Alterations of mitochondrial biogenesis and alterations of mitochondrial antioxidant defense in Friedreich's ataxia

Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis.

Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2).

Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation.

In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription.

In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells.

Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low.

Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency.

Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1 and frataxin reduced levels. Our finding confirm previous publications showing that PGC-1a directly regulate SOD2 levels in vitro and in vivo. We then tested the response to oxidative stress induced by the addition of hydrogen peroxide (H2O2) at different time and doses. Our data show that H2O2 directly increase PGC-1a and SOD2 levels (p<0.01 and p<0.05) in control cells; no effect was observed in FRDA cells, suggesting a lack in the activation of this response. Moreover, PGC-1α direct silencing, using a specific siRNA, in control fibroblasts led to a similar loss of SOD2 response (p<0.001) to oxidative stress as observed in FRDA fibroblasts, confirming its crucial role in this response (3). We then measured the same parameters after pharmacological manipulations of PGC-1a. PGC-1a activation with the PPAR agonist (Pioglitazone) or with a cAMP-dependent protein kinase (AMPK) agonist (AICAR) restored normal SOD2 induction (4) in FRDA cells (p<0.01). In vivo treatment of the KIKO mice (35-40% of wiled-type frataxin) with Pioglitazone significantly up-regulate SOD2 (5) in cerebellum (p<0.01) and spinal cord (p<0.05), two primary affected tissues in patients.

The search for experimental drugs increasing the amount of frataxin is a very active and timely area of investigation. In cellular and in animal model systems, the replacement of frataxin function seems to alleviate the symptoms or completely reverts the phenotype. Therefore, drugs that are able to increase directly the amount of frataxin, at least up to the level of an asymptomatic carrier, are attractive candidates for new approaches to the therapy of FRDA. Our findings show (1) that a potent PPAR-gamma agonists can increase frataxin expression. We do also show a regulatory loop between frataxin and PGC-1a. Thus, we suggest that this loop could play a critical role in the pathogenesis of the disease and breaking this loop could help to slow down the pathological phenotype observed in FRDA patients. Particularly, PGC-1α down-regulation (3) is likely to contribute to the blunted antioxidant response observed in cells from FRDA patients. This response can be restored by AMPK and PPAR agonists in vitro (4) and in vivo, as shown by Pioglitazone treatment (5) in a mouse model for the disease. To conclude, our study provide evidences that PPAR-gamma agonists are a potential treatment for Friedreich’s ataxia, consisting with their action on both mitochondrial biogenesis and oxidative stress defenses.