Novel Nrf2-Inducer Prevents Mitochondrial Defects and Oxidative Stress in Friedreich's Ataxia Models

Abstract

Friedreich's Ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, affecting dorsal root ganglia (DRG), cerebellar dentate nuclei and heart. It is caused by a GAA repeat expansion mutation within the frataxin gene (FXN). This impedes FXN transcription resulting in a progressive decrease of the mitochondrial protein, frataxin. Increased oxidative stress leading to a chronic depletion of endogenous antioxidants affects the survival of the cells and causes neurodegeneration. In particular, cerebellar granule neurons (CGNs) show a significant increase of reactive oxygen species (ROS), lipid peroxidation and lower level of reduced glutathione (GSH). In FRDA, one of the major pathways of oxidant scavengers, the Nrf2 antioxidant pathway, is defective. Previous studies on FRDA-like CGNs showed that the reduced level of frataxin and the oxidative stress induce mitochondrial impairments. By triggering the Nrf2 endogenous pathway pharmacologically we determined whether this could promote mitochondrial fitness and counteract oxidative stress. In this work, we sought to investigate the beneficial effect of a promising Nrf2-inducer, omaveloxolone (omav), in CGNs from two FRDA mouse models, KIKO and YG8R, and human fibroblasts from patients. We found that CGNs from both KIKO and YG8R presented Complex I deficiency and that omav was able to restore substrate availability and Complex I activity. This was also confirmed in human primary fibroblasts from FRDA patients. Although fibroblasts are not the major tissue affected, we found that they show significant differences recapitulating the disease; this is therefore an important tool to investigate patients' pathophysiology. Interestingly, we found that patient fibroblasts had an increased level of endogenous lipid peroxidation and mitochondrial ROS (mROS), and lower GSH at rest. Omav was able to reverse this phenotype, protecting the cells against oxidative stress. By stimulating the cells with hydrogen peroxide (H₂O₂) and looking for potential mitochondrial pathophysiology, we found that fibroblasts could not maintain their mitochondrial membrane potential (ΔΨ_m). Remarkably, omav was protective to mitochondrial depolarization, promoting mitochondrial respiration and preventing cell death. Our results show that omav promotes Complex I activity and protect cells from oxidative stress. Omav could, therefore, be used as a novel therapeutic drug to ameliorate the pathophysiology of FRDA.

Keywords: Friedreich’s ataxia; human fibroblasts; lipid peroxidation; mitochondrial dysfunction; nuclear factor (erythroid-derived 2)-like 2; reactive oxygen species.

Figure 1

Omaveloxolone (Omav) prevents Complex I inhibition in friedreich’s ataxia (FRDA) fibroblasts and neurons. (A) Representative trace of nicotinamide adenine dinucleotide (NADH) autofluorescence where the basal level refers to the NADH redox state and the response to NaCN subtracting the response to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) represents the NADH pool. (B–D) The histograms represent the calculation total NADH pool in KIKO, YG8R cerebellar granule neurons (CGNs) and Patients compared to their controls and without/with omav (**p < 0.005; ***p < 0.0005). (E–G) The histograms represent the calculation total NADH redox state in KIKO, YG8R CGNs and Patients compared to their controls and without/with omav (*p < 0.05; ***p < 0.0005).

Figure 2

Oxidative stress in FRDA fibroblasts is prevented by omav. (A) Lipid peroxidation was measured in Control and FRDA cells with C11 BODIPY (581/591) using live imaging over time. (B) The ratio of the fluorophore was calculated (488/562 nm) and analyzed the rate of increase in percentage (%; taken Control as 100%). Patient 1 and 2 fibroblasts untreated showed a significant increase compared to Control (***p < 0.0005) and a significant decrease in rate after omav compared to untreated cells (**p < 0.005). (C) The curve shows the kinetic curve of CM-H₂Xros generated by Control and FRDA cells. (D) The rate of increase was expressed in ArbU/min. The rates in untreated patients’ cells were significantly increased compared to Control (*p < 0.05) and patients’ cells untreated and treated with omav showed a significant difference (Patient 1 *p < 0.05; Patient 2 **p < 0.005). (E) Glutathione (GSH) was measured with monochlorobimane (MCB) and was lower in patients untreated compared to Control (***p < 0.0005) and the amount seemed to be restored in patients’ cells treated with omav (*p < 0.05).

Figure 3

Omav protected ΔΨ_m maintenance in stressed fibroblasts. (A) Basal ΔΨ_m was measured with 25 nM tetramethyl rhodamine, methyl ester (TMRM). Untreated and cells pre-treated with omav did not show significant differences. (B) We, therefore, treated patients’ fibroblasts with 1 mM H₂O₂ for 2 h and assessed the maintenance of ΔΨ_m with 1 μM TMRM (De-quencing mode) and challenged the cells with 2 μg/mL oligomycin. (C) The increase of TMRM after oligomycin administration was then calculated in %. Patients’ cells showed a stable line if untreated, however, those treated with H₂O₂ showed a marked depolarization which was significantly prevented with omav (**p < 0.005).

Figure 4

Omav prevents cell death in FRDA fibroblasts. Cells were stained with DAPI for total cells counting and propidium iodide (PI) to visualize dead cells. The % of dead cells was calculated for each case. Hydrogen peroxide (H₂O₂) treated cells in red (Carrier, Patients 1 and 2) showed a % of cell death greater than the Control. This was prevented by omav (***p < 0.0005).

Figure 5

Interaction between omav and Nrf2 pathway. The Nrf2 transcription factor in cells is regulated by ubiquitination and proteosomal degradation through Keap1 protein. The increase of reactive oxygen species (ROS) in cell promotes its translocation to the nucleus where it binds the antioxidant responsive elements (ARE) favoring the transcription of the genes involved in the antioxidant response. Omav is a Nrf2 inducer promotes its activity by inhibiting Keap1 ubiquitinating activity and preventing Nrf2 degradation.