Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disorder with severely debilitating effects and no current cure. FRDA is mainly caused by the hyper-expansion of a GAA repeat present in intron 1 of the FXN gene, which results in decreased gene expression and consequently a deficiency of the mitochondrial protein frataxin. In the first instance, frataxin deficiency renders an impaired protection from oxidative stress. Antioxidant therapy with cannabinoids (CBD and THC) and CTMIO was investigated in GAA repeat FXN YAC transgenic mouse models of FRDA, but no significant improvements were detected on functional measurements such as rotarod performance and locomotor activity. Additionally such compounds failed to protect the brain of treated mice from oxidative insults. Therefore, the use of such antioxidant compounds cannot be advocated for FRDA therapy. Recent findings indicate that FXN silencing in FRDA may be mediated by repressive heterochromatin, suggesting the use of histone deacetylase inhibitors (HDACi) as FXN up-regulators. Therefore, therapy with a benzamide-type HDACi (106) was similarly investigated on the FXN YAC GAA mouse model. No significant improvements were detected by functional and histochemical analysis. However, significant changes were produced in global acetylation levels of H3 and H4 in the brain of treated mice, suggesting that the drug is capable of crossing the blood-brain barrier and producing an effect. Additionally, significant increases in frataxin expression were detected in the brain of treated mice. To identify further FRDA disease mechanisms, characterization of the FXN gene for the presence of the CCCTC-binding factor (CTCF) was also performed on FRDA patient cerebellum samples. Overall, lower levels of CTCF were detected in FRDA-associated FXN alleles, suggesting the potential involvement of CTCF in the regulation of FXN transcription.

Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin. 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.

Iron-sulfur clusters are protein cofactors that are critical for all life forms. Elaborate multi-component systems have evolved for the biosynthesis of these cofactors to protect organisms from the toxic effects of free iron and sulfide ions. In eukaryotes, the Fe-S cluster assembly machinery operates in the matrix space of the mitochondria and contains a myriad of proteins that mediate sulfur, iron, and electron transfer to assemble Fe-S clusters on the scaffold protein ISCU2 and then distribute these clusters to target proteins. Our lab has recently described stable 3, and 4-protein complexes composed of the cysteine desulfurase NFS1, the co-chaperone ISD11, and ISCU2 (SDU), and NFS1, ISD11, ISCU2, and FXN (SDUF) subunits. In the latter, SDUF, FXN functions as an allosteric activator switching this assembly complex on for Fe-S cluster biosynthesis. Insufficient expression of the mitochondrial protein FXN leads to a progressive neurodegenerative disease, Friedreich's Ataxia (FRDA). In ~2% of patients, FRDA is caused by one of 15 known missense mutations on one allele accompanied by the GAA repeat on the other leading to a complicated phenotype that includes loss of Fe-S clusters. Here we present in vitro evidence that FRDA FXN variants are deficient in their ability to bind the SDU complex, their ability to stimulate the sulfur transfer reaction from NFS1 to ISCU2, and in their ability to stimulate the rate of cluster assembly on ISCU2. Here, in vitro evidence is presented that FXN accelerates the sulfur transfer reaction from NFS1 to ISCU2. Additionally, we present kinetic evidence that identifies the most buried cysteine residue, C104 on ISCU2 as the sulfur acceptor residue suggesting, FXN stabilizes a conformational change to facilitate sulfur delivery. Subsequent mutational studies suggest FXN binding to SDU results in a helix to coil transition in ISCU2 exposing C104 to accept the persulfide sulfur and thereby accelerating the rate of sulfur transfer. We further provide the first biochemical evidence that the persulfide transferred to ISCU2 from NFS1 is viable in Fe-S cluster formation. In contrast to human FXN, the Escherichia coli FXN homolog CyaY has been reported to inhibit Fe-S cluster biosynthesis. To resolve this discrepancy, a series of inter-species enzyme kinetic experiments were performed. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Friedreich ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide repeat expansion mutation within the first intron of the FXN gene. Normal individuals have 5 to 30 GAA repeats, whereas affected individuals have from approximately 70 to more than 1,000 GAA triplets. In addition to progressive neurological disability, FRDA is associated with cardiomyopathy and an increased risk of diabetes mellitus. Currently there is no effective therapy for FRDA and this is perhaps due to the lack of an effective system to test potential drugs. Therefore, the main aim of this thesis is to develop a novel cell culture system, to aid in rapid drug screening for FRDA. Firstly, I have demonstrated the establishment of novel cell culture systems, including primary fibroblasts, neural stem cells (NSC) and splenocytes, from FRDA YAC transgenic mouse models (YG8 and YG22). Then, I have shown the differentiation of NSCs into neurons, oligodendrocytes and astrocytes. The presence of these cells was confirmed by using cell specific immunofluorescence assays. I have also shown that both YG8 and YG22 rescue mice have less tolerance to hydrogen peroxide induced oxidative stress than WT mice, as similarly seen in FRDA patient fibroblasts. Recent findings indicate that FRDA is associated with heterochromatin-mediated silencing of the FXN gene accompanied by histone changes, flanking the GAA repeats. This suggested potential therapeutic use of compounds which can reduce the methylation and increase the acetylation of histone proteins. Therefore, using human and mouse primary fibroblast cell lines I have investigated the efficacy and tolerability of various DNA demethylating agents, GAA interacting compounds and class III histone deacetylase (HDAC) inhibitors. Although DNA demethylating agents showed increased FXN expression, no correlation between the level of DNA methylation and FXN expression was identified. Nevertheless, the use of GAA interacting compounds, particularly DB221, and the HDAC inhibitor, nicotinamide, have shown encouraging results, provoking us to use such compounds in future long-term in vivo studies. In addition, I have also investigated the long-term efficacy of two benzamide-type HDAC inhibitors, RGFA 136 and RGFP 109, on the FRDA YAC transgenic mice. No overt toxicity was identified with either drug, indicating a safe administration of these compounds. Both compounds produced improved functional analysis together with significantly reduced DRG neurodegeneration. However, neither of these compounds was shown to significantly increase the FXN mRNA expression. Nevertheless, elevated levels of frataxin protein in the brain tissues were obtained with RGFP 109, suggesting that RGFP 109 is capable of crossing the blood-brain barrier. I have also found increased levels of global acetylated H3 and H4 histone proteins in brain tissues, along with significant increase in aconitase enzyme activity, particularly with RGFP 109 treatments. Overall, these results support future clinical trial development with such compounds.

