Anatomy and Neuroscience - Theses
Permanent URI for this collection
Search Results
1 - 5 of 5
-
ItemTAM signalling in CNS demyelination and multiple sclerosis( 2015)Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Involvement of the immune system in the pathogenesis of MS is a key feature of the disease, and an understanding of the mechanisms underlying how immune responses are shaped during CNS demyelination will provide insight into the development of new therapeutic strategies. The TAM (Tyro3, Axl, Mertk) family of receptor tyrosine kinases and their ligands Growth Arrest-Specific 6 (Gas6) and Protein S (ProS) have been shown to modulate many immunological processes important during central demyelination. The major aim of this thesis is to provide further insight into TAM biology in the context of both an animal model of inflammatory demyelination and human MS. By conducting a study examining MS patients and common genetic variations within TAM genes, I identified the MERTK gene as a novel MS susceptibility gene. Examination of plasma from MS patients revealed that levels of the TAM ligand PROS are decreased in MS and that low PROS levels are associated with increased MS disease severity. To interrogate the role of TAM signalling in modulating disease severity during inflammatory demyelination, I used the experimental autoimmune encephalomyelitis (EAE) animal model and observed major changes in TAM gene expression within the CNS and peripheral immune cells during EAE. Examination of Gas6-/- mice during EAE showed that absence of the TAM receptor ligand Gas6 results in both attenuated microglial/macrophage responses and disease severity during the effector phase of EAE. Conditional deletion of Mertk from dendritic cells (DC) resulted in worse disease during the effector phase of EAE. Stratification by sex revealed sexual dimorphism in TAM gene expression and also in the outcome of EAE in both Gas6-/- mice and mice with DC-specific deletion of Mertk. In summary, the data presented in this thesis suggest that the TAM family plays key roles in MS susceptibility and modulating innate immune responses during inflammatory demyelination, providing evidence for members of the TAM family as either markers of disease severity and/or therapeutic targets for the treatment of MS.
-
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.
-
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.
-
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.
-
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.