Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders. To date, ten SCA loci have been described (SCA1-SCA8, SCA10 and SCA11), with six genes having been cloned (SCA1, SCA2, SCA3/MJD, SCA6, SCA7 and SCA8) and shown to contain CAG/CTG repeats. / This study investigated various aspects of the SCA2, SCA6, and SCA7 subtypes. Haplotype analysis in our panel of SCA2 families identified multiple ancestral mutation events to be responsible for disease in this group. Screening for the newly identified SCA6 and SCA7 mutations in our large collection of SCA families and patients revealed that these mutations are rare in our panel, each accounting for less than 1% of our ataxia samples. Finally, the CAG repeat-containing locus hGT1 was found to be associated with residual age at onset variability in our SCA2 families. / Together, these results add to our growing understanding of the SCAs, and bring us a few steps closer to effective diagnoses of, and treatments for, these devastating diseases.

No description available.

No description available.

Iron-sulfur clusters are protein cofactors that are critical for all life forms. Elaborate multi-component systems have evolved for the biosynthesis of these cofactors to protect organisms from the toxic effects of free iron and sulfide ions. In eukaryotes, the Fe-S cluster assembly machinery operates in the matrix space of the mitochondria and contains a myriad of proteins that mediate sulfur, iron, and electron transfer to assemble Fe-S clusters on the scaffold protein ISCU2 and then distribute these clusters to target proteins. Our lab has recently described stable 3, and 4-protein complexes composed of the cysteine desulfurase NFS1, the co-chaperone ISD11, and ISCU2 (SDU), and NFS1, ISD11, ISCU2, and FXN (SDUF) subunits. In the latter, SDUF, FXN functions as an allosteric activator switching this assembly complex on for Fe-S cluster biosynthesis. Insufficient expression of the mitochondrial protein FXN leads to a progressive neurodegenerative disease, Friedreich's Ataxia (FRDA). In ~2% of patients, FRDA is caused by one of 15 known missense mutations on one allele accompanied by the GAA repeat on the other leading to a complicated phenotype that includes loss of Fe-S clusters. Here we present in vitro evidence that FRDA FXN variants are deficient in their ability to bind the SDU complex, their ability to stimulate the sulfur transfer reaction from NFS1 to ISCU2, and in their ability to stimulate the rate of cluster assembly on ISCU2. Here, in vitro evidence is presented that FXN accelerates the sulfur transfer reaction from NFS1 to ISCU2. Additionally, we present kinetic evidence that identifies the most buried cysteine residue, C104 on ISCU2 as the sulfur acceptor residue suggesting, FXN stabilizes a conformational change to facilitate sulfur delivery. Subsequent mutational studies suggest FXN binding to SDU results in a helix to coil transition in ISCU2 exposing C104 to accept the persulfide sulfur and thereby accelerating the rate of sulfur transfer. We further provide the first biochemical evidence that the persulfide transferred to ISCU2 from NFS1 is viable in Fe-S cluster formation. In contrast to human FXN, the Escherichia coli FXN homolog CyaY has been reported to inhibit Fe-S cluster biosynthesis. To resolve this discrepancy, a series of inter-species enzyme kinetic experiments were performed. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 79-97.

Friedreich's ataxia is a neurodegenerative disorder caused by the low expression of a mitochondrial protein called frataxin. Studies in the yeast Saccharomyces cerevisiae have unraveled a role for the frataxin homologue (Yfh1p) in iron-sulfur cluster (Fe/S) biosynthesis, probably by interacting with the scaffold protein, Isu1p, and providing iron to the machinery. Yfh1p possesses a large â-sheet platform that may be involved in the interaction with other proteins through conserved residues at its surface. We have used directed mutagenesis associated with polymerase chain reaction (PCR) to study conserved residues localizing either at the surface of the protein, Thr110, Thr118, Val120, Asn122, Gln124, Gln129, Trp131, Ser137 and Arg141, or buried in the core of the protein, Ile130 and Leu132. Mutants T110A, T118A, V120A, N122A, Q124A, Q129A, I130A, W131A, L132A, S137A and R141A were generated in yeast. Growth on iron- or copper-containing medium was severely impaired for mutants Q129A, I130A, W131A and R141A. Others were roughly growing as well as the wild-type strain. We assessed the efficiency of Fe/S biosynthesis by measuring aconitase activity. The results confirmed those obtained on metal-containing medium: mutants Q129A, I130A, W131A and R141A showed a high decrease in their aconitase activity that dropped to the deleted strain level. Moreover, S137A showed also a decreased aconitase activity. We monitored the interaction between Yfh1p and Isu1p by co-immunoprecipitation and it turned out that only the W131A mutation affects directly this interaction. Even if the amount of Yfh1p determined by western blot analysis was highly decreased for several mutants, it is not sufficient to explain the phenotypes as they were poorly restored by overexpression of the mutant proteins to wild-type levels, except for W131F. We have concluded that Gln129, Trp131, and Arg141 are important for Yfh1p function, while Ile130 and Ser137 are required for the folding of the protein. All these residues cluster to the 4th and 5th â-strand of the protein. Our work has demonstrated for the first time the importance of this area for Yfh1p function and shows that Trp131 is involved in the interaction with Isu1p.

