| dc.description.abstract | Friedreich’s ataxia (FA) is the most common autosomal recessive ataxia, and patients of the disease are severely afflicted with progressive neuro- and cardio-degeneration. The main cause of FA is due to the deficient expression of the mitochondrial protein, frataxin, and its deficiency has been well reported to be associated with oxidative stress and losses in energy metabolism. The major aim of this thesis was to elucidate the molecular mechanisms involved in the dysregulated anti-oxidative response in frataxin deficiency, which is responsible for the exacerbation of oxidative stress in FA. In light of the disease-associated deficits in mitochondrial bioenergetics and stress, this thesis then sought to explore the involvement of mitochondrial dynamics in the pathogenesis of FA. Considering these two pathological aspects of the disease, the thesis further assessed the efficacy of two different treatments aimed at restoring antioxidant defence and energy metabolism in vivo in frataxin deficiency. Results from these investigations are significant due to their potential applications and relevance to the development of future therapeutics for FA patients. This dissertation is comprised of 6 chapters: a comprehensive literature review (Chapter 1 Introduction); a materials and methods chapter (Chapter 2 Materials and Methods); 3 results chapters (Chapter 3 – 5); and a general discussion of principle findings and future directions chapter (Chapter 6 Discussion and Future Directions). viii Chapter 3: Various studies in models of FA have previously reported a decrease in the expression of the master regulator of antioxidant response, nuclear factor-erythroid 2-related factor-2 (Nrf2), due to unknown mechanisms. This chapter herein, examined the Nrf2-Keap1 signalling pathway using a mouse conditional frataxin knockout (KO) model of FA, by comparing the heart and skeletal muscle in these mice. The frataxin KO hearts exhibited fatal cardiomyopathy, while the skeletal muscle was asymptomatic. These two tissue-types demonstrated contrasting molecular alterations. In the KO heart, protein oxidation and decreased GSH:GSSG ratio were observed, as well as decreased total and nuclear Nrf2 expression and increased Keap1 levels. However, the skeletal muscle did not demonstrate these alterations. Notably, for the first time, a mechanism involving Gsk3β-signalling in the activation of nuclear Nrf2 export and/or degradation machinery was demonstrated in the KO heart. This process involved the increased activation of Gsk3β, increased Fyn phosphorylation, and the nuclear accumulation of β-TrCP to facilitate Nrf2 nuclear export. Furthermore, Nrf2-DNA-binding activity and the mRNA expression of Nrf2-targets were decreased in the frataxin KO mice. However, certain Nrf2 antioxidant targets, namely, NADPH quinone oxidoreductase-1 (Nqo1), glutathione-S-transferase-Mu1 (Gstm1), and thioredoxin reductase 1 (TxnRD1), demonstrated increased protein levels in the KO heart. In general, two potential mechanisms could be responsible for the reduced Nrf2 levels in the frataxin-deficient hearts: (1) increased cytosolic Keap1 levels, and (2) the activation of Gsk3β-signalling or the Gsk3β-Fyn axis that decreases nuclear Nrf2 levels. On the other hand, the frataxin-deficient skeletal muscle had no decrease in Nrf2 levels and had contrasting results to the heart. Collectively, these findings have revealed tissue-specific ix alterations in frataxin deficiency, but more importantly, the data have uncovered potential mechanisms that could significantly dysregulate the anti-oxidative response in FA. Chapter 4: The mitochondrion is an essential organelle that maintains cellular function and health through its role in energy production. The mitochondrion protects the cell from oxidative stress by maintaining its homeostasis with critically dynamic processes of mitochondrial biogenesis, fusion/fission, and mitophagy. An imbalance between oxidative stress formation and endogenous antioxidant processes can induce mitochondrial protein defects that can severely disrupt mitochondrial homeostasis. This can lead to mitochondrial dysfunction, which is accompanied by mitochondrial protein oxidation and mitochondrial DNA (mtDNA) damage, culminating in the depletion of ATP and NAD+, apoptosis and organ failure. The heart and the nervous systems, which have an abundance of mitochondria, are most vulnerable to mitochondrial protein dysfunction, as evident in a number of belligerent human disease states. FA is also regarded as a mitochondrial disease, due to the role of frataxin in mitochondrial functions. Frataxin deficiency leads to a defect in mitochondrial iron metabolism and oxidative stress that potentiates the pathology of the disease. However, alterations to mitochondrial homeostasis have not been fully elucidated in the pathogenesis of FA. Using the aforementioned frataxin KO mice model of FA that develops dilated cardiomyopathy, a number of key observations were found in the KO hearts relative to their wild-type littermates: (1) irregular mitochondrial morphologies and abnormal structure of cristae; (2) increased Parp activation, decreased NAD+:NADH ratio and reduced Sirt1 activity, (3) increased protein markers of mitochondrial biogenesis and dynamics (both fusion and fission), and (4) increased autophagic x flux at 10-weeks of age. These novel findings demonstrate significant changes to mitochondrial homeostasis in the condition of frataxin deficiency. Not only does this illustrate the importance of maintaining mitochondrial homeostasis in cardio-degenerative diseases, but it offers the potential for the development of new treatments that target mitochondrial function. Chapter 5: Results from Chapter 3 have found multiple molecular mechanisms involved in the dysregulation of the Nrf2 signalling pathway that negatively affects the anti-oxidative response in frataxin-deficient condition. Data from Chapter 4 have demonstrated significant alterations to mitochondrial morphologies, dynamics, and function in the frataxin KO mice with age. Taken together, these results indicate the critical involvement of oxidative stress, mitochondrial dysfunction and decreased bioenergetics in the pathogenesis of FA. Since there are currently limited treatments available for FA patients, there is an urgent need to develop new therapies that focuses on ameliorating these pathological deficits of the disease. This chapter herein examined the potential therapeutic effects of two agents, namely, N-acetylcysteine (NAC) in the supplementation of the antioxidant glutathione, and the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazine (SNH6), developed in our laboratory that has a dual-mechanism of action mediated by NAD+ supplementation and iron chelation. Using the previously described MCK mouse model of FA, these animals were treated from the asymptomatic age of 4-weeks-old, up until 9-weeks of age, where the animal displays an overt dilated cardiomyopathy. In general, iron deposits, interstitial fibrosis, and enlargement of cardiac muscle fibre size were observed in histological examinations of the KO hearts treated with either NAC or SNH6. Hence, the treatments did not attenuate disease progression or prevent the xi development of cardiac hypertrophy. Despite these observations, the treatment with SNH6 did significantly increase NAD+ levels, and as a result, there was increased sirtuin 1 and Parp activities in the SNH6-treated KO hearts. Hence, SNH6-supplementation of NAD+ potentially restored, in part, mitochondrial function and dynamics, despite that it did not increase the NAD+:NADH ratio and ATP levels. Collectively, increasing endogenous antioxidant levels and NAD+ supplementation are two different, but important therapeutic strategies that deserves further investigation. Particularly, the therapeutic use of the novel agent, SNH6, and the supplementation of NAD+, holds promise for the development of novel therapeutic strategies for FA patients. In conclusion, this dissertation has elucidated the molecular mechanisms involved in the dysregulation of anti-oxidative response and mitochondrial dynamics in the condition of frataxin deficiency of FA. The research in this thesis has enhanced our understanding of the pathophysiology of the disease and its associated cardiomyopathy, which offers new insights into the development of potential therapeutics. Thus, the significance of this dissertation is in its relevance to the advancement of future therapies for effective FA treatment. | en |
