Fabio Acquaviva

Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPARγ pathway as a therapeutic target in Friedreich’s ataxia

Friedreich’s ataxia (FRDA), the most common inherited ataxia, is characterized by focal neurodegeneration, diabetes mellitus and life-threatening cardiomyopathy. Frataxin, which is significantly reduced in patients with this recessive disorder, is a mitochondrial iron-binding protein, but how its deficiency leads to neurodegeneration and metabolic derangements is not known. We performed microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency, and found molecular evidence of increased lipogenesis in skeletal muscle, and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of the PPARγ coactivator Pgc1a and transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Finally, we show that genetic modulation of the PPARγ pathway affects frataxin levels in vitro, supporting PPARγ as a novel therapeutic target in FRDA.

PGC-1alpha Down-Regulation Affects the Antioxidant Response in Friedreich's Ataxia

Background Cells from individuals with Friedreichs ataxia (FRDA) show reduced activities of antioxidant enzymes and cannot up-regulate their expression when exposed to oxidative stress. This blunted antioxidant response may play a central role in the pathogenesis. We previously reported that Peroxisome Proliferator Activated Receptor Gamma (PPARγ) Coactivator 1-alpha (PGC-1α), a transcriptional master regulator of mitochondrial biogenesis and antioxidant responses, is down-regulated in most cell types from FRDA patients and animal models. Methodology/Principal Findings We used primary fibroblasts from FRDA patients and the knock in-knock out animal model for the disease (KIKO mouse) to determine basal superoxide dismutase 2 (SOD2) levels and the response to oxidative stress induced by the addition of hydrogen peroxide. We measured the same parameters after pharmacological stimulation of PGC-1α. Compared to control cells, PGC-1α and SOD2 levels were decreased in FRDA cells and did not change after addition of hydrogen peroxide. PGC-1α direct silencing with siRNA in control fibroblasts led to a similar loss of SOD2 response to oxidative stress as observed in FRDA fibroblasts. PGC-1α activation with the PPARγ agonist (Pioglitazone) or with a cAMP-dependent protein kinase (AMPK) agonist (AICAR) restored normal SOD2 induction. Treatment of the KIKO mice with Pioglitazone significantly up-regulates SOD2 in cerebellum and spinal cord. Conclusions/Significance PGC-1α down-regulation is likely to contribute to the blunted antioxidant response observed in cells from FRDA patients. This response can be restored by AMPK and PPARγ agonists, suggesting a potential therapeutic approach for FRDA.

DNA methylation in intron 1 of the frataxin gene is related to GAA repeat length and age of onset in Friedreich ataxia patients

Background: The most frequent mutation of Friedreich ataxia (FRDA) is the abnormal expansion of a GAA repeat located within the first intron of FXN gene. It is known that the length of GAA is directly correlated with disease severity. The effect of mutation is a severe reduction of mRNA. Recently, a link among aberrant CpG methylation, chromatin organisation and GAA repeat was proposed. Methods: In this study, using pyrosequencing technology, we have performed a quantitative analysis of the methylation status of five CpG sites located within the region upstream of GAA repeat, in 67 FRDA patients. Results: We confirm previous observation about differences in the methylation degree between FRDA individuals and controls. We showed a direct correlation between CpG methylation and triplet expansion size. Significant differences were found for each CpG tested (ANOVA p<0.001). These differences were largest for CpG1 and CpG2: 84.45% and 76.80%, respectively, in FRDA patients compared to 19.65% and 23.34% in the controls. Most importantly, we found a strong inverse correlation between CpG2 methylation degree and age of onset (Spearman’s ρ  =  −0.550, p<0.001). Conclusion: Because epigenetic changes may cause or contribute to gene silencing, our data may have relevance for the therapeutic approach to FRDA. Since the analysis can be performed in peripheral blood leucocytes (PBL), evaluation of the methylation status of specific CpG sites in FRDA patients could be a convenient biomarker.

Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2.

