Cerebellum (2008) 7:360–365 DOI 10.1007/s12311-008-0036-x
Recombinant Human Erythropoietin Increases Frataxin Protein Expression Without Increasing mRNA Expression Fabio Acquaviva & Imma Castaldo & Alessandro Filla & Manuela Giacchetti & Daniele Marmolino & Antonella Monticelli & Michele Pinelli & Francesco Saccà & Sergio Cocozza
Published online: 26 June 2008 # Springer Science + Business Media, LLC 2008
Abstract Friedreich’s ataxia is an autosomal recessive neurodegenerative disease that is due to the loss of function of the frataxin protein. The molecular basis of this disease is still a matter of debate and treatments have so far focused on managing symptoms. Drugs that can increase the amount of frataxin protein offer a possible therapy for the disease. One such drug is recombinant human erythropoietin (rhu-EPO). Here, we report the effects of rhu-EPO on frataxin mRNA and protein in primary fibroblast cell cultures derived from Friedreich’s ataxia patients. We observed a slight but significant increase in the amount of frataxin protein. Interestingly, we did not observe any increase in the messenger RNA expression at any of the times and doses tested, suggesting that the regulatory effects of rhu-EPO on the frataxin protein was at the posttranslational level. These findings could help the evaluation of the treatment with erythropoietin as a potential therapeutic agent for Friedreich’s ataxia. Keywords Friedreich’s ataxia . Frataxin . Erythropoietin
F. Acquaviva (*) : I. Castaldo : M. Giacchetti : D. Marmolino : A. Monticelli : M. Pinelli : S. Cocozza Medical Genetics Unit, Department of Cellular and Molecular Biology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy e-mail:
[email protected] A. Filla : F. Saccà Department of Neurological Sciences, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy
Introduction Friedreich’s ataxia (FA) is an autosomal recessive neurodegenerative disease caused by the loss of function of a small protein named frataxin. Affected individuals are either homozygous for a GAA triplet repeat expansion in the first intron of the FXN gene (95%) or compound heterozygous for the expansion and a point mutation (5%) [1]. The exact function of frataxin is still unknown. However, it is known that the frataxin protein is targeted to the mitochondrial matrix [2] where it plays a key role in the maintenance of iron homeostasis, mainly by acting as a component of the iron–sulfur cluster (ISC) assembly machinery [3]. In addition, an extra-mitochondrial localization of frataxin and its physical interaction with IscU1, a cytosolic component of the human ISC assembly machineries, has been demonstrated [4]. No treatments are available for FA and therapeutic strategies mainly act on the symptoms of frataxin deficiency. However, it is unlikely that gene therapy alongside substitutive treatments will be in use any time soon. The phenotype of cells lacking frataxin can be rescued by the expression of frataxin [5]. Therefore, drugs that are able to increase the amount of frataxin are excellent candidates for FA therapy. Several drugs have been assessed for their ability to increase the amount of cellular frataxin, including 3nitropropionic acid (3-NP) [6], butyric acid and hemin [7]. More recently, recombinant human erythropoietin (rhu-EPO) has been shown to increase frataxin protein levels in cultured human lymphocytes derived from FA patients and other cell types such as neuronal cell lines, and cardiac cells [8]. For many years, erythropoietin (EPO) was thought to be exclusively produced by the kidney and only acted upon erythropoietin receptor (EPOR) present in the bone marrow, however, it is now well established that EPOR is also
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expressed in other tissues, including the nervous tissue [9]. Moreover, it is noticeable that rhu-EPO crosses the blood– brain barrier [10, 11], and has been shown to have neuroprotective affects [12, 13]. Recombinant human erythropoietin is a commercial drug that has been assessed for safety and has been approved for use in patients with nephrological or hematological diseases. For these reasons rhu-EPO is considered an excellent candidate for the treatment of FA and, thus, several clinical trials are currently ongoing. The treatment of Friedreich’s ataxia with erythropoietin has a patent application associated with it and encouraging results from one clinical trial are already available [14]. The aim of this study was to investigate whether the administration of rhu-EPO had an effect on frataxin expression either at the transcriptional or post-transcriptional level. Several primary fibroblast cell cultures were established from skin biopsies of FA patients and healthy controls. We investigated the effects of different doses and times of exposure of rhu-EPO treatment on frataxin mRNA and protein levels. The doses of rhu-EPO and times of rhuEPO exposure that were used in this study are compatible with concentrations that can be reached in acute administration in patients. We found that whilst the amount of frataxin protein was increased by rhu-EPO, frataxin mRNA levels did not show any variation. Thus, we suggest a posttranslational regulation of frataxin by rhu-EPO.
