The following issue is excerpted from a more comprehensive overview of major issues in current frataxin research. For the complete issue, please click here.

August 12, 2009

Edited by Dr. Michael Marusich.

Past issues are available for review in the archives.

Table of Contents





The mitochondrial protein frataxin is one of a growing number of proteins where the evidence of involvement in disease (FA or Friedreich’s Ataxia) preceded any detailed understanding of its functioning. We now know that frataxin is essential for synthesis of iron-sulfur clusters (ISC) and hemes, prosthetic groups familiar to mitochondriacs because of their essential function in redox reactions of mitochondria specifically, and the cell in general. Frataxin appears to have additional functions not yet well defined. Moreover, the consequences of altered iron metabolism on energy production, on potentially toxic oxidative stress, and in many other cellular processes remains poorly defined. This lack of the “big picture” is not stopping rapid advance in developing ways to treat FA. A major reason is the innovative development of cell and animal models for studying the function of the protein and its role in the disease. In many ways the progress being made is a model for what is needed and what can be achieved for other mitochondrial diseases, and hence the brief overview here.


A number of exciting new cell models are being developed, most notably to prepare experimentally accessible cell populations differentiated into the tissue and cell types strongly affected in FA, for example cerebellar neurons and primary sensory neurons of the dorsal root ganglia, as well as differentiated cardiac cells. Such lines would allow more detailed examination of tissue-specific relevant pathophysiology of frataxin deficiency and would also serve as more appropriate models for high-throughput screening of new therapeutic drugs than current cell lines which are either fibroblast or lymphoblast derived, i.e., not from clinically affected tissues. The obvious holy grail here is to utilize stem cells that could be differentiated on command into the desired cell types, and the first reported establishment of multipotent stem cell lines containing defined frataxin genes has now been published (Zaibak F., et al. Gene Ther. 2009 Mar;16(3):404-14. Epub 2009 Jan 29. Integration of functional bacterial artificial chromosomes into human cord blood-derived multipotent stem cells.).

A further limitation of previous cell models has been their unsuitability for high throughput screening (HTS) due to issues of stability (patient-derived cells) and/or the sub-optimal GAA/TTC constructs in reporter cell lines that fail to fully express the known molecular defects of patient-derived expansions. Therefore it is good to report that new cell model cell lines have been described that address these issues and offer new opportunities for HTS to identify new therapeutic agents. These include a set of reporter cell lines carrying varying levels of GAA repeats in the introns of GFP reporter minigenes (Soragni E., et al. Nucleic Acids Res. 2008 Nov;36(19):6056-65. Epub 2008 Sep 27. Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia.). These reporter constructs show molecular characteristics of heterochromatin reporter silencing near the GAA repeat (histone hypoacetylation and DNA hypermethalation) that is responsive to known regulatory molecules. Interestingly, these conditional reporters constructs are stable when maintained in a transcriptionally silent mode, but unstable when driven to transcribe. Expression of the reporter construct can be turned on when needed to assess effects of therapeutic drugs in high throughput screens.

A second set of novel cell lines has been constructed by specifically knocking out the endogenous normal (conditional) mouse frataxin gene from mouse fibroblasts transfected stably with various human frataxins, including wild-type frataxin and frataxins with pathogenic point mutations found in human compound heterozygous affected FA patients (Calmels N., et al. PLoS One. 2009 Jul 24;4(7):e6379. The first cellular models based on frataxin missense mutations that reproduce spontaneously the defects associated with Friedreich ataxia.). Clones of these “humanized” mouse cell lines were shown to recapitulate spontaneously for the first time in vitro all biochemical hallmarks of frataxin deficiency, including iron accumulation in mitochondria. Importantly, the clones are stable in culture and the degree of downstream biochemical effects, i.e., iron accumulation, reduction in levels and activities (but not transcripts) of ISC enzymes correlated well with the pathogenic severity of the mutation in the FA patients from which the mutated frataxin genes were derived. Therefore they will be useful models for function-based HTS of drugs that target downstream effects of frataxin deficits, and also to monitor efficacy of frataxin protein replacement therapeutic approaches.


A wide range of FA/frataxin animal models have been developed over the past few years and these continue to provide important insights into frataxin function and FA pathogenesis. These models and work accomplished with them is summarized in an excellent up to date review by Puccio (Puccio H. J Neurol. 2009 Mar;256 Suppl 1:18-24. Multicellular models of Friedreich ataxia.). These models have proven invaluable in studies of the pathogenesis of FA, to gain insight into new therapeutic targets and as experimental models to assess efficacy of new therapies.

