Anatomy and Neuroscience - Theses
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ItemEarly neuronal and glial cell changes in diabetic retinopathy(University of Melbourne, 2010)
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ItemModulation of the intermediate-conductance calcium-activated potassium (IKca) channel by protein kinase a (PKA)(University of Melbourne, 2010)
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ItemNeural plasticity and gene-environment interactions in the PLC-?1 knockout mouse(University of Melbourne, 2007)
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ItemPyroglutamate-Aß in the pathogenesis of Alzheimer's disease( 2013)Background: Amyloid-beta (Aβ) peptides are central contributors to Alzheimer’s disease (AD) pathogenesis. Although Aβ peptides are present in all human brains, the AD brain is particularly enriched for oligomeric Aβ species and Aβ peptides containing post-translational modifications such as oxidation, amino-truncation and pyroglutamate (pE). Metal interactions are a critical aspect of Aβ-induced neurotoxicity, however the effects of pE formation on Aβ-metal reactions such as nucleated oligomerisation, redox cycling and the production of reactive oxygen species (ROS) have not been investigated. Recent reports have indicated that pE-Aβ peptides are more neurotoxic than full-length Aβ, although a mechanistic difference in the toxic properties of these peptides has yet to be established. Increased levels of glutaminyl cyclase (QC) are thought to be responsible for the abundance of pE-Aβ in the AD brain, via the cyclisation of exposed N-terminal glutamate to pyroglutamate on amino-truncated Aβ. The relative levels of QC protein and mRNA are reported to be elevated in the temporal cortex and peripheral blood of individuals with AD compared to healthy controls. However, there are no published values of QC enzymatic activity in human central nervous system tissues. The involvement of QC in pE-Aβ formation and AD pathogenesis has led to the recent generation of QC inhibitors as a potential therapeutic intervention for AD. For this reason, there is a critical need to establish standardised levels of QC protein and activity in populations of healthy individuals and people with AD. Furthermore, there is a lack of animal models of pE-Aβ expression, thus the generation of simple pE-Aβ expression models may facilitate the study of potential QC inhibitors as an AD therapeutic. Objectives: I aimed to assess potential differences between synthetic pE-Aβ and full-length Aβ peptides in terms of their oligomerisation rate, fibril ultrastructure, cellular life-span and neurotoxicity. I also sought to compare the Aβ variants for their capacity to undergo nucleated polymerisation in the presence of Cu2+ or Zn2+, in addition to the generation of ROS and oxidative modifications such as dityrosine via redox cycling reactions with Cu2+ and ascorbate. To determine whether changes in soluble QC (sQC) expression and activity are a feature of AD pathogenesis, I aimed to establish standardised ranges of sQC protein and activity in the human brain through analysis of post-mortem cortical tissue samples from a cohort of AD and control brains. Finally, I sought to generate a Caenorhabditis elegans nematode model of pE-Aβ expression for in vivo comparisons of Aβ variant cytotoxicity and cellular interactions. Results: Vastly different rates of fibrilisation and fibril ultrastructures were observed for amino-truncated and pE-Aβ peptides compared with full-length peptides. Amino-truncated Aβ showed accelerated fibril seeding compared to full-length Aβ, while further addition of Cu2+ inhibited fibrilisation and produced aggregates of different ultrastructures between the seeded mixtures. In contrast, Zn2+ promoted fibrilisation but was also found to rapidly and reversibly aggregate Aβ peptides in short incubation periods. Redox-cycling reactions of Aβ, Cu2+ and ascorbate demonstrated significant differences between full-length Aβ and pE-Aβ peptides in the profiles of oligomers produced as well as the rate of hydroxyl radical production and dityrosine formation. The reaction of Aβ1-40 with Cu2+ and ascorbate was further found to cause amide-bond hydrolysis and the formation of amino-truncated Aβ peptides. Both the Aβ1-42 and Aβ3pE-42 peptides were toxic to cortical neurons and inhibited hippocampal long-term potentiation, however methodological differences in the preparation of peptides were found to significantly alter the relative Aβ neurotoxicity. Aβ1-42 was the only peptide to significantly increase neuronal ROS levels, suggesting that the toxicity observed for Aβ3pE-42 was ROS-independent. The levels of Aβ3pE-42 were much higher than Aβ1-42 following 48 h treatment of the peptides on cortical neurons, indicating that Aβ3pE-42 is highly resistant to proteolysis in neurons. Mean levels of sQC protein were modestly, though significantly, elevated in the frontal cortex of individuals with AD compared with healthy controls. No significant difference in the mean levels of total sQC activity or specific activity were observed between AD and control subjects. Gel electrophoresis and mass-spectrometry