Frataxin is Reduced in Friedreich Ataxia Patients and is Associated with Mitochondrial Membranes

Abstract

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.

Lateral-flow immunoassay for the frataxin protein in Friedreich’s ataxia patients and carriers

“…It should be noted that this individual (GM 16216) had the smallest amount of total expanded GAA triplet repeats within the FA patient cohort and it only overlapped a single FA carrier -the one that has the most GAA repeats (GM 15849) ( Table 2). Finally, we demonstrate that there is an inverse correlation between the number of triplet repeats in the frataxin gene and the levels of the protein, which is consistent with current understanding of the pathology of FA and GAA repeats [4,15]. Previous studies which quantified either frataxin protein levels by Western blot analysis or measured frataxin mRNA levels by real time PCR have found that frataxin levels in FA patients are between 6% and 30% of controls for protein levels and between 13% and 30% for mRNA levels [2,17].…”

“…Previously, frataxin protein levels could only be estimated by Western blot, which is cumbersome and semi-quantitative at best [15]. In contrast, the lateral flow immunoassay described here is quantitative, accurate and reproducible, with low intra and inter-assay error throughout a wide working range.…”

“…This could be achieved by simply adding a second detector zone specific for another mitochondrial protein to normalize mitochondrial load and serve as an internal control. It would of course be critical to determine empirically that FA does not affect levels of the chosen mitochondrial marker since levels of many nuclear-encoded mitochondrial proteins and mRNAs are altered in FA [15,[38][39][40][41][42].…”

“…At the molecular level the large expanded GAA repeats (>66) in intron 1 of the FXN gene interfere with FXN transcription, resulting in reduced frataxin mRNA and protein levels in FA patients [14][15][16][17]. The decrease in frataxin protein has broad, far-reaching effects because the protein is an essential iron chaperone required for the biogenesis of iron-sulfur clusters, aconitase activation, and heme biosynthesis [18][19][20][21], and further performs a critical role in iron detoxification and anti-oxidant protection [22][23][24].…”

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia

“…F riedreich's ataxia (FRDA) is a neurodegenerative disorder that arises from a genetic deficit of the mitochondrial iron chaperone protein, frataxin (1)(2)(3). The progressive loss of coordination of limb movements, dysarthria, nystagmus, scoliosis, and diabetes characteristic of FRDA usually manifest before adolescence.…”