Anatomy and Neuroscience - Theses
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ItemUsing induced pluripotent stem cell-derived retinal pigment epithelium cells to characterise phenotypic differences associated with reticular pseudodrusen in age-related macular degeneration( 2024-02)Age-Related Macular Degeneration (AMD) is one of the leading causes of severe vision loss in individuals aged over fifty in Western populations. A hallmark of AMD pathogenesis is the accumulation of lipid and protein deposits, termed drusen, in the macula. Past studies have established that retinal pigment epithelium (RPE) dysfunction alone initiates drusen-like deposit formation, with disease lines exhibiting greater volume and number with respect to deposit formation in vitro. Despite this, automated quantification methods for these deposits were lacking in the literature. While conventional drusen on the basal side of the RPE is a hallmark of AMD pathogenesis, the recognition of reticular pseudodrusen (RPD) on the apical side indicates a distinct AMD phenotype. This thesis focuses on the modelling of RPD using human induced pluripotent stem cells (hiPSCs) as part of this extensive investigation into molecular distinctions between conventional AMD and RPD. Initial identity confirmation of hiPSC-derived RPE cells involved rigorous characterisation and functional assays to establish baseline phenotypic presentations. Subsequent investigations delved into pathological AMD signatures, initially confirming the appearance of hallmark drusen deposits in culture. Recognising the time-intensive and manual nature of existing quantification methods, significant efforts were made to develop and publish two semi-automated quantification tools, fostering standardised approaches in the scientific community. These tools facilitated stressor experiments with N-Retinylidene-N-Retinylethanolamine (A2E), revealing its potential exacerbation of AMD phenotypes in vitro. A large-scale proteomics study aimed to identify differentially expressed proteins between AMD and RPD samples uncovered distinctions in extracellular matrix proteins, the spliceosome complex, and cellular homeostasis, including mitochondrial health. Validation of mitochondrial hits involved assays comparing baseline oxidative states. Utilising the developed pipelines for quantifying drusen-like deposits at baseline and post-stressor treatment, and investigating baseline oxidative stress in RPE cultures derived from patient-specific iPSCs revealed differences between AMD cohorts with and without RPD. This study underscores the utility of patient-specific iPSCs for in vitro modelling of AMD pathogenesis, elucidating variations in phenotypic presentations within stratified AMD disease cohorts. The data suggests discernible differences in disease profiles concerning mitochondrial dysfunction and drusen accumulation between AMD samples with or without RPD, emphasising the potential of iPSCs in unravelling the complexities of AMD.
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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.