analyses of a C. elegans strain designed to express Aβ1-42 (CL2120) unexpectedly revealed that the predominant peptide expressed was actually Aβ3-42 – a precursor substrate for pE-Aβ formation. Genetic manipulation of this strain to co-express human sQC resulted in production of an additional Aβ species in these worms with hydrophobic properties consistent with Aβ3pE-42. Conclusions: Previous studies have established that amino-truncation and pE formation greatly enhance the oligomerisation and fibrilisation of Aβ peptides. The data presented here demonstrate that these modifications also affect the capacity of Aβ to undergo facile redox cycling with Cu2+, thus altering the relative production of cytotoxic ROS and oxidative protein modifications such as dityrosine. Aβ3pE-42 showed either comparable or enhanced toxicity to cortical neurons compared with Aβ1-42, although different peptide dissolution methods were seen to skew the relative toxicity of each peptide. Aβ1-42 greatly increased cytosolic ROS in neurons, whereas pE-Aβ peptides did not, suggesting that pE-Aβ induced neurotoxicity is ROS-independent. Furthermore, pyroglutamate formation renders Aβ resistant to proteolysis in neurons, indicating that pE-Aβ peptides are both neurotoxic and biologically persistent. The reported abundance of pE-Aβ in AD brains is not however a function of increased QC activity in the frontal cortex, suggesting that the rate of pE-Aβ formation is either due to regional-specific changes in QC activity or increased production of amino-truncated Aβ precursors, or both. The process of Aβ amino-truncation in vivo may also be due to multiple processes such as aminopeptidase activity and the interactions of Aβ with Cu2+. C. elegans models of pE-Aβ expression may facilitate further studies into the biological properties of these amyloidogenic peptides and the screening of potential therapeutics to inhibit their formation.
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ItemAnatomical changes at the axon initial segment in neuronal hyperexcitability( 2013)The axon initial segment (AIS) is an important sub-cellular region in neurons, playing diverse and critical roles in neuronal excitability, the maintenance of neuronal polarity, and the regulation of cytoplasmic trafficking. Previously thought to be a uniform, static structure, it is now apparent that the AIS exhibits greater levels of complexity and plasticity than previously predicted, and is an increasingly interesting and relevant focus of research in neuroscience. A range of proteins are expressed at high densities at the AIS, some exclusively, including structural molecules, ion channels and cell adhesion molecules. The molecular composition and structural characteristics of the AIS vary by neuronal subtype, brain region and developmental stage, resulting in differences in functional phenotypes of these neurons, although the more subtle aspects of this are yet to be elucidated. The important roles played by AIS-localised proteins, along with the potential consequences of disruption to AIS integrity, composition or structure, make this an incredibly important neuronal region to consider in a variety of pathophysiological pathways in the brain. Many AIS proteins have been implicated in CNS disease; in particular a large number of AIS ion channels are implicated in epilepsy. Additionally, the emerging phenomenon of AIS plasticity, by which neuronal excitability is altered as a result of changes in the gross structural anatomy of the AIS, could potentially play a role in epilepsy. In this thesis I explore two aspects of AIS involvement in disorders of neuronal hyperexcitability using immunohistochemistry and high-resolution confocal microscopy. The first study analyses the effects of seizures on AIS structure in two animal models of neuronal hyperexcitability, in which I have identified structural changes in the position of the AIS relative to the soma in animals experiencing seizures. This is the first study to demonstrate plasticity of the AIS in epilepsy, and the results suggest differing roles of this phenomenon in established genetic epilepsy and in the pathogenesis of acquired seizure disorders. The second study describes the AIS localisation of an ion channel subtype – the β1 subunit of the voltage-gated sodium channel – in both health and disease states, using a genetic mouse model of a human epilepsy syndrome. I have demonstrated the endogenous localisation of this subunit to the AIS and revealed its disruption in genetic epilepsy, an important finding complementing functional studies in elucidating the pathogenic mechanisms in this type of epilepsy. These studies reveal the novel involvement of AIS structural plasticity in neuronal hyperexcitability as well as a mechanism of AIS dysfunction in genetic epilepsy, together highlighting the ubiquitous influence of AIS function on neurological health. The linking of genetic mutations, environmental conditions and anatomical AIS phenotypes will further enhance our understanding of the pathophysiological basis of disorders of neuronal hyperexcitability and aid identification of novel therapeutic targets for neurological disease.