Friedreich ataxia (FRDA) is a lethal autosomal recessive neurodegenerative disorder caused by expanded GAA repeats in the FXN gene, resulting in local epigenetic changes and reduced expression of the mitochondrial protein frataxin. The disease is characterised by neurodegeneration of large sensory neurons of the dorsal root ganglia and spinocerebellar tracts. It has been recently reported that a novel frataxin antisense transcript, FAST-1, is overexpressed in FRDA patient derived fibroblasts. However, the lack of fundamental information about FAST-1 gene such as size, sequence, length and its origin has hindered the understanding of its interactions with FXN gene. Therefore, I proposed to investigate these characteristics of FAST-1 in a panel of FRDA cells and mouse models. Firstly, using Northern blot hybridisation with small and large riboprobes, I identified two bands with different sizes (~500 bp and 9 kb size), representing potential FAST-1 transcripts. Then to confirm the exact size and the location of the FAST-1 gene, I performed 5’- and 3’ RACE experiments, followed by cloning and sequencing. This analysis resulted in identification of the 5’- and 3’-ends of FAST-1, which mapped to nucleotide positions ‘-359’ and ‘164’ of the FXN gene, giving the total length of FAST-1 as 523 bp size. Strikingly, the full-length 523 bp FAST-1 transcript also corresponds to one of the Northern blotting results where I identified a band at approximately 500 bp size, indicating that the Northern blotting may have correctly identified the same full-length FAST-1 transcript. Subsequently, by optimising number of experimental parameters within our lab, I developed a robust qRT-PCR method to quantify FAST-1 expression levels. Using this technique, I analysed the expression pattern of this antisense transcript in various FRDA cell lines and mouse models. I confirmed the original finding of increased FAST-1 levels in human FRDA fibroblasts, and further quantified FAST-1 levels in FRDA mouse model cell lines and tissues. However, no consistently altered patterns of FAST-1 expression were identified in relation to FXN expression. Therefore, either they are not directly connected, as originally reported by De Biase et al., or their relationship varies between cell and tissue types. Lastly, improved understanding of epigenetic changes in FRDA and growing evidence on long-gene regulation led me to study the ‘neighbouring genes’ rather than just focusing on the FXN gene. Therefore, I studied a region of approximately 750 kb on both sides of the FXN and quantified the expression levels of two genes (PGM5 and PIP5K1β) on 5’- end and four genes (TJP2, FAM189A2, APBA1 and PTAR1) on 3’- end of FXN gene in human primary fibroblasts. I found that PGM5 and PIP5K1β genes, located at 5’- end of the FXN genes, were downregulated in FRDA fibroblasts and these findings coincide with the recent epigenetic changes identified in FRDA, where significant enrichment of gene repressive histone marks and increased DNA methylation were shown in upstream region of GAA repeats in intron 1 of the FXN gene. Out of four genes that were studied in the 3’- end of the FXN gene, only one gene (APBA1) was downregulated, which suggests that there are fewer repressive epigenetic marks downstream of the GAA repeat.

