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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ItemPsi regulates transcription of the MYC oncogene in Drosophila melanogaster( 2014)The MYC oncogene drives cell growth and consequently, increased MYC is associated with most human cancers. Thus tight regulation of MYC is critical and this thesis investigates one mechanism for controlling MYC abundance at the transcriptional level. In all multicellular animals, cells and tissues must have the capacity to quickly respond to the environment in order to activate MYC transcription and cell growth. Based on in vitro studies we predict that the presence of paused, but transcriptionally active, RNA polymerase II in the MYC promoter will be required for a rapid response to the cellular environment. Of particular interest to this project, human tissue culture studies from David Levens group (NIH, Bethesda) have shown that in response to serum the single stranded DNA binding proteins FUBP and FIR are recruited to the promoter of the MYC oncogene, and are associated with release of paused RNA Polymerase II. Moreover, the FIR protein is required for repression of MYC transcription, which occurs via interaction with the general transcription factor TFIIH complex. This mechanism for MYC repression appears to be conserved between mammals and Drosophila, as the Quinn lab has demonstrated that the homolog of FIR (Hfp) represses Drosophila MYC in a manner dependent on interaction with the TFIIH complex. However, dissecting the role of the FUBP protein (FUBP1) in MYC regulation has been complicated by the fact that mammals have multiple FUBP family members (FUBP 1,2 and 3), which bind highly overlapping transcriptional targets. Originally, based on the finding that FUBP1 binds the MYC promoter prior to FIR, we predicted FUBP1 might play a role in activating RNA Polymerase II release and MYC transcript elongation. This thesis has used the Drosophila melanogaster model system to answer the question of whether the sole FUBP family member, Psi, is required for dMYC transcription in vivo. Here we demonstrate that Psi knockdown significantly reduced dMYC mRNA levels, which suggests Psi may be normally required for maintaining dMYC transcription in vivo. In line with a direct role in regulating MYC transcription, Chromatin Immunoprecipitation (ChIP) revealed enrichment for Psi on the dMYC promoter. Moreover, ChIP for poised RNA Pol II (Serine 5) proximal to the dMYC transcription start site, revealed that Psi knockdown results in a reduced RNA Polymerase II Serine 5 enrichment, consistent with the reduction in dMYC mRNA in the Psi knockdown. Together the experiments detailed in this thesis provide the first evidence that MYC is regulated by RNA Polymerase II pausing in vivo. Moreover, we demonstrate that Psi, the ortholog of mammalian FUBP1, is normally required for accumulation of the activated (Ser 5 phosphorylated) RNA Polymerase II on the dMYC promoter and, thus, for the regulation dMYC transcription.
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ItemStriatal neurogenesis under physiological and pathological conditions in the neonate and adult brain( 2014)Post-natal neurogenesis within the mammalian brain primarily occurs within the subventricular zone lining the lateral ventricles and the dentate gyrus of the hippocampus. Previous studies have demonstrated the crucial role of post-natal neurogenesis for correct neuronal functionality including formation of subcortical brain structures, ongoing cortical development, spatial memory, olfaction, mating behaviour, and self-repair upon injury. Substantial advances have been made in the last decade on our understating of the basic physiology underlying neurogenesis in the postnatal mammalian brain. The bulk of the work in this area has been based on analysis of the adult brain. Relatively less is known about the capacity for neurogenesis in specific structures within the neonatal brain. My PhD aims to address a number of questions focused on striatal neurogenesis in the neonate and adult brain under normal physiological conditions and also acute pathological conditions afflicting the striatum. The second chapter characterises and quantitatively assesses the therapeutic potential of meteorin, a soluble factor shown to have biological efficacy after striatal damage, on exploiting various aspects of adult neurogenesis in the SVZ after forebrain injury via over-expression post-injury. The third chapter examines neonatal striatal development during the first week of birth, focusing on the continued production of striatal neurons. Characterisation of these neonatally born neurons reveals a large number obtain a medium spiny projection neuron identity and are able to innervate their correct biological targets in the brain. Further adding to this aspect of neonatal development, the fourth chapter looks at the development of a novel neonatal forebrain ischemia model through delivery of endothelin-1, a vasoconstriction protein. This novel neonatal stroke model is able to mimic a number of clinical hallmarks including atrophy of forebrain structures, white matter damage and motor impairment. Using this model we also assessed how neonatal stroke effects striatal neurogenesis. Using birth-dating techniques we showed that production of new striatal neurons and specifically new medium spiny projection neurons was unaffected after ET-1 induced neonatal stroke.
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ItemInvestigation of the mechanisms BDNF utilises to promote central nervous system myelination( 2014)Brain-derived neurotrophic factor (BDNF) promotes CNS myelination, which is crucial for normal CNS function. This thesis investigates the influence BDNF and its signalling exerts upon myelination. Surprisingly, I found that conditional deletion of TrkB in oligodendroglia exerted no effect on myelination in vivo. However, over-expression of Erk2, a molecule activated by BDNF signalling, promotes myelination in vitro. I found that Erk1/2 interacts with and phosphorylates the oligodendroglial transcription factors Olig1 and Olig2, which may contribute to Erk1/2’s promyelinating effects.
