Medicine (RMH) - Theses
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ItemInvestigation of the synergy between Alzheimer’s Disease and epilepsy through data-driven molecular networks( 2023-03)Overview: In the recent years, a bi-directional association between Alzheimer’s Disease (AD) and epilepsy has been observed, with AD-like cognitive impairments often presenting in epilepsy patients, and high rates of epileptic seizures seen in a sub-set of AD patients. These seizure-prone AD patients reportedly show accelerated cognitive decline and more aggressive disease progression compared to those without seizures. The mechanism and primary mode of action of this association remains unknown, although a synergistic interaction has been proposed. The general aim of this doctoral research was to investigate the electrical and molecular properties of the above-mentioned pathophenotypes and elucidate the mechanisms underlying the potential synergy between AD-like amyloid pathology and epileptiform activity, and their role in accelerated cognitive decline. Introduction and literature review: The introductory sections of Chapter 1 provide an overview of the current literature on epilepsy and Alzheimer’s Disease, focusing on pathophysiological mechanisms commonly implicated in both syndromes. The subsequent sections discuss several benchmark studies that first reported on the increased co-occurrence of seizures among AD patients, followed by a critical review of the most prominent as well as recently emerged hypotheses that aim to provide mechanistic insight into the nature of the proposed bi-directional association between AD and acquired epilepsy. The concluding sections provide a gentle introduction into the emerging field of network medicine, systems- based analysis, interrogation methods of high-throughput biological data and the general framework of computational models and methodology that was implemented throughout this work. Experimental chapters: The first and second experimental chapters aim to characterize the molecular signature of a brain affected by amyloid pathology and seizures. Utilizing proteomic and metabolomic data from two collaborative studies as well as publicly available transcriptomic data, Chapter 2 describes the molecular signature of human AD and that of most widely used mouse models of AD, while Chapter 3 captures the molecular profile of well- established rat models of genetic (GAERS) and acquired (post SE) epilepsies. Informed by the insight gained from Chapters 2 and 3, the third experimental chapter (Chapter 4) aimed to capture the shared molecular signature associated with AD and temporal lobe epilepsy (TLE) – the most common type of epilepsy comorbid with AD. A hypothesis-free, systems-level approach was used to characterize the pathophysiological state of each disease on a molecular level by constructing data-driven gene coexpression networks representing the respective pathologies. The topology and architecture as well as the preservation of functional gene modules between the two networks were compared through network preservation analysis, identifying two clusters of synaptic reorganization and signalling-associated genes as highly preserved between AD and TLE. The fourth and final experimental chapter (Chapter 5) aims to investigate the mechanism and potential mediators of the bi-directional relationship between amyloid pathology and epilepsy by examining the effect of recurrent seizures on hallmark features of AD pathology such as amyloid plaque deposition and cognitive performance. RNA sequencing and bioinformatic analysis of mouse hippocampal tissue was conducted in order to investigate the molecular mechanisms of synergy between recurrent seizures and already- present AD pathology as well as identify key mediators of accelerated disease progression, which could serve as promising targets for intervention. Discussion and conclusions: Informed by computational analysis from chapters 2, 3 and 4, and reinforced by experimental evidence from chapter 5, the final chapter of this thesis (Chapter 6) provides a synthesis of the newly gained insights into the strong synergistic nature of the relationship between amyloid pathology and recurrent seizures. A subsequent extensive review of the most current molecular neuroscience research facilitated interpretation of our results, leading to the proposal of a “dual-pathology” disease model for epilepsy and AD. In this paradigm, the synergistic self-propagating interaction between epileptiform activity and amyloid pathology defines a distinct subpopulation of “dual-pathology” patients, characterized by faster disease progression and more severe cognitive decline. Furthermore, I describe specific cellular pathways mediating the synergy between amyloid pathology and recurrent seizure activity and introduce a mechanistic framework underlying the chain of events through which this synergy leads to accelerated cognitive deterioration. Each step in this framework or chain of events is reinforced by a benchmark proof-of-concept study published in leading peer- reviewed journals and which, with the exception of the most recent 2022-2023 studies, have been independently replicated by other research groups. The concluding sections of this chapter emphasize the utility of integrating phenotypic and electroencephalographic data from in vivo studies with high-throughput “omics” data into network-based computational models for a holistic examination of pathophysiological mechanisms underlying complex diseases and identification of novel therapeutic targets.