Friedreichs ataxia is a recessive neurodegenerative disease due to insufficient expression of the mitochondrial protein frataxin. Although it has been shown that frataxin is involved in the control of intracellular iron metabolism, by interfering with the mitochondrial biosynthesis of proteins with iron/sulphur (Fe/S) clusters its role has not been well established. We studied frataxin protein and mRNA expression and localisation during cellular differentiation. We used the human colon adenocarcinoma cell line Caco-2, as it is considered a good model for intestinal epithelial differentiation and the study of intestinal iron metabolism. Here we report that the protein, but not the mRNA frataxin levels, increase during the enterocyte-like differentiation of Caco-2 cells, as well as in in-vivo-differentiated enterocytes at the upper half of the crypt-villus axis. Furthermore, subcellular fractionation and double immunostaining, followed by confocal analysis, reveal that frataxin localisation changes during Caco-2 cell differentiation. In particular, we found an extramitochondrial localisation of frataxin in differentiated cells. Finally, we demonstrate a physical interaction between extramitochondrial frataxin and IscU1, a cytoplasmic isoform of the human Fe/S cluster assembly machinery. Based on our data, we postulate that frataxin could be involved in the biosynthesis of iron-sulphur proteins not only within the mitochondria, but also in the extramitochondrial compartment. These findings might be of relevance for the understanding of both the pathogenesis of Friedreichs ataxia and the basic mechanism of Fe/S cluster biosynthesis.

Recombinant human erythropoietin increases frataxin protein expression without increasing mRNA expression.

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 post-translational level. These findings could help the evaluation of the treatment with erythropoietin as a potential therapeutic agent for Friedreich’s ataxia.

PPAR-γ Agonist Azelaoyl PAF Increases Frataxin Protein and mRNA Expression. New Implications for the Friedreich’s Ataxia Therapy

Friedreich’s ataxia is a neurodegenerative disease due to frataxin deficiency, and thus, drugs increasing the frataxin amount are excellent candidates for therapy. By screening Gene Expression Omnibus profiles, we identified records showing a frataxin response to the peroxisome proliferator-activated receptors gamma (PPAR-γ) agonist rosiglitazone. We decided to investigate the effect of the PPAR-γ agonist Azelaoyl PAF on the frataxin protein and mRNA expression profile. We treated human neuroblastoma cells SKNBE and primary fibroblasts from skin biopsies from Friedreich’s ataxia (FRDA) patients and healthy controls with the PPAR-γ agonist Azelaoyl PAF. We show in this paper for the first time that Azelaoyl PAF significantly increases the intracellular frataxin levels by twofold in the neuroblastoma cell line SKNBE and fibroblasts from FRDA patients and that Azelaoyl PAF increases frataxin protein through a transcriptional mechanism. PPAR-γ agonist Azelaoyl PAF increases both messenger RNA and protein levels of frataxin. We hypothesize that PPAR-γ agonists could play a role in the treatment of FRDA, and our results offer the logical bases to further investigate the usefulness of this group of agents for the treatment of the FRDA.

Epoetin alfa increases frataxin production in Friedreich's ataxia without affecting hematocrit

Objective of the study was to test the efficacy, safety, and tolerability of two single doses of Epoetin alfa in patients with Friedreichs ataxia. Ten patients were treated subcutaneously with 600 IU/kg for the first dose, and 3 months later with 1200 IU/kg. Epoetin alfa had no acute effect on frataxin, whereas a delayed and sustained increase in frataxin was evident at 3 months after the first dose (+35%; P < 0.05), and up to 6 months after the second dose (+54%; P < 0.001). The treatment was well tolerated and did not affect hematocrit, cardiac function, and neurological scale. Single high dose of Epoetin alfa can produce a considerably larger and sustained effect when compared with low doses and repeated administration schemes previously adopted. In addition, no hemoglobin increase was observed, and none of our patients required phlebotomy, indicating lack of erythropoietic effect of single high dose of erythropoietin.