Friedreich’s ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart disease. It is caused by a pathological intronic hyperexpansion of a GAA repeat in the FXN gene, encoding the essential mitochondrial protein frataxin. At the homozygous state, the GAA expansion induces a heterochromatin state with decreased histone acetylation and increased methylation, resulting in a partial deficiency of frataxin expression. This was established in cells from FRDA patients. We showed that the same chromatin changes exist in a GAA based mouse model, KIKI, generated in our laboratory. Furthermore, treatment of KIKI mice with a novel Histone Deacetylase Inhibitor (HDACi), 106, a pimelic diphenylamide that increases frataxin levels in FRDA cell culture, restored frataxin levels in the nervous system and heart of KIKI mice and induced histone hyperacetylation near the GAA repeat. As shown by microarrays, most of the differentially expressed genes in KIKI were corrected towards wild type. In an effort to improve the pharmacological profile of compound 106, we synthesized more compounds based on its structure and specificity. We characterized two of these compounds in FRDA patients’ peripheral blood lymphocytes and in the KIKI mouse model. We observed a sustained frataxin upregulation in both systems, and, by following the time course of the events, we concluded that the effects of these compounds last longer than the time of direct exposure to HDACi. Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACis for FRDA. Laboratory tools to follow disease progression and assess drug efficacy are needed in a slowly progressive neurodegenerative disease such as FRDA. We used microarrays to characterize the gene expression profile in peripheral lymphocytes from FRDA patients, carriers and controls. We identified gene expression changes in heterozygous, clinically unaffected GAA expansion carriers, suggesting that they present a biochemical phenotype, consistent with data from animal models of frataxin deficiency. We identified a subset of genes changing in patients as a result of pathological frataxin deficiency establishing robust gene expression changes in peripheral lymphocytes. These changes can be used as a biomarker to monitor disease progression and potentially assess drug efficacy. To this end, we used he same methodology to characterize the gene expression profiles in peripheral lymphocytes after treatment with pimelic diphenylamide HDACi. This treatment had relevant effects on gene expression on peripheral patients’ blood lymphocytes. It increased frataxin levels in a dose-dependent manner, and partially rescued the gene expression phenotype associated with frataxin deficiency in the tested cell model, thus providing the first application of a biomarker gene set in FRDA.

With cardiovascular disease, diabetes mellitus, and neurodegenerative conditions on the rise, understanding their pathogenesis is paramount to tackling this public health crisis. Current research indicates that the primary cause of these diseases is mitochondrial dysfunction in the affected patients. While genetics plays a role in these conditions, lifestyle choices and exposure to toxins also significantly contribute to their development. Unfortunately, early-stage diagnosis can be difficult due to overlapping symptoms with other diseases. Developing innovative therapies that can prevent or reverse the deterioration of metabolic dysfunctions is critical to establishing early intervention. My research focused on investigating molecular targets linked with Friedrich's Ataxia, an inherited metabolic disorder, through conducting functional in-vitro studies using human-derived cell samples, as well as developing inventive animal models created via Xenopus laevis tadpoles to evaluate the effects of environmental stressors. My investigations have uncovered promising treatment options that improve mitochondrial function, mitigate oxidative stress, and elucidate critical mechanisms involved in environmentally induced disruptions to mitochondria. / Doctor of Philosophy / Metabolic dysfunction is a widespread health issue that affects millions of individuals each day. Its associated disorders, such as cardiovascular disease, diabetes, and neurodegenerative conditions, are rising due to various factors ranging from genetic predispositions to environmental and lifestyle-related risks. Therefore, there's an urgent need to identify this disorder early on and develop innovative treatment options. Considering this growing public health concern, it has become imperative to establish new methods for detecting metabolic dysfunction at its nascent stage while also exploring potential therapeutic interventions. Our research utilized cells derived from affected patients and animal models in devising novel approaches toward understanding the molecular mechanisms underpinning metabolic dysfunction. Our findings revealed several pathways and molecular targets contributing significantly to this condition, which could effectively be leveraged to develop targeted therapeutic strategies to combat its effects. Expanding our knowledge base will enable us to stay updated with emerging insights on treating metabolic dysfunction effectively while substantially improving patient outcomes.

Friedreichs ataxia (FRDA) is a progressive spinocerebellar ataxia (SCA) inherited as an autosomal recessive trait. The neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities characteristic to FRDA result from a deficiency in the mitochondrial protein frataxin. Frataxin chaperones iron to heme and iron-sulfur clusters and its deficiency causes mitochondrial iron accumulation and oxidative stress.<p> To address the effect of frataxin deficiency on mitochondrial iron chemistry, mitochondria were isolated from FRDA and control fibroblasts. X-ray absorption spectroscopy showed that ferrihydrite was the predominant form of iron in both. Near edge analysis showed that the ferrihydrite in the FRDA mitochondria resembled the highly organized ferrihydrite of ferritin. Western blotting confirmed that FRDA mitochondria had 3-fold more holoferritin containing stainable iron. I conclude that mitochondria from FRDA fibroblasts mineralize excess iron as ferrihydrite within mitochondrial ferritin.<p> To address how cellular iron dysregulation affected metal distribution in brain and spinal cord, a new synchrotron imaging technique, rapid-scanning x-ray fluorescence (RS-XRF) was employed and validated. Brain structures were readily identified by their unique metal content and distribution. This showed that RS-XRF could be used to reveal metal pathologies associated with diseases of metal metabolism such as FRDA. Since human FRDA tissues were not available for a detailed study, RS-XRF was employed to study the distribution of metals in normal cerebellum, a major site of FRDA-associated neurodegeneration, and to localize and quantify metals in the brain and spinal cord from a patient with a SCA of unknown aetiology. The motivation for this work is the prospect of future systematic studies on metal pathology in neurodegenerative diseases with direct application to FRDA. Novel findings arising from this work were the metal segmentation of the dentate nucleus, the high copper content of the olivary region and the different metal content of lesions at different stages of neurodegeneration. My results suggest that not only iron, but also copper and zinc may play a role in the physiopathology of neurodegeneration. Therefore, all three metals should be investigated in FRDA and other SCA of both known and unknown aetiologies to identify possible new therapeutic targets.