Investigating the therapeutic effects of different antioxidants in Friedreich’s Ataxia

Abstract

Friedreich’s Ataxia (FRDA) is a lethal autosomal neurological disorder caused by the abnormal expansion of Guanine-Adenine-Adenine (GAA) repeat in intron of the FXN gene. This leads to the downregulation of the FXN gene and reduced levels of the mitochondrial protein frataxin. Unaffected individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have ~70 to more than 1000 GAA triplets. The disease affects the brain, spinal cord, heart and skeletal muscles thereby characterizing the disease by progressive neurodegeneration, cardiomyopathy, musculoskeletal deformities and diabetes mellitus. The low levels of frataxin protein result in increased oxidative stress and mitochondrial dysfunction thereby driving disease progression. Different antioxidants have been tested in the past with quite satisfying and encouraging results in vitro and in vivo. So far, no cure has been found for this disease. This project aimed at investigating the effect of antioxidants on mitochondrial redox homeostasis and metabolism in FRDA. Candidate compounds selected include Dimethylfumarate (DMF), N-acetylcysteine (NAC), Resveratrol (RESV), L-Ascorbic acid (LAA) and Alpha tocopherol (AT) and Alpha Lipoic acid (ALA), Indole-3-propionic acid (I3PA), Trolox (TX), Xanthohumol (XAN), Ginsenoside RB1 (GnRB1) and Orotic acid (OA). Galleria mellonella model was used to determine the LD50 of potential antioxidants and proof-of-concept study was conducted using Juglone. LAA, NAC, DMF, RESV and AT were observed to rescue the larvae from oxidative toxicity induced by Juglone. Toxicity of compounds in FRDA cells was assessed using PrestoBlue® cell viability assay. Mitochondrial ROS (mROS)-decreasing capacity of compounds in FRDA fibroblast cells and 2D sensory neurons were assessed by MitoSOX-based flow cytometric assay, and FXN and NRF2 gene expression levels and mitochondrial copy number were assessed using qRT-PCR. ELISA was used to quantify the levels of frataxin and NRF2 proteins in the fibroblast and sensory neurons. Significant reduction in mROS, cytoplasmic ROS, and increase FXN, NRF2 gene expression, frataxin and NRF2 proteins were observed after treatment with LAA, NAC and DMF. Also, a significant improvement in aconitase/citrate synthase, GSH/GSSG and Mitochonsrial membrane potential were observed following treatment in LAA, NAC and DMF. Therapeutic combination of LAA and NAC was shown to consistently improve the disease characteristics in FRDA which were investigated in this study. It is therefore proposed that synergistic combination of LAA and NAC can be a viable therapeutic modality which could blunt FRDA disease progression.