Friedreich’s Ataxia: From the (GAA)n Repeat Mediated Silencing to New Promising Molecules for Therapy

Friedreich’s ataxia (FRDA) is a neurodegenerative disease due to a pathological expansion of a GAA triplet repeat in the first intron of the FXN gene encoding for the mitochondrial protein frataxin. The expansion is responsible for most cases of FRDA through the formation of a nonusual B-DNA structure and heterochromatin conformation that determine a direct transcriptional silencing and the subsequent reduction in frataxin expression. Among other functions, frataxin is an iron chaperone central for the assembly of iron–sulfur clusters in mitochondria; its reduction is associated with iron accumulation in mitochondria, increased cellular sensitivity to oxidative stress and cell damage. There is, nowadays, no effective therapy for FRDA and current therapeutic strategies mainly act to slow down the consequences of frataxin deficiency. Therefore, drugs that are able to increase the amount of frataxin are excellent candidates for a rational approach to FRDA therapy. Recently, several drugs have been assessed for their ability to increase the amount of cellular frataxin, including human recombinant erythropoietin, histone deacetylase inhibitors, and the PPAR-γ agonists.

β2-adrenergic receptor and UCP3 variants modulate the relationship between age and type 2 diabetes mellitus

BackgroundIt is widely accepted that Type 2 Diabetes Mellitus (T2DM) and other complex diseases are the product of complex interplay between genetic susceptibility and environmental causes. To cope with such a complexity, all the statistical and conceptual strategies available should be used. The working hypothesis of this study was that two well-known T2DM risk factors could have diverse effect in individuals carrying different genotypes. In particular, our effort was to investigate if a well-defined group of genes, involved in peripheral energy expenditure, could modify the impact of two environmental factors like age and obesity on the risk to develop diabetes. To achieve this aim we exploited a multianalytical approach also using dimensionality reduction strategy and conservative significance correction strategies.MethodsWe collected clinical data and characterised five genetic variants and 2 environmental factors of 342 ambulatory T2DM patients and 305 unrelated non-diabetic controls. To take in account the role of one of the major co-morbidity conditions we stratified the whole sample according to the presence of obesity, over and above the 30 Kg/m2 BMI threshold.ResultsBy monofactorial analyses the ADRB2-27 Glu27 homozygotes had a lower frequency of diabetes when compared with Gln27 carriers (Odds Ratio (OR) 0.56, 95% Confidence Interval (CI) 0.36 – 0.91). This difference was even more marked in the obese subsample.Multifactor Dimensionality Reduction method in the non-obese subsample showed an interaction among age, ADRB2-16 and UCP3 polymorphisms. In individuals that were UCP3 T-carriers and ADRB2-16 Arg-carriers the OR increased from 1 in the youngest to 10.84 (95% CI 4.54–25.85) in the oldest. On the contrary, in the ADRB2-16 GlyGly and UCP3 CC double homozygote subjects, the OR for the disease was 1.10 (95% CI 0.53–2.27) in the youngest and 1.61 (95% CI 0.55–4.71) in the oldest.ConclusionAlthough our results should be confirmed by further studies, our data suggests that, when properly evaluated, it is possible to identify genetic factors that could influence the effect of common risk factors.

Aspirin reduces the outcome of anticancer therapy in Meth A–bearing mice through activation of AKT-glycogen synthase kinase signaling

Aspirin displays, at millimolar concentrations, several mechanisms independent from its ability to inhibit cyclooxygenases. Occasionally, the mechanisms displayed in vitro have been clearly related to an effect of clinical relevance in vivo. An expanding literature has been focusing on the cytoprotective effect of aspirin in neurodegenerative disorders and the activation of AKT pathway in neuroprotection and induction of resistance to anticancer drugs. In this work, we tested the ability of aspirin to activate the AKT survival pathway in methylcholanthrene-induced fibrosarcoma cells (Meth A) transplanted into BALB/c nude mice and the clinical effect of aspirin cotreatment during etoposide (VP-16)–based anticancer therapy. We found that cotreatment with aspirin reduced VP-16-induced apoptosis and activated AKT in vitro and in vivo. In Meth A–bearing mice, aspirin administration also activated glycogen synthase kinase-3 and reduced the activity and the efficacy of anticancer therapy in VP-16 cotreated animals. Our data suggest that the antiapoptotic effect of aspirin operates in vivo through the activation of AKT-glycogen synthase kinase pathway causing a decrease in the outcome of VP-16-based therapy. These findings could have clinical relevance in treatment of human malignancies. [Mol Cancer Ther 2006;5(5):1318–24]