Interferon Gamma Enhances Cytoprotective Pathways via Nrf2 and MnSOD Induction in Friedreich's Ataxia Cells.

Friedreich's ataxia (FRDA) is a rare monogenic disease characterized by multisystem, slowly progressive degeneration. Because of the genetic defect in a non-coding region of FXN gene, FRDA cells exhibit severe deficit of frataxin protein levels. Hence, FRDA pathophysiology is characterized by a plethora of metabolic disruptions related to iron metabolism, mitochondrial homeostasis and oxidative stress. Importantly, an impairment of the antioxidant defences exacerbates the oxidative damage. This appears closely associated with the disablement of key antioxidant proteins, such as the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the mitochondrial superoxide dismutase (MnSOD). The cytokine interferon gamma (IFN-γ) has been shown to increase frataxin expression in FRDA cells and to improve functional deficits in FRDA mice. Currently, IFN-γ represents a potential therapy under clinical evaluation in FRDA patients. Here, we show that IFN-γ induces a rapid expression of Nrf2 and MnSOD in different cell types, including FRDA patient-derived fibroblasts. Our data indicate that IFN-γ signals two separate pathways to enhance Nrf2 and MnSOD levels in FRDA fibroblasts. MnSOD expression increased through an early transcriptional regulation, whereas the levels of Nrf2 are induced by a post-transcriptional mechanism. We demonstrate that the treatment of FRDA fibroblasts with IFN-γ stimulates a non-canonical Nrf2 activation pathway through p21 and potentiates antioxidant responses under exposure to hydrogen peroxide. Moreover, IFN-γ significantly reduced the sensitivity to hydrogen peroxide-induced cell death in FRDA fibroblasts. Collectively, these results indicate the presence of multiple pathways triggered by IFN-γ with therapeutic relevance to FRDA.

Hsa-miR223-3p circulating level is upregulated in Friedreich's ataxia and inversely associated with HCLS1 associated protein X-1, HAX-1.

Frataxin (FXN) deficiency is responsible for Friedreich's ataxia (FRDA) in which, besides the characteristic features of spinocerebellar ataxia, two thirds of patients develop hypertrophic cardiomyopathy that often progresses to heart failure and premature death. Different mechanisms might underlie FRDA pathogenesis. Among them, the role of miRNAs deserves investigations. We carried out an miRNA PCR-array analysis of plasma samples of early-, intermediate- and late-onset FRDA groups, defining a set of 30 differentially expressed miRNAs. Hsa-miR223-3p is the only miRNA shared between the three patient groups and appears upregulated in all of them. The up-regulation of hsa-miR223-3p was further validated in all enrolled patients (n = 37, Fc = +2.3; P < 0.0001). Using a receiver operating characteristic curve analysis, we quantified the predictive value of circulating hsa-miR223-3p for FRDA, obtaining an area under the ROC curve value of 0.835 (P < 0.0001) for all patients. Interestingly, we found a significant positive correlation between hsa-miR223-3p expression and cardiac parameters in typical FRDA patients (onset < 25 years). Moreover, a significant negative correlation between hsa-miR223-3p expression and HAX-1 (HCLS1-associated protein X-1) at mRNA and protein level was observed in all FRDA patients. In silico analyses suggested HAX-1 as a target gene of hsa-miR223-3p. Accordingly, we report that HAX-1 is negatively regulated by hsa-miR223-3p in cardiomyocytes (AC16) and neurons (SH-SY5Y), which are critically affected cell types in FRDA. This study describes for the first time the association between hsa-miR223-3p and HAX-1 expression in FRDA, thus supporting a potential role of this microRNA as non-invasive epigenetic biomarker for FRDA.

Frataxin deficiency in Friedreich's ataxia is associated with reduced levels of HAX-1, a regulator of cardiomyocyte death and survival.

Frataxin deficiency, responsible for Friedreich's ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient's lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.

Drug repositioning screening identifies etravirine as a potential therapeutic for friedreich's ataxia.

BACKGROUND: Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the frataxin gene, resulting in decreased frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase frataxin levels. OBJECTIVES: With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase frataxin levels. METHODS: Using a cell-based reporter assay to monitor variation in frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS: Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing frataxin messenger RNA translation. Importantly, frataxin accumulation in treated patient cell lines is comparable to frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS: Considering its excellent safety profile along with its ability to increase frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.