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ItemSodium channels and epilepsy: neuronal dysfunction in genetic mouse models( 2014)Mutations in sodium channels have long been linked to inherited epilepsies. Recent clinical findings identified patients with Dravet syndrome that were homozygous for a mutation in SCN1B which encodes the β1 auxiliary subunit of sodium channels. Dravet syndrome is a severe childhood epileptic encephalopathy, and patients commonly present with frequent seizures, developmental regression, ataxia with associated gait abnormalities, and shorter lifespans. We have engineered a mouse model based on the human C121W epilepsy mutation (β1-C121W). Mice homozygous for this C121W mutation displayed similar deficits in health and motor skills to Dravet syndrome. Our experiments showed that β1-C121W homozygous neurons fired more action potentials per current injection, had significantly higher membrane resistance, and were more prone to demonstrate a bursting subtype. These hallmarks of neuronal excitability may contribute to the increased sensitivity to thermal seizures in the homozygous mice. Neuron morphology analysis also revealed that neurons within the subiculum of these animals were significantly smaller in size, consistent with the observed increased input resistance. Application of a new anti-epileptic drug, retigabine, successfully reversed the input resistance in homozygous animals down to wildtype levels, and dampened neuronal excitability. Retigabine injected intraperitoneally into homozygous mice was extremely efficient at reducing thermal seizure susceptibility. These findings highlight the potential utility of applying disease-mechanism based strategies to aid anti-epileptic therapy. In order to examine network excitability in another genetic model of epilepsy, the function of the Nav1.2 sodium channel alpha subunit during development was studied. The NaV1.2 gene has two developmentally regulated splice variants; the ‘neonatal’ and ‘adult’ isoforms. A mutation discovered in patients with benign familial neonatal-infantile epilepsy (BFNIE) increases the excitability of the ‘neonatal’ isoform such that it resembles the adult isoform. Moreover, previous work from the current laboratory using human NaV1.2 expressed in HEK293 cells showed that the ‘neonatal’ form is less excitable than the ‘adult’ form. Based on these data and because the proportion of the neonatal Nav1.2 mRNAs gradually decreases with age during development we hypothesize that the ‘neonatal’ NaV1.2 isoform reduces neuronal excitability in infant brain and therefore plays a protective physiological role. To test this the current laboratory engineered a mouse line which continuously expresses the adult form of Nav1.2 from birth (NaV1.2adult) and investigated seizure susceptibility and neuronal phenotypes. Homozygous NaV1.2adult mice were of normal size and had no obvious seizures under observation during routine video analysis. However, NaV1.2adult mice had increased susceptibility to PTZ-induced seizures, suggesting that the neonatal isoform of NaV1.2 may confer an a novel form of seizure protection. Pyramidal neurons recorded from cortical layers 2/3 of postnatal day 3 (P3) Nav1.2adult neonates show heightened excitability reflected by the presence of a fast-firing neuronal population, which was not seen in the wild-type. At P15, the differences between Nav1.2adult and wildtype at a single neuron level were no longer evident. Interestingly, we also identified an increase in the amplitude of miniature inhibitory post synaptic currents in Nav1.2adult mice compared to the wildtype mice. These results suggest that inherent changes in the neuronal networks occur as a consequence of continuous expression of the adult isoform of NaV1.2 during development. Although further investigation is required to fully understand the biological roles of the two NaV1.2 isoforms, it is predicted that the neonatal isoform of NaV1.2 confers seizure protection in the NaV1.2 mouse model of BFNIE.