Materials and Methods
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Primary fibroblasts were incubated with 5 IU/ml and 10 IU/ml of rhu-EPO for 3 and 24 h. These concentrations are similar to in-vivo serum levels after subcutaneous administration of 600–2,400 IU/kg of rhu-EPO [15]. As a negative control, the primary fibroblast cultures were grown without rhu-EPO. The rhu-EPO used was EPREX® (B03XA01 epoetin alpha; Janssen-Cilag, Cologno Monzese (MI), Italy). Western Blot Cells were lysed in a buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X100, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml of leupeptin/aprotinin for 30 min on ice. Cell lysates were clarified by centrifugation for 15 min at 14,000 rpm and the protein concentration in supernatants was measured using the Bradford assay (Bio-Rad). An equal amount of protein (∼100 µg) was denatured and electrophoresed on 14% SDS-polyacrylamide gel, transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA) by electroblotting and blocked in 10% non-fat dry milk for 1 h. The antibodies used were: anti-frataxin monoclonal antibody 1G2 (Chemicon International, Temecula, CA, USA) and anti-α-tubulin antibody (Sigma, St Louis, MO, USA) and anti-mouse IgG (Sigma, St Louis, MO, USA) with a peroxidase conjugate. The ECL kit (Amersham Biosciences, Piscataway, NJ, USA) was used according to the manufacturer’s instructions. The protein bands were scanned, the relative intensities of each signal
Patients and Ethics This study examined six Friedreich’s ataxia patients, from the outpatient Clinic of Neurology Department of the “Federico II” Medical School and six healthy controls. The patients were homozygous for the GAA repeat expansion and contained between 500 and 1,200 repeats. A description of the age, the gender and the GAA triplet expansion is presented in Table 1. All of the patients enrolled for the study were being treated with idebenone 5 mg/kg and were following a standard protocol of physiotherapy. The local ethics committee approved the study; all the individuals were informed about the aim of the study and gave their written consent. Cell-culture and Treatments Primary fibroblast cultures were established from skin biopsies. Fibroblasts were grown in DMEM supplemented with 20% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin and streptomycin. All experiments were conducted on fibroblasts between seven and 11 passages.
Table 1 Subject description Subjects
Age
FA 590 C 590 FA 305 C 305 FA 584 C 584 FA 303 C 303 FA 171 C 171 FA 728 C 728
24 30 40 49 38 42 25 30 35 33 31 32
Sex
GAA repeats
M M M M F F F F F F F F
795/1182 N.R. 847/928 N.R. 527/634 N.R. 655/974 N.R. 983/1093 N.R. 503/1057 N.R.
Fold changea 2.89 1.83 1.49 1.84 1.73 1.62 2.16 1.23 1.84 1.14 1.92 1.82
The ages indicated correspond to the age of the individual when the skin biopsies were taken (2006). The GAA repeat number refers to shorter/longer allele and NR indicate a number of repeats within the normal range. FA Friedreich’s ataxia patients, C relative healthy controls, M male, F female a After 24 h of treatment with 10 IU/ml of rhu-EPO
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was quantified using the NIH Image Software and all values normalized to the intensity of tubulin. Real-time PCR Total mRNA from fibroblasts was extracted with TriPure Isolation Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions and reverse transcribed with 100 U of Superscript II RNaseH reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Approximately 50 ng of cDNA were amplified by real-time PCR using primers and TaqMan MGB probes (Applied Biosystems, Foster City, CA, USA) for the frataxin gene (FXN) and for three different reference genes (hypoxanthine phosphoribosyl-transferase 1 HPRT1, human β-actin and glyceraldehyde-3-phosphate dehydrogenase GAPDH). Each sample ran in triplicate in a 20 µl reaction using TaqMan Universal PCR Master Mix. All the reactions were performed in an ABIPrism 7000 sequence detector system. Quantitative real-time PCR analyses were carried out using the 2(−Delta Delta C(t)) method (2−ΔΔCt) [16].
