A new FRDA mouse model [ Fxn null:YG8s(GAA) > 800] with more than 800 GAA repeats

Abstract

Introduction: Friedreich's ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing.

Methods: We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxn ^null:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models.

Results: We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls.

Discussion: These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.

Keywords: FRDA; FXN; Friedreich’s ataxia; GAA repeat; Y47JR; YG8JR; frataxin; mouse model.

FIGURE 1

Phenotypic and behavioral analysis of YG8JR FRDA mouse models. (A) Body weight (M, male; F, female) and (B) Rotarod analysis of YG8JR compared to Y47JR control mice at age 3–4 and 4–5 months old. (C) Beam-walk analysis of YG8JR compared to Y47JR control mice at age 3–4 and 4–5 months old using 22 and 12-mm beams. Locomotor activity analysis of YG8JR mice compared with Y47JR control mice assaying. (D) Ambulatory distance, (E) vertical time, (F) vertical count, (G) jump count, and (H) average velocity. Values represent mean ± SD. Asterisks indicate significant differences between YG8JR and Y47JR, assessed by unpaired two-tailed Student’s t-test (n = 5 mice per genotype) on time points 3–4 and 4–5 months old (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 2

Intergenerational and somatic guanine-adenine-adenine (GAA) repeat analysis. (A) Representative example of GelRed stained 1.5% agarose gel determines the GAA PCR sizes of skeletal muscle tissues from parents and offspring YG8JR FRDA mice indicating a range from approximately 820 to 900 GAA repeat units in size within FXN gene. Parent 1: male, 7 months old. Parent 2: female, 6 months old. Offspring 1: males, 8–9 months old. Offspring 2: females, 6–9 months old. (B) DNA samples from different tissues of YG8JR FRDA mice. YG8JR 1: female, 9 months old. YG8JR 2: male, 9 months old. YG8JR 3: male, 8 months old. YG8JR 4: male, 9 months old. B, brain; C, cerebellum; H, heart. 1 Kb plus DNA ladder was used as the molecular marker (M).

FIGURE 3

Frataxin expression levels. (A) Quantitative RT-PCR analysis of FXN mRNA extracted from Y47JR control and YG8JR FRDA mouse brain tissues (n = 3 per genotype) using mouse-human specific primers. The experiments were repeated 2–3 times for each genotype and performed in triplicates. Data were normalized to the mean frataxin level of Y47JR samples taken as 100%. Values represent mean ± SD. Asterisks indicate significant differences between YG8JR and Y47JR, assessed by unpaired two-tailed Student’s t-test (**p < 0.01). (B) Representative Western blot images and relative densitometric bar graphs of Frataxin in brain tissues of YG8JR FRDA and Y47JR control mice (n = 3, 2–3 independent experiments). Tubulin was used as protein loading control. Data were normalized to the mean frataxin level of Y47JR samples taken as 100%. Values represent mean ± SD. Asterisks indicate significant differences between YG8JR and Y47JR, assessed by unpaired two-tailed Student’s t-test (*p < 0.05).

FIGURE 4

Frataxin expression levels. (A) Quantitative RT-PCR analysis of FXN mRNA extracted from FRDA and control mouse brain tissues (n = 3) using FXN mRNA with mouse-human specific primers. The experiments were repeated 2–3 times for each genotype and performed in triplicates. Data were normalized to the mean frataxin level of Y47JR samples taken as 100%. Values represent mean ± SD. Asterisks indicate significant differences compared to Y47JR mice, assessed by unpaired two-tailed Student’s t-test (*p < 0.05, **p < 0.01, and ***p < 0.001). (B) Relative densitometric bar graphs of Frataxin in brain tissues of FRDA and control mice (n = 3, 2–3 independent experiments). Tubulin was used as protein loading control. Data were normalized to the mean frataxin level of Y47JR samples taken as 100%. Values represent mean ± SD. Asterisks indicate significant differences compared to Y47JR control mice, assessed by unpaired two-tailed Student’s t-test (*p < 0.05). Two control (Y47R and Y47JR) and four FRDA [YG8JR (>800 GAA), YG8sR (>700 GAA), YG8sR (∼450 GAA), and YG8sR (∼200 GAA)] mice were used.

FIGURE 5

Epigenetic changes within the FXN gene and aconitase activity assay. (A–F) ChIP quantitative PCR analysis on cerebellum tissues of YG8JR and Y47JR tissues for (A–C) acetylated (H3K9ac) and (D–F) methylated (H3K9me3) residues within FXN (A,D) promoter/exon1 (PRO), GAA (B,E) upstream (UP), and (C,F) downstream (DOWN) region (n = 4 mice per group analyzed in triplicates). Results are represented as the relative amount of immunoprecipitated DNA compared with input DNA. YG8JR values were normalized to the Y47JR values that were set as 100%. DNA methylation analysis of CpG U13 in FXN GAA repeat region of DNA from YG8JR FRDA and Y47JR control mice in (G) brain (n = 2 each group), (H) cerebellum (Y47JR n = 3, YG8JR n = 4), and (I) heart (n = 2 each group) tissues. DM, densely methylated; IM, intermediately methylated; UM, unmethylated. Asterisks indicate significant differences between YG8JR and Y47JR, assessed by unpaired two-tailed Student’s t-test (*p < 0.05, **p < 0.01).

FIGURE 6

Aconitase activity. Aconitase activity assay on (A) cerebellum and (B) heart tissues of YG8JR and Y47JR control mice. Relative aconitase activity normalized to citrate synthase (CS) activity from cerebellum and heart tissues (n = 8 per group). YG8JR values were normalized to the Y47JR values that were set as 100%. Asterisks indicate significant differences between YG8JR and Y47JR, assessed by unpaired two-tailed Student’s t-test (*p < 0.05).