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ItemGenetic and environmental modulation of neuronal excitability( 2014)Genetic epilepsy is the cause of many of the epilepsies. How identified epilepsy mutations alter function at the single neuron and whole animal levels is relatively well understood, however how neuronal networks are affected is not. Considering ~30% of epilepsy patients do not sufficiently control their seizures with current anti epileptic drugs, a deeper understanding of how epilepsy mutations change network level activity may enable the development of more efficacious treatment options. Interestingly, recent work has shown that an acidic shift in brain pH induced by respiration of carbogen gas (5% CO2 – 95% O2) is a rapid and extremely effective intervention to halt seizures. Despite various ion channels and receptors being implicated in reducing neuron excitability, we do not fully understand the mechanism(s) by which an acidic pH prevents seizures. Seizures are defined by hyper excitable and hyper synchronous network activity, therefore the anti seizure effect of carbogen gas may occur by a reduction in neuron excitability and/or synchrony. Synchronous network activity is generated in part by gap junction couplings between inhibitory interneurons. While non-mammalian gap junctions are modulated by pH this has never been shown between mammalian neurons by direct electrophysiological recordings. Therefore the aims of this thesis are: I. To investigate the neuronal network dysfunction and anti epileptic drug sensitivity in a mouse model of human genetic epilepsy, II. To investigate pH modulation of neuronal excitability with regard to carbogen gas’ anti seizure effect, and III. To investigate the role of gap junction couplings between inhibitory interneurons and to determine if these mammalian gap junction are modulated by pH. A NaVβ1(C121W) mouse model of human genetic epilepsy developed by this laboratory, has enhanced neuronal excitability and temperature sensitivity attributed to a decreased threshold for action potential firing in the axon initial segment. To investigate the network consequences of this neuronal dysfunction and to establish a genetic disease state model we developed an in vitro assay to investigate CA1 network properties and anti epileptic drug sensitivity. Tetanic stimulation reliably induced local CA1 oscillations that required excitatory and inhibitory synaptic activity and pacemaker currents for their generation. Slices from NaVβ1(C121W) heterozygous mice displayed several hallmarks of increased temperature dependent network excitability. Anti epileptic drugs were more effective in reducing network excitability in slices from NaVβ1(C121W) heterozygous mice and were more effective in suppressing time to thermogenic seizures in NaVβ1(C121W) heterozygous mice compared to WT controls. Hippocampal networks of the NaVβ1(C121W) heterozygous mouse model of genetic epilepsy show enhanced excitability consistent with earlier single neuron studies bridging important scales of brain complexity relevant to seizure genesis. Altered pharmacosenstivity further suggests that genetic epilepsy models may be useful in the development of novel anti epileptics that target disease state pathology. Recent studies have shown that an acidic shift in brain pH induced by application of carbogen gas is a rapid and extremely effective treatment to halt seizures in animal models and in human patients. However, we do not fully understand the mechanism(s) by which an acidic pH induced by carbogen gas halts seizures. Thus the aim of this study was to investigate the biophysical mechanisms underpinning the seizure protective effect of an acidic pH. Using a thermogenic seizure assay, we show that respiration of carbogen gas prevents seizures and that an acidic shift in pH reduces in vitro local CA1 network activity. Furthermore, using whole-cell patch-clamp recordings we show that there is a selective reduction in action potential firing in CA1 pyramidal neurons following an acidic shift in pH. Whereas, inhibitory interneuron action potential firing is not reduced. These data indicate that rather than an enhancement of inhibitory activity, the anti seizure effect of an acidic pH is driven by a reduction in excitatory drive. Gap junction coupled inhibitory interneurons are important for creating synchronous network activity. Recent experimental data and computer modeling predicts that pH modulation of gap junction couplings, potentially via changes in gap junction conductance, can underlie seizures, however this has not been directly demonstrated in a mammalian system. Therefore the aim of these experiments was to investigate pH modulation of gap junction coupled inhibitory interneurons in mammalian brain slices. Simultaneous whole-cell recordings from up to four neurons showed that gap junction connectivity enhances neuron excitability and synchrony. Furthermore, an acidic shift in pH reduced the gap junction conductance of connected neurons and reduced synchronous neuronal activity. Demonstrating that gap junction coupling increases microcircuit synchrony and excitability and that a physiological acidification reduces gap junction conductance that is well positioned to impact network function.
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ItemThe effects of stress on the onset and progression of Huntington's disease in a transgenic mouse model( 2014)Huntington’s disease (HD) is a neurodegenerative disorder largely governed by genetics. The cause of the disease is a fully penetrant gene mutation, inherited by autosomal dominant transmission. The length of this mutation also predicts the age of disease onset, which can range from childhood to late adulthood. Work from our lab on the R6/1 transgenic mouse model of HD was the first to show that environmental factors can alter symptom progression. Environmental enrichment and voluntary wheel running delayed or ameliorated the triad of motor, affective and cognitive dysfunctions in HD mice. Recent clinical studies also suggest that lifestyle factors can affect the age of onset. Currently, there are no treatments to slow or change the course of HD so environmental interventions may offer a feasible approach to extend the symptom-free years in HD gene-positive individuals. There is evidence to suggest that the stress response is abnormal in HD mice and patients. The present study is the first to investigate the impact of stressors on the onset and progression in an animal model of HD. We used an acute (Chapter 3) and two chronic stress paradigms (Chapters 4 and 6) to assess the impact on characteristic symptoms of HD. We also extended the phenotyping of R6/1 HD mice to include behaviours of ethological relevance (Chapter 5). All 3 stress protocols were able modify various functions in R6/1 HD mice, notably accelerating cognitive decline and further impairing olfactory deficits. This work contributes data for sex differences in the HD phenotype and to the general stress literature. Importantly, we show that stress is not only able to modulate specific behaviours in HD mice, but that the gene mutation may confer a susceptibility to the negative effects of stress. Therefore, behavioural management therapy in combination with other lifestyle changes may help manage the course of the disease in gene positive individuals.
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