Fig. 1. Frataxin protein expression after rhu-EPO treatment. Representative western blot analysis of primary fibroblasts from a Friedreich’s ataxia patient (FA) and a healthy control before and after treatment with 5 IU/ml and 10 IU/ml of recombinant human erythropoietin for 3 and 24 h
Statistical Analysis Statistical analyses were performed used SPSS 13.0 (SPSS Inc., Chicago, IL, USA) software. The Shapiro–Wilk test was used to determine that the data were normally distributed, then statistical significance was calculated using the one sample T-test. Differences with p values less than 0.05 (p0.05 for the healthy controls and 1.34 and 1.05 p>0.05 for the FA patients). However, a statistically significant increase in frataxin protein was observed after 24 h of treatment with 10 IU/ml of rhuEPO. The fibroblasts from the healthy controls showed a 1.58-fold increase (p=0.007) in frataxin protein, and the fibroblasts from the FA patients showed a 2-fold increase (p=0.004). Incubation with 5 IU/ml rhu-EPO for 24 h increased the amount of frataxin protein by 1.46 and 1.37 fold in controls and FA patients, respectively (p>0.05). This analysis seems to confirm that the increase in frataxin protein after 24 h of treatment with rhu-EPO was greater in the FA fibroblast primary cell cultures than the control fibroblast cultures although no statistical significance (p>0.05) was detected. To gain further insight into the mechanism by which rhuEPO exerts its effect on the levels of frataxin protein, we investigated frataxin mRNA levels by real-time PCR. We obtained total mRNA from the same fibroblast primary cell culture lines and then quantified the frataxin mRNA in the presence of two different doses of rhu-EPO, for either 3 or 24 h. This was repeated for the six control- and six-FA patient fibroblast primary cell cultures. In comparison to HPRT1, no increase in mRNA levels was observed in any of
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Fig. 2. Frataxin protein and mRNA expression after rhu-EPO treatment. Densitometric scan analysis of six independent western blots from healthy controls (a) and FRDA patients (b). The relative intensities of the bands were quantified using the NIH Image Software and all the values were normalized to the intensities of the respective tubulin signal. Results are expressed as a fold increase of the means (+/ −SE) over the value of expression in the untreated cells arbitrary set as
1. Frataxin mRNA quantification via real-time PCR analysis of primary fibroblasts from healthy controls (c) and FA patients (d) before and after treatment with 5 IU/ml and 10 IU/ml of recombinant human erythropoietin for 3 and 24 h. Frataxin mRNA values were normalized to the reference gene HPRT1 and are reported here as fold increase of the means of six independent experiments, over the value of expression in the untreated cells arbitrary set as 1 (+/−SE)
the control cell lines or the FA patient cell culture lines (Fig. 2c and d). After 3 h of incubation with 5 or 10 IU/ml of rhu-EPO the frataxin mRNA was unaffected (fold-change: 0.92 and 0.89 in the controls, p>0.005 and 0.94 and 0.91 for the FA patients, p>0.005). Similarly, after 24 h of treatment, frataxin mRNA levels remained unchanged (0.93 and 0.77 in the healthy controls, p>0.005, and 0.84 and 1.1 fold in the FA patients, p>0.005, with 5 IU/ml and 10 IU/ml, respectively). Similar results were obtained using two other reference genes (GAPDH and β-actin; data not shown).
