth in culture. (A) Representative phase-contrast images of WT, HET and MUT MEFs at days 1 and 3 in culture after equal plating. Three independent growth curve experiments were performed using two independent MEF lines per genotype (n=2 biological replicates). 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 in duplicate wells at day 0, then counted every 24 h. The experiment was repeated three times using two independent MEF lines per genotype (n=2 biological replicates), and a representative curve is shown as the mean±s.d. (C) WT, HET and MUT MEF 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 determined using ordinary one-way ANOVA (*P<0.05).

Fig. 4.

Fxn^G127V MUT MEFs proliferate more slowly after cell cycle synchronization. (A) A schematic representation of the cell cycle synchronization protocol is shown, whereby quiescence is induced by serum deprivation for 72 h, followed by serum restoration and progression into the cell cycle. (B) Flow cytometry analysis of cell cycle distribution of WT, HET and MUT MEFs (at passage 4) monitored from 16 to 26 h after serum restoration. Each time point is an average of two replicates.

Fig. 5.

Fxn^G127V MUT MEFs are prone to senescence and cell death. (A) Representative phase-contrast images of WT, HET and MUT MEFs stained for detection of SA-β-galactosidase (blue cells) taken at ×200 total magnification. Scale bars: 200 μm. (B) Quantification of senescent cells in WT, HET and MUT MEF cultures 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 by ordinary 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 are divided into quadrants (Q1-Q4). Ten thousand events were collected for each measurement, and measurements were repeated in two independent experiments with 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; total measurements per bar=4. Significant differences were determined between MUT and WT, HET groups by two-way ANOVA using Tukey's method for multiple comparisons (***P<0.001, ****P<0.0001). Cells used for all staining experiments were early passage (passage 3 or 4).

Fig. 6.

Increased mitochondrial damage in Fxn^G127V MUT MEFs. (A) Representative confocal images of WT and MUT passage (p) 4 and 6 MEFs stained with MitoTracker DeepRed FM are shown (z-stack maximum intensity projections). Cells were imaged using an oil immersion ×63 objective. Three independent experiments were conducted (staining and imaging) using two independent MEF lines per genotype (n=2 biological replicates). (B) Fields from ten images per group (A) were analyzed, and mitochondrial network filament lengths were calculated from an average of 415 measurements per field using IMARIS Filament Tracer software. Each bar represents the mean±s.d. of the averaged measurements per field; total averaged measurements per bar=6-9. The significant difference between MUT and WT filament lengths was determined by Student's unpaired t-test (*P<0.05). (C) Relative quantitation of mitochondrial DNA lesions in 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=2 biological replicates, total measurements per bar=4. Significant differences were determined by Student's unpaired t-tests (*P<0.05, **P<0.01). (D) Relative quantitation of mitochondrial DNA copy number normalized to genomic DNA is plotted as the mean±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).

Fig. 7.

Bioenergetic characteristics of Fxn^WT and Fxn^G127V MUT MEFs. (A-J) Shown are OCRs and mitochondrial function indices recorded during mitochondrial 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 MEFs with 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. Comparisons were made with Student's unpaired t-tests between WT and MUT MEFs for each condition. Three independent experiments were performed for panels A-J, each with 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.

Fig. 8.

Viability of Fxn^G127V MUT MEFs can be rescued by mitigating oxidative stress. (A) Growth analysis of WT, HET and MUT MEFs grown in hypoxic conditions 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 twice using 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 MEF population doubling times as calculated from the growth curves shown in A. Bars represent the mean±s.d.; total measurements per bar=4. (C-E) XTT assays were performed after 24 h treatments of WT and MUT MEFs with respective compounds. Absorbances were recorded (specific A_{465 nm} and background A_{660 nm}), and data 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). Significant differences were determined by ordinary one-way ANOVA comparing each treatment with untreated WT (*P<0.05, **P<0.01, ****P<0.0001).