Aim of this work was to find out how mitochondrial processing peptidases are working in the mitochondrion of Trypanosoma brucei. I have shown by RNA interference that mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) are essential for procyclic stages. Moreover, processing of human frataxin in T. brucei has a similar pattern as in human cells.

Friedreich's ataxia is a neurodegenerative disorder caused by the low expression of a mitochondrial protein called frataxin. Studies in the yeast Saccharomyces cerevisiae have unraveled a role for the frataxin homologue (Yfh1p) in iron-sulfur cluster (Fe/S) biosynthesis, probably by interacting with the scaffold protein, Isu1p, and providing iron to the machinery. Yfh1p possesses a large â-sheet platform that may be involved in the interaction with other proteins through conserved residues at its surface. We have used directed mutagenesis associated with polymerase chain reaction (PCR) to study conserved residues localizing either at the surface of the protein, Thr110, Thr118, Val120, Asn122, Gln124, Gln129, Trp131, Ser137 and Arg141, or buried in the core of the protein, Ile130 and Leu132. Mutants T110A, T118A, V120A, N122A, Q124A, Q129A, I130A, W131A, L132A, S137A and R141A were generated in yeast. Growth on iron- or copper-containing medium was severely impaired for mutants Q129A, I130A, W131A and R141A. Others were roughly growing as well as the wild-type strain. We assessed the efficiency of Fe/S biosynthesis by measuring aconitase activity. The results confirmed those obtained on metal-containing medium: mutants Q129A, I130A, W131A and R141A showed a high decrease in their aconitase activity that dropped to the deleted strain level. Moreover, S137A showed also a decreased aconitase activity. We monitored the interaction between Yfh1p and Isu1p by co-immunoprecipitation and it turned out that only the W131A mutation affects directly this interaction. Even if the amount of Yfh1p determined by western blot analysis was highly decreased for several mutants, it is not sufficient to explain the phenotypes as they were poorly restored by overexpression of the mutant proteins to wild-type levels, except for W131F. We have concluded that Gln129, Trp131, and Arg141 are important for Yfh1p function, while Ile130 and Ser137 are required for the folding of the protein. All these residues cluster to the 4th and 5th â-strand of the protein. Our work has demonstrated for the first time the importance of this area for Yfh1p function and shows that Trp131 is involved in the interaction with Isu1p.

Friedreich’s ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart disease. It is caused by a pathological intronic hyperexpansion of a GAA repeat in the FXN gene, encoding the essential mitochondrial protein frataxin. At the homozygous state, the GAA expansion induces a heterochromatin state with decreased histone acetylation and increased methylation, resulting in a partial deficiency of frataxin expression. This was established in cells from FRDA patients. We showed that the same chromatin changes exist in a GAA based mouse model, KIKI, generated in our laboratory. Furthermore, treatment of KIKI mice with a novel Histone Deacetylase Inhibitor (HDACi), 106, a pimelic diphenylamide that increases frataxin levels in FRDA cell culture, restored frataxin levels in the nervous system and heart of KIKI mice and induced histone hyperacetylation near the GAA repeat. As shown by microarrays, most of the differentially expressed genes in KIKI were corrected towards wild type. In an effort to improve the pharmacological profile of compound 106, we synthesized more compounds based on its structure and specificity. We characterized two of these compounds in FRDA patients’ peripheral blood lymphocytes and in the KIKI mouse model. We observed a sustained frataxin upregulation in both systems, and, by following the time course of the events, we concluded that the effects of these compounds last longer than the time of direct exposure to HDACi. Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACis for FRDA. Laboratory tools to follow disease progression and assess drug efficacy are needed in a slowly progressive neurodegenerative disease such as FRDA. We used microarrays to characterize the gene expression profile in peripheral lymphocytes from FRDA patients, carriers and controls. We identified gene expression changes in heterozygous, clinically unaffected GAA expansion carriers, suggesting that they present a biochemical phenotype, consistent with data from animal models of frataxin deficiency. We identified a subset of genes changing in patients as a result of pathological frataxin deficiency establishing robust gene expression changes in peripheral lymphocytes. These changes can be used as a biomarker to monitor disease progression and potentially assess drug efficacy. To this end, we used he same methodology to characterize the gene expression profiles in peripheral lymphocytes after treatment with pimelic diphenylamide HDACi. This treatment had relevant effects on gene expression on peripheral patients’ blood lymphocytes. It increased frataxin levels in a dose-dependent manner, and partially rescued the gene expression phenotype associated with frataxin deficiency in the tested cell model, thus providing the first application of a biomarker gene set in FRDA.

No description available.