The disease is caused by the loss of function of frataxin; the pathological phenotype of the affected cells is rescued by complementing with frataxin [5]. Current treatments mainly act by suppressing the downstream effects of frataxin deficiency; however, a more attractive therapeutic approach would be to use drugs that increase the amount of frataxin protein. Approaches that can increase frataxin protein levels in defective cells need careful validation because, in theory, it could be possible to re-establish several cellular functions by doubling the residual amount of frataxin. It has been demonstrated that recombinant human erythropoietin (epoetin-β) causes an increase in frataxin protein expression in lymphocytes derived from FA patients and also in cardiac and neuronal cell lines [8]. This finding has now resulted in great interest for this drug as a possible therapeutic agent in FA patients. In this study, we investigated the mechanism by which rhu-EPO acts on frataxin by assessing the effect of the drug on frataxin mRNA and protein levels. We found that whilst rhu-EPO increased the amount of frataxin protein, it had no effect on frataxin mRNA. This suggests that the observed increase in frataxin protein is attributable to a posttranslational mechanism. It is possible to hypothesize that frataxin undergoes a stabilization process, which results in
Discussion Friedreich’s ataxia (FA) is a progressive degenerative disease for which there is currently no effective treatment. FA patients have impaired frataxin production, probably due to either the formation of an aberrant triplex DNA structure [17], to the abnormal CpG methylation status of the region flanking the expansion [18], or, as a consequence of an acetylation-mediated DNA silencing mechanism [19]. The amount of frataxin protein in affected individuals range from 4% to 29% of normal levels, whereas asymptomatic carriers have about 50% of the normal levels [2].
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the increase of its intracellular half-life. However, the pathway potentially involved in this phenomenon remains unknown. EPO/EPOR signaling is well studied. The main effector of EPO action is the Jak/STAT5 pathway but several other pathways are also activated after EPO binding to its receptor, among them PI3K/Akt, MAPK, and cAMP/pkA [reviewed in 20]. Further studies are required to further elucidate the possible mechanism by which rhu-EPO acts to stabilize and increase frataxin protein. In a pilot clinical trial, a reduction in oxidative stress markers such as urinary 8-hydroxydeoxyguanosine and serum peroxides was observed after 5,000 IU rhu-EPO administration for 8 weeks, three times a week [14]. The concentration of rhu-EPO tested in our study (5 IU/ml and 10 IU/ml) corresponds to 600–2,400 IU/kg of rhu-EPO, which are doses currently in use in clinical practice [15]. It may be possible to increase the dosage in future clinical trials with rhu-EPO for FA, but the overall clinical evaluation of each FA patient will allow correct evaluation of the best usable dose. It is important to note that no new frataxin will be translated in rhu-EPO-treated cells and similarly, no new frataxin mRNA will be either transcribed or stabilized. Moreover, it should also be determined whether the increased intracellular frataxin is a truly useful protein or merely an accumulation of a non-functional protein. Finally, our data suggests that the level of frataxin mRNA should be not considered a useful endpoint in evaluating the effectiveness of rhu-EPO treatment during the ongoing clinical trials. Therefore, other cellular markers need to be examined as the primary endpoint, one such marker is the level of frataxin protein. Further studies into the effectiveness and validation of treatment with rhu-EPO can be focused on the evaluation of oxidative stress markers alongside the improvement in the grading of the neurological parameters. Longer trials will be required to obtain real significant endpoints.
Authors’ contributions F.A. participated in conceiving the study, performed the cell cultures, the protein and mRNA studies, and prepared the manuscript; D.M. and I.C. participated in the cell cultures in the mRNA studies; A.F. and F.S. selected the patients and performed the skin biopsies; M.G., M.P., and A.M participated in the design of the study and to the analysis of the Real Time experiments, and performed the statistical analysis; S.C. conceived the study, and participated in its design and coordination, and helped draft the manuscript.
Cerebellum (2008) 7:360–365 Acknowledgements This work was supported in part by a grant from Friedreich’s Ataxia Research Alliance (FARA, USA). We would like to thank Leeanne McGurk for critical reading of this manuscript.
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