Interferon gamma upregulates frataxin and corrects the functional deficits in a Friedreich ataxia model

Human Molecular Genetics, 2012, Vol. 21, No. 13 doi:10.1093/hmg/dds110 Advance Access published on March 23, 2012 2855–2861 Interferon gamma upregulates frataxin and corrects the functional deficits in a Friedreich ataxia model Barbara Tomassini1, Gaetano Arcuri1, Silvia Fortuni1, Chiranjeevi Sandi2, Vahid Ezzatizadeh2, Carlo Casali3, Ivano Condò1, Florence Malisan1, Sahar Al-Mahdawi2, Mark Pook2 and Roberto Testi1,∗ 1 Laboratory of Immunology and Signal Transduction, University of Rome ‘Tor Vergata’, 00133 Rome, Italy, 2Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge UB8 3PH, UK and 3Department of Neurology, University of Rome ‘La Sapienza’, Polo Pontino, 04100 Latina, Italy Received January 14, 2012; Revised and Accepted March 15, 2012 INTRODUCTION Friedreich’s ataxia (FRDA) is a devastating orphan disease. Symptoms usually appear late in the first decade or early in the second decade of life, and include features of both peripheral and cerebellar ataxia. Cardiac involvement is very frequent and premature death is often caused by cardiac insufficiency due to dilated cardiomyopathy. Approximately 10% of patients also develop diabetes mellitus (1). FRDA is caused by defective frataxin expression. Frataxin is a mitochondrial protein, synthesized as a 210-amino acid precursor that is proteolytically processed into a 130-amino acid mature polypeptide (2,3). Frataxin binds iron and it is involved in the assembly of iron-sulfur clusters (ISC) (4,5), prosthetic groups incorporated into several key metabolic enzymes (6). Frataxin-defective cells in fact have reduced activity of ISC-containing enzymes, such as aconitase and succinate dehydrogenase, a general imbalance in intracellular iron distribution and increased sensitivity to oxidative stress. The cells mostly affected by frataxin reduction are the large sensory neurons of dorsal root ganglia (DRG) (7). There is currently no specific therapy to prevent the progression of the disease (8). Here, we show that frataxin can be upregulated by interferon gamma (IFNg), a cytokine involved in multiple aspects of iron metabolism and the immune response (9). Most importantly, in vivo treatment with IFNg increases frataxin levels in DRG neurons and substantially prevents DRG neuronal degeneration and neurological dysfunction in FRDA mice. RESULTS During the course of a proteomic screening for proteins differentially expressed in cells derived from FRDA patients, a serendipitous observation suggested that IFNg might upregulate frataxin. Different IFNg-responsive cell lines were then exposed to recombinant IFNg and frataxin accumulation was quantitated after 24 h by sodium dodecyl sulfate–polyacrylamide ∗ To whom correspondence should be addressed. Tel: +39 0672596503; Email: # The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Friedreich’s ataxia (FRDA) is the most common hereditary ataxia, affecting ∼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNg) in a variety of cell types, including primary cells derived from FRDA patients. IFNg appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNg increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNg in cellular metabolism and have direct implications for the treatment of FRDA. 2856 Human Molecular Genetics, 2012, Vol. 21, No. 13 gel electrophoresis (SDS – PAGE) and immunoblot analysis. As shown in Figure 1, IFNg induces the accumulation of frataxin in human cervical carcinoma HeLa cells (Fig. 1A) and in the monocytic leukemia cell line U937 (Fig. 1B) in a dosedependent manner. Similarly, IFNg can promote frataxin expression in the human glioblastoma cell line U118 (Fig. 1C). To verify that IFNg could induce frataxin accumulation in non-transformed cells, resting peripheral blood mononuclear cells (PBMC) from normal individuals were exposed Figure 1. IFNg induces frataxin accumulation in multiple cell types. HeLa cells (A), U937 cells (B), U118 cells (C) and PBMC isolated from healthy donors (D) were cultured for 24 h in the presence of the indicated concentrations of IFNg, and then whole cell lysates were analyzed by SDS– PAGE and blotted with anti-frataxin and anti-actin mAbs. Representative blots are shown, three to six independent experiments for each cell type were performed. to IFNg and frataxin accumulation was quantitated by SDS– PAGE and immunoblot analysis. Figure 1D shows that IFNg can induce frataxin accumulation in resting PBMC in a dosedependent manner. Together, these data indicate that IFNg is able to upregulate frataxin levels in a variety of cell types. We then tested whether IFNg can upregulate frataxin in cells derived from FRDA patients. FRDA-derived GM03816 fibroblasts were exposed for 24 h to different doses of IFNg, and then frataxin was quantitated by SDS – PAGE and immunoblot analysis. Figure 2A shows that IFNg can induce the upregulation of frataxin in frataxin-defective fibroblasts, in a dose-dependent manner. To verify that IFNg could be effective on primary FRDA cells, freshly isolated PBMC from several FRDA patients were exposed to different doses of IFNg for 24 h. Frataxin was then quantitated by SDS – PAGE and immunoblot analysis. As shown in Figure 2B, PBMC isolated from a FRDA patient, and treated for 24 h with IFNg, exhibit significantly increased levels of frataxin expression, in a dose-dependent manner. Comparison with the levels of frataxin present in a healthy control (a brother of the patient) indicates that IFNg induces a substantial recovery of frataxin levels. PBMC isolated from 9 out of 10 FRDA patients tested gave similar results. To gain insight into the mechanism of frataxin upregulation, we investigated whether IFNg treatment modulated frataxin mRNA levels. Quantitative RT – PCR analysis showed that a significant increase in frataxin mRNA can be detected in FRDA fibroblasts as early as 1 h after exposure to IFNg, with peak accumulation at 2 h and return to baseline levels after 4 h (Fig. 2C). Moreover, pre-treatment with actinomycin D completely prevented IFNg-induced frataxin mRNA accumulation (Fig. 2D). The mRNA ac (...truncated)