Frataxin deficiency impairs mitochondrial biogenesis in cells, mice and humans

Abstract

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by inherited deficiency of the mitochondrial protein Frataxin (FXN), which has no approved therapy and is an area in which biomarkers are needed for clinical development. Here, we investigated the consequences of FXN deficiency in patient-derived FRDA fibroblast cell models, the FRDA mouse model KIKO, and in whole blood collected from patients with FRDA. We observed decreased mitochondrial copy number in all the three FRDA models tested: cells, mice and patient blood. In addition, we observed 40% residual mitochondrial gene expression in FRDA patient blood. These deficiencies of mitochondrial biogenesis in FRDA cells and patient blood are significantly correlated with FXN expression, consistent with the idea that the decreased mitochondrial biogenesis is a consequence of FXN deficiency. The observations appear relevant to the FRDA pathophysiological mechanism, as FXN-dependent deficiency in mitochondrial biogenesis and consequent mitochondrial bioenergetic defect could contribute to the neurodegenerative process. The observations may also have translational potential, as mitochondrial biogenesis could now be followed as a clinical biomarker of FRDA as a correlate of disease severity, progression, and therapeutic effect. Also, mitochondrial copy number in blood is objective, scalar and more investigator-independent than clinical-neurological patient rating scales. Thus, FXN deficiency causes mitochondrial deficiency in FRDA cells, the KIKO mouse model, and in whole blood of patients with FRDA, and this deficiency could potentially be used in clinical trial design.

Figure 1

There is decrease in mitochondrial mass in FRDA fibroblast cell model. Human fibroblast cells were cultured in 96 well plate and stained with DAPI, phalloidin and mitotracker deep red FM. (A) Representative Image obtained from MetaXpress® Hi-content image acquisition, Molecular Devices. (B) Amount of mitochondrial mass measured as mitotracker intensity per cell. Bars represent averages ± standard deviations (n = 8, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

Frataxin expression is correlated to mitochondrial biogenesis marker in human fibroblast cell model. Linear regression analysis of FXN mRNA expression with (A) mitochondrial DNA to nuclear DNA ratio (mtTL1/B2M) (B) NRF1 mRNA expression and (C) TFAM mRNA expression.

Figure 3

Frataxin knockdown in healthy fibroblasts decreases mitochondrial DNA copy number. Human fibroblast cells are treated with FXN siRNA and CTL siRNA. (A) qPCR analysis of mt/nDNA ratio 5 days post transfection in GM3440 and GM8402. (B) qPCR analysis of mt/nDNA ratio at different time points in GM3440. Bars represent averages ± standard deviations (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Frataxin over-expression in patient fibroblast increases mitochondrial protein expression. Human fibroblast cells are treated with (pcDNA3.1-frataxin-flag) or (pcDNA3.1). (A, B) Western blot analysis of FXN and VDAC normalized to tubulin. Bars represent averages ± standard deviations (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Mitochondrial copy number decreases in brain and skeletal muscle tissue of KIKO mice model of FRDA. DNA was extracted from brain and skeletal muscle tissues of KIKO and WT mice followed by qPCR analysis of mitochondrial DNA copy number over nuclear DNA copy number (mt-ND1/Cftr). Bars represent averages ± standard deviations (n = 7, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Mitochondrial biogenesis defect can be determined in whole blood of patients with FRDA. DNA and RNA was extracted from whole blood of patients with FRDA and age-matched controls. (A) qPCR analysis of mitochondrial DNA copy number over nuclear DNA copy number (mtTL1/B2M). (B) Linear regression analysis of mitochondrial copy number to FXN mRNA expression. (C) qRT-PCR analysis of mitochondrial subunit gene expression-mtND6 (complex-I), mtCO2 (complex-II) and mtATP6 (complex-V). Bars represent averages ± standard deviations (n = 7, *P < 0.05, **P < 0.01, ***P < 0.001).