Gene expression profiling of heart, skeletal muscle and liver from KIKO model mice identified the PPAR-γ pathway as a new therapeutic target in FA (Coppola G., et al. Hum Mol Genet. 2009 Jul 1;18(13):2452-61. Epub 2009 Apr 17. Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPARgamma pathway as a therapeutic target in Friedreich's ataxia.). KIKO mice are heterozygote knockout/knockin animals that carry a single frataxin allele with 230 GAA repeats, resulting in residual frataxin expression at 25-36% of normal levels. Coppola et al., observed tissue-specific alterations in metabolic pathways consistent with increased lipogenesis, insulin resistance and cardiomyopathy. These alterations suggested PPAR-γ co-activator PGC1-α down-regulation and transcription factor Srebp1 up-regulation in skeletal muscle and liver in these animals. Subsequent analysis of fibroblasts and lymphoblast cells derived from FA patients, and neural precursor cells from KIKO mice revealed a strong positive correlation between PGC1-α levels and frataxin levels while experimental knockdown of PGC1-α in normal human cultured fibroblasts resulted in a coordinate decrease in frataxin mRNA and protein levels, consistent with PGC1-α’s role as the master regulator of the PPAR-γ pathway and mitochondrial biogenesis. The authors suggest a positive feedback loop between PGC1-α and frataxin in these tissues and cells. The therapeutic potential of manipulating this pathway with drug treatment was “confirmed” by the publication a few months earlier of a report that the PPAR-γ agonist Azelaoyl PAF induced up-regulation of frataxin mRNA and protein levels approximately two-fold in FA patient-derived fibroblasts and in a neuroblastoma cell line (Marmolino D., et al. Cerebellum. 2009 Jun;8(2):98-103. Epub 2008 Dec 23. PPAR-gamma agonist Azelaoyl PAF increases frataxin protein and mRNA expression: new implications for the Friedreich's ataxia therapy.). Interestingly, Marmolino et al., (who also collaborated on the Coppola et al., study) initially identified the PPAR-γ pathway as a potential therapeutic target in FA by reviewing archived records of HTS transcription profiles of experimental therapeutic drugs in the Gene Expression Omnibus Dataset at NCBI/NIH and identifying two cases in which treatment with the (FDA-approved) PPAR-γ agonist rosiglitazone had been reported to increase frataxin levels (among many other proteins) in mice and humans. These compounds are now of intense interest as potential FA therapeutics as they are already approved for other conditions and are generally regarded as safe.

Sutak et. al., used a muscle-specific creatine kinase frataxin conditional knockout mouse to perform the first extensive proteomic analysis (2D electrophoresis) of the downstream effects of frataxin deficiency (Sutak R., et al. Proteomics. 2008 Apr;8(8):1731-41. Proteomic analysis of hearts from frataxin knockout mice: marked rearrangement of energy metabolism, a response to cellular stress and altered expression of proteins involved in cell structure, motility and metabolism.). They observed significant changes in more than 50 cardiac proteins, 50% of which are involved in energy metabolism, most notably striking reductions in levels of complex I and complex II subunit proteins, both of which are ISC containing enzyme complexes of the respiratory chain previously shown to be adversely affected in FA. Interestingly, there was also evidence of metabolic compensation in the form of up-regulation of multiple non-ISC enzymes involved in energy metabolism pathways. Also consistent with the known sensitivity of FA cells and tissues to oxidative stress, approximately 20% of the altered proteins are involved in stress or anti-oxidation protection. In summary, this protoeomic study confirmed some known downstream effects of frataxin deficiency and identified additional molecular details, some of which may serve as biomarkers to monitor in FA therapies.

A better understanding of the molecular response to oxidative stress caused by frataxin deficits was also provided by Anderson et al., (Anderson P.R., et al. PNAS. 2008 Jan 15;105(2):611-6. Epub 2008 Jan 9. Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia.) who used a drosophila model of FA to show that boosting levels of enzymes that scavenge H202 reversed the FA phenotype and in particular restored aconitase activity, resistance to exogenous oxidative stress and a normal lifespan (Anderson et al., PNAS 2008, 105, 611-616). In contrast, increasing expression of superoxide scavengers had no effect. These results thus support the continued development of therapeutics designed to limit reactive oxygen species, and in particular to target management of H202.

Finally, Ventura, et al. (Ventura N., et al. Aging Cell. 2009 Apr 22. [Epub ahead of print]. p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress.) reported that metabolic control is the key to understanding why “that which doesn’t kill you makes you strong” (Ventura et al., Aging Cell 2009, 8, 380-393). These authors showed that cep-1, the C. elegans p53 ortholog is necessary both for lifespan extension induced by mild suppression ion of frataxin or OXPHOS enzymes and also the execution of lifespan arrest when these enzymes are markedly reduced. This outwardly incongruous dual role is actually consistent with p53’s known roles as a metabolic checkpoint and regulator of mitochondrial metabolism (which can be subverted by mutations in cancer to promote tumor proliferation –the Warburg effect), activation of antioxidant defenses, and its executioner role as a tumor suppressor. These pathways now offer new potential approaches for therapy in FA and other mitochondrial diseases.

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