Die Krebsentstehung wurde vor rund 80 Jahren auf veränderten zellulären Energiestoffwechsel zurückgeführt. Diese Hypothese konnte bisher weder experimentell bewiesen noch widerlegt werden. Durch den Einsatz zweier Modellsysteme mit unterschiedlicher Expression des mitochondrialen Proteins Frataxin konnte in der vorliegenden Arbeit <br> gezeigt werden, dass der mitochondriale Energiestoffwechsel einen Einfluss auf die Tumorentstehung zu besitzen scheint. Eine Reduktion des mitochondrialen Energiestoffwechsels wurde durch die hepatozytenspezifische Ausschaltung des mitochondrialen Proteins Frataxin in Mäusen erreicht. Der durch das Cre-/loxP-Rekombinasesystem erreichte organspezifische Knock-out wurde auf Transkriptions- und Translationsebene nachgewiesen. Anhand verminderter Aconitaseaktivität, geringeren Sauerstoffverbrauches und reduzierten ATP-Gehaltes im Lebergewebe wurde ein signifikant verminderter Energiestoffwechsel dargestellt. Zwar entsprach die Genotypenverteilung in den Versuchsgruppen der erwarteten Mendelschen Verteilung, dennoch war die mittlere Lebenserwartung der <br> Knock-out-Tiere mit ca. 30 Wochen stark reduziert. Bereits in jungem Alter war bei diesen Tieren die Ausbildung von präneoplastischen Herden zu beobachten. Mit proteinbiochemischen Nachweistechniken konnte in Lebergewebe 4-8 Wochen alter Tiere eine verstärkte Aktivierung des Apoptosesignalweges (Cytochrom C im Zytosol, <br> verstärkte Expression von Bax) sowie eine Modulation stressassoziierter Proteine (geringere Phosphorylierungsrate p38-MAPK, vermehrte Expression HSP-25, verminderte Expression HSP-70) aufgezeigt werden. Im inversen Ansatz wurde eine Steigerung des mitochondrialen Energiestoffwechsels durch stabile transgene Frataxinüberexpression in zwei Kolonkarzinomzelllinien erreicht. Diese Steigerung zeigte sich durch erhöhte Aconitaseaktivität, erhöhten Sauerstoffverbrauch, gesteigertes mitochondriales Membranpotenzial und erhöhten ATP-Gehalt in den Zellen. Die frataxinüberexprimierenden Zellen wuchsen signifikant langsamer als Kontrollzellen und zeigten im Soft-Agar-Assay und im Nacktmausmodell ein deutlich geringeres Potenzial zur Ausbildung von Kolonien bzw. Tumoren. Mittels Immunoblot war hier eine vermehrte Phosphorylierung der p38-MAPK festzustellen. <br> Die zusammenfassende Betrachtung beider Modelle zeigt, dass ein reduzierter mitochondrialer Energiestoffwechsel durch Regulation der p38-MAPK und apoptotischer Signalwege ein erhöhtes Krebsrisiko zu verursachen vermag. / Eigthy years ago, it was suggested that impaired energy metabolism might cause cancer. Compelling experimental evidence for this hypothesis is lacking. By use of two different model systems here we show that impaired expression of the mitochondrial protein frataxin leading to impaired mitochondrial energy metabolism appears to be <br> inversely related to tumour growth. To generate mice with reduced mitochondrial energy metabolism the expression of mitochondrial protein frataxin was disrupted in a hepatocyte-specific manner by using the cre/loxP-system. Presence, efficiency and specificity of disruption were shown at transcriptional and translational levels. Decreased activity of aconitase, reduced oxygen consumption and diminished ATP level in the liver revealed diminished energy <br> metabolism. Although knock-out mice were born in the expected Mendelian frequency, they exhibited a significantly decreased life expectancy. Young mice exhibited hepatic preneoplasia. The use of proteinbiochemical techniques revealed activation of apoptotic <br> pathways (cytochrome c in the cytosol, increased expression of bax) and modulation of stress-associated cascades (decreased phosphorylation of p38-MAPK, increased expression of HSP-25 and diminished expression of HSP-70). Inversely, transgenic overexpression of frataxin in colon cancer cell lines lead to increased mitochondrial energy metabolism as demonstrated by elevated activity of aconitase, increased oxygen consumption, elevated mitochondrial membrane potential and increased ATP levels. Frataxin-overexpressing colon cancer cells exhibit a <br> concurrent decrease in replication rate. The colony forming capacity in soft-agar-assay and tumour formation in nude mice were clearly decreased. Immunoblotting revealed elevated phosphorylation of p38-MAPK. Taken together, these models suggest that reduced mitochondrial energy metabolism may promote cancer through regulation of p38-MAPK and apoptotic pathways.