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ItemFlamingo/Starry Night in embryonic abdominal sensory axon development of Drosophila( 2008)The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.
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ItemEnteric serotonin interneurons: connections and role in intestinal movement( 2008)5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.
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ItemCloning and characterisation of gripe: a novel interacting partner of e12 during brain development( 2002-10)The mammalian cerebral cortex is a remarkable product of brain evolution, and is the structure that most distinctively delineates the human species from others (Northcutt and Kaas, 1995; Rakic, 1988). Neurons in the adult brain are organised into cytoarchitectonic areas, defined by distinct biochemical, morphological and physiological characteristics (Rakic 1988). Remarkably, this complex structure is generated from a simple neuroepithelium. What are the signalling mechanisms that direct neuron formation and subsequent functional-parcellation of the cerebral cortex? Key to the study of this process is an understanding of neuronal fate determination. Available evidence demonstrates an intrinsic programming potential by neuronal progenitors within subdomains of the developing cerebral cortex that is instructive for proper corticogenesis. These regional domains are demarcated by expression of certain transcription factors, including members of the Helix-Loop-Helix (HLH) family of proteins. The HLH family of transcription factors are key contributors to a wide array of developmental processes, including neurogenesis and haematopoiesis. These factors are thought to exert their regulatory influences by binding to cognate promoter-DNA sequences as dimers. While studies in mice have convincingly demonstrated that neurogenic HLH proteins such as NeuroD (Lee et al., 1995; Miyata et al., 1999; Liu et al., 2000) and Mash1 (Casarosa et al., 1999) are intimately involved in neuronal fate determination and terminal differentiation, the role of the ubiquitously expressed HLH protein, E12, in mammalian neurogenesis remains ambiguous. Originally discovered as an important regulator of lymphopoiesis, expression studies revealed its widespread expression in proliferative zones of multiple nascent organs of the embryo, including the developing cerebral cortex; implying a role for E12 in development of the nervous system. Since the function of E12 is, in part, coded by its capacity for protein dimerisation, a search was undertaken for binding partners in developing mouse brain, and using a yeast 2-hybrid assay. Yeast 2-hybrid prey libraries were constructed using complementary DNA (cDNA) isolated from embryonic mouse forebrain tissue at early (embryonic day e11.5) and peak (e15.5) stages of neurogenesis. Screening of these libraries for binding partners to an E12 bait resulted in cloning of HLH factors, such as Mash1, NSCL and Id2. Importantly, a novel binding partner, named GRIPE, was cloned as a novel GAP Related Interacting Protein to E12. GRIPE binds to the HLH region of E12, and may require E12 for nuclear import. Furthermore, GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidently detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were reconfirmed through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein whose dimerisation with E12 has important consequences for cells undergoing neuronal differentiation. A model is proposed to suggest how neurogenic HLH proteins that dimerise to E12 may promote signalling cascades driving early neuroblast differentiation.
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ItemA morphological characterisation of central neural pathways to the kidney( 2005-04)This study was undertaken to locate and characterise the neurons in the central nervous system that project to the kidney. In particular, the aim was to illustrate and characterise the neural link between regions in the hypothalamus known to influence renal function and fluid balance, and nerves known to innervate the kidney.