Mechanism of erythropoietin-dependent increase in frataxin

Friedreich`s Ataxia (FRDA) is the most common inherited ataxia. It affects one in 50.000 indoeuropean people. Clinically, FRDA is characterized by multiple symptoms including progressive spinocerebellar ataxia, diabetes mellitus and hypertrophic cardiomyopathy. FRDA is caused by a GAA-trinucleotide expansion in the first intron of the frataxin gene and this expansion results in reduced expression of frataxin, a small mitochondrial protein. Frataxin seems to be involved in mitochondrial iron homeostasis and/or assembly of iron-sulfur (FeS) proteins and heme synthesis. Intramitochondrial iron accumulation in FRDA has been postulated to lead to oxidative stress, finally resulting in cell death. There is currently no effective treatment of FRDA available, however antioxidants and iron chelators have been considered to reduce some clinical features of FRDA. Some stimulation of frataxin production has been observed with exogenous substances, but recently the coapplicant found that recombinant human erythropoietin (rhuEPO) significantly increased frataxin expression and in clinical studies could show the effectiveness of such treatment. Nevertheless, the mechanism of this rhuEPO-dependent increase in frataxin protein remains elusive. However, due to our recent finding that the MID1/alpha4 protein complex is able to bind and enhance the translation of G-quartet containing mRNAs, and frataxin mRNA harbours several G-quartets, we hypothesize that this rhuEPO-dependent increase is based on enhanced translation efficiency. This hypothesis is supported by a recently reported observation, that the rhuEPO-dependent increase in frataxin is not based on elevated mRNA levels. Additionally, there are several reports that link rhuEPO with translation regulation, strikingly also one that observed a severalfold rhuEPO-dependent enhanced translation of alpha4, which is an important effector of the translational master regulator mTOR. Alpha4 reduces protein phosphatase 2A activity towards the key translational regulators and mTOR targets S6-kinase and 4E-BP1 and thereby favors mTOR signalling to boost translation. We therefore want to analyse in detail the effect of rhuEPO on the production of FRDA-relevant proteins (frataxin, ferritin, ...) by profiling of polysome-associated mRNAs +/- rhuEPO in FRDA cells and by analysing its implication with MID1/alpha4. In preliminary experiments we could observe an association of frataxin mRNA with the MID1 complex as well as a MID1/alpha4 dependency of the cellular frataxin levels. This provides a sound basis for detailed analyses of a role of MID1 in the erythropoietin-dependent frataxin increase. Elucidation of the mechanism of the clinically observed beneficial rhuEPO effect by our "from bedside back to bench" approach will finally pave the way to improved FRDA treatments.

Friedreichs ataxia (FRDA) is an inherited movement disorder leading to early death due a lack in the mitochondrial protein frataxin. Treatment of patients with the hormone erythropoietin can lead to an improvement of symptoms by increasing the levels of frataxin protein. This project deals with the mechanisms that underlie the increase in frataxin protein in FRDA patients treated with erythropoietin. The working hypothesis underlying this project assumes that the observed erythropoietin-dependent increase in frataxin is based on enhanced production of the protein and that this correlates with a protein called alpha4, which is implicated in protein production, and which is induced by erythropoietin. The preliminary experiments showed that a reduction of alpha4 and its binding partner MID1 resulted in a reduction of frataxin protein and that the frataxin mRNA binds to MID1. Thus we hypothesized that the two proteins MID1 and alpha4 drive the erythropoietin-dependent increased production of frataxin. We set out to identify the precise binding determinants on mRNAs that trigger the association with MID1/alpha4 to understand how the production of specific proteins is triggered. A putative mechanism could be the reduction of a certain enzyme that inhibits protein production, namely the catalytic subunit of protein phosphatase 2A (PP2Ac). We could identify several structural determinants on mRNAs that bind MID1/alpha4 and found a very prominent group of proteins harboring such determinants Two of those are especially interesting, namely a mutated protein involved in Chorea Huntington another devastating neurodegenerative disease and the androgen receptor, one of the crucial players in prostate cancer. We could show, that the production of these proteins is dependent on MID1/alpha4 binding to their mRNAs and that high levels of MID1/alpha4 can lead to a massive production of these proteins. This is an important finding as the levels of both target proteins are directly correlated with the respective diseases suggesting that drugs inactivating MID1/alpha4 could be very promising drugs to treat these diseases. However, we could neither find strong binding motifs on the frataxin mRNA, nor verify the working hypothesis, because erythropoietin treatment of cells from FRDA patents increased PP2Ac, the inhibitor of protein production. Thus the hypothesis is falsified and had to be dismissed. Nevertheless, a reduction in MID1/alpha4 leads to a reduced production of frataxin, which means that MID1/alpha4 do have some role in this respect. To provide a strong basis for the elucidation of the underlying mechanism we therefore studied the MID1/alpha4 complex in detail, identified new players and developed methods to identify novel modulators. One of which turned out to be a promising candidate to treat another neurodegenerative disease, namely Alzheimers disease. Furthermore, we managed to reprogram skin cells from FRDA patients to cell types that are most severely afflicted in the patients (but which cannot be taken from patients) to be able to study the pathologic mechanisms leading to the deadly cell defects of this disease in follow up projects.