Nature of the genetic defect in Ataxia Oculomotor Apraxia Type 3 (AOA3)

Nor Abdul Murad (2011). Nature of the genetic defect in Ataxia Oculomotor Apraxia Type 3 (AOA3) PhD Thesis, School of Medicine, The University of Queensland.

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Author Nor Abdul Murad
Thesis Title Nature of the genetic defect in Ataxia Oculomotor Apraxia Type 3 (AOA3)
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2011-08
Thesis type PhD Thesis
Supervisor Professor Martin F Lavin
Dr. Olivier Becherel
Total pages 330
Total colour pages 60
Total black and white pages 270
Subjects 11 Medical and Health Sciences
Abstract/Summary Ataxia Oculomotor Apraxia Type 3 (AOA3) is a novel form of AOA disease clinically characterized by cerebellar degeneration, oculomotor apraxia, cerebellar ataxia and childhood onset. Mitochondrial dysfunction and increased levels of oxidative stress were observed in AOA3 cells. The first evidence of mitochondrial dysfunction in AOA3 was reported by Gueven and colleagues (Gueven et al., 2007). This included a depolarization defect of the mitochondrial membrane after DNA damage, defective DNA-damage induced apoptosis, and low levels of ATP. Here the mitochondrial dysfunction was further characterized and revealed that AOA3 had increased mitochondrial mass compared to controls. The increased mitochondrial mass could be due to compensation mechanisms for the presence of defective mitochondria. In addition, these cells also showed low mitochondrial membrane potential which subsequently led to the failure of DNA damage-induced apoptosis. Low mitochondrial membrane potential inhibits the release of cytochrome C and Apoptosis inducing factor (AIF) and leads to failure of apoptosis induction. An overall mitochondrial dysfunction in these cells was indicated by decreased mitochondrial respiration compared to controls. Oxidative stress has been implicated in many neurodegenerative diseases such as Alzheimer’s, Freidriech’s Ataxia, Parkinson’s and Amyotrophic Lateral Sclerosis (ALS). Immunofluorescence for oxidative stress markers such as nitrotyrosine (protein oxidation), 8-oxo-dG (oxidative DNA damage) and 4-HNE (lipid peroxidation) also revealed oxidative stress in AOA3. Further investigations revealed increased 4-HNE lipid peroxidation of mitochondrial proteins. Several mitochondrial proteins were modified that include ATP synthase, mitochondrial acetylaceto-CoA thiolase, calreticulin, elongation factor Tu and heat shock protein 60 (HSP 60). These proteins are involved in several mechanisms that include ATP synthesis, Ca2+ regulation, RNA metabolism and protein refolding. Oxidized ATP synthase and mitochondrial acetylaceto-CoA thiolase may lead to decreased levels of ATP as observed in AOA3 cells. Furthermore, an increase in mitochondrial superoxide production was also observed. Mitochondrial dysfunction and oxidative stress suggest that AOA3 could be a novel form of mitochondrial disease. Sequencing analysis of the AOA3 mitochondrial genome revealed a maternal inheritance of cytochrome B gene (I349T) mutation in AOA3. Autosomal recessive cerebellar ataxia type 2 (ARCA2) is a novel form of cerebellar ataxia caused by mutations in the Aarf domain containing kinase 3 (ADCK3) gene, which displays similar clinical characteristics with AOA3. To determine whether AOA3 may result from mutation in ADCK3, we also sequenced this gene and identified a paternal inheritance of Y429C mutation. The absence of Y429C in the AOA3’s siblings suggests that it could be pathogenic. PMut analysis revealed that both mutations were pathogenic with NN output of 0.68 for I349T and 0.78 for Y429C respectively (NN>0.5 is pathogenic). Given that both ADCK3 and cytochrome B are components of complex III, the stability and activity of the mitochondrial complexes were determined. BN-PAGE failed to reveal any gross-abnormalities in the mitochondrial supercomplex. Overall, the mitochondrial complexes activities were normal. Therefore, it can be concluded that both mutations did not affect the mitochondrial complex formation and activity. The pathogenicity of the ADCK3 Y429C mutation was studied using small interfering RNA (siRNA) against the ADCK3 gene and expression of ADCK3-WT and ADCK3-Y429C cDNAs into controls and ARCA2 cells. Following transfection, mitochondrial function assays were performed. However the pathogenicity of this mutation was not conclusively determined. Nevertheless, we postulated that these mutations may lead to neurodegeneration through the increased levels of ROS and decreased mitochondrial membrane potential observed in these cells. High levels of ROS can cause oxidative stress in cells and subsequently lead to further damage to macromolecules such as DNA, proteins, lipids, and cell membranes. Oxidative stress can also alter the mitochondrial membrane potential resulting in neuronal depolarization, activation of excitatory amino acid receptors, impaired intracellular Ca2+ homeostasis, protease activation and finally cell death. Since mitochondria are regulated by both, the nuclear and mitochondrial genomes, mitochondrial dysfunction and oxidative stress can also trigger mitochondrial and nuclear genome instability, chromosomal abnormalities, irregular-nuclear communication, inhibition of the nuclear DNA repair pathways, and inhibition of mitochondrial homeostasis. In conclusion, AOA3 may represent a novel mitochondrial disease associated with ataxia and characterized by mitochondrial dysfunction and oxidative stress which may result from mtDNA and nuclear gene mutations encoding for mitochondrial component.
Keyword Ataxia Oculomotor Apraxia Type 3 (AOA3), Autosomal Recessive Cerebellar Ataxia Type 2 (ARCA2), Neurodegeneration, Ataxia, Mitochondrial diseases, Oxidative stress, Mitochondrial dysfunction, Cytochrome B gene, Aarf-domain containing kinase 3 (ADCK3)
Additional Notes Colour pages: 7,10,14, 32, 36, 38, 40, 41, 42, 44, 45, 46, 47, 51, 55, 72, 80, 81, 83, 84, 88, 90, 92, 93, 94, 97, 98, 100, 103, 108, 137, 138, 140, 141, 144, 146, 147, 148, 150, 152, 154, 157, 158, 177, 187, 189, 190, 191, 192, 195, 197, 200, 201, 229, 230, 301, 302, 303, 304, 305 Landscape : 17, 26, 27, 282, 298, 299, 300

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Created: Thu, 08 Sep 2011, 17:48:08 EST by Ms Nor Abdul Murad on behalf of Library - Information Access Service