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ItemAntiepileptic drug teratogenicity: a human and laboratory translational study( 2018)Antiepileptic drug (AED) associated teratogenicity has been well documented in the literature. The risk of physical birth defects during the first trimester of pregnancy is increased threefold for most AEDs and over ten-fold for the most teratogenic AED, valproate. Despite this risk, women require long term treatment to stop or reduce the occurrence of seizures and the consequent harm to both mother and foetus, including the possibility of sudden unexpected death in epilepsy. The mechanism resulting in this teratogenicity, and in particular why some women are more susceptible to have children with AED induced birth defects is incompletely elucidated. In recent years there has been emerging evidence that AEDs may be interacting with genomic factors to result in birth defects. These genomic factors may be susceptibility alleles in the mother or father, de novo mutations in the child or epigenetic factors such as alterations in DNA methylation in the mother or child. The studies reported in this thesis aim to a) develop an animal model of valproate induced defects that closely mimics a human clinical setting and can be used to better understand the pathogenesis of AED induced defects, and b) identify genomic markers of AED induced defects using whole genome analysis of human samples and determining if having epilepsy is a contributing factor to the onset of these defects. For aim a) the development of the animal model entailed using an epileptic strain of rats, Genetic Absence Epilepsy Rats from Strasbourg, determining a dose at which dietary valproate is therapeutic, mating the rats and conducting a morphological assessment of both internal and external defects. For aim b) human samples were collected and subjected to whole genome analysis, including whole exome sequencing and DNA methylation scans. Additionally, birth defect rates for non-epileptic women in the Australian Pregnancy Register were also separately quantified. The human samples for investigations were collected from participants and their families enrolled in the Register. Using both human and animal models this study aimed to generate new knowledge, which could ultimately lead to a pharmacogenomic approach to the selection of AEDs for women who wish to become pregnant. This would allow women to make more informed decisions, reduce the risk of having a baby with a birth defect and potentially assist in the formation of new AEDs with lower teratogenic risk.
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ItemGenetic and molecular determinants of acquired and genetic epilepsy( 2016)Introduction: Despite the differences in the pathophysiological mechanisms and clinical features of both genetic and acquired epilepsies, dysfunction of both voltage- and ligand-gated ion channels have been highlighted as major contributors of the epileptic phenotype. In this thesis, the molecular mechanism by which T-type Ca2+ channels, AMPAR and TARPs lead to the development of epilepsy was evaluated. In addition, whole genome sequencing was utilised to identify novel candidate genes that could play a role in the epilepsy phenotype. Results: Anti-epileptogenic effects of selective blocking of T-type calcium channels in models of acquired epilepsy. The anti-epileptogenic effects of Z944, a novel, potent and highly selective T-type Ca2+ was used in the amygdala kindling and post-status epilepticus (SE) model. In the amygdala kindling model, Z944 a novel, potent and highly selective T-type Ca2+, did not suppress seizures in fully kindled rats. However, treatment with Z944 delayed de progression of kindling. In the post-SE model, treatment with Z944 after SE massively reduced the number of spontaneous seizures in comparison to vehicle and levetiracetam treated animals. Moreover, treatment with Z944 showed a strong comorbidity modifier effect in depressive like behaviour and may improve cognition after SE. Effects of the T-type calcium channel CaV3.2 R1584P mutation on seizure susceptibility in congenic rats. The CaV3.2 R1584P mutation was not enough to cause absence seizures in a seizure resistant background in the congenic animals but may be associated with the anxiety phenotype. However, the genetic background of the NEC congenic might contain genes that may suppress the pro-epileptic effect of the R1584P mutation. Role of AMPAR and TARPs in the pathogenesis of genetic generalised epilepsy and acquired epilepsy. The results of this chapter indicate a temporal association between the increased TARPs, Stargazin, γ3, γ4, γ5 and γ8, mRNA expression and the development of absence seizures in GAERS. In the post-SE epilepticus model, there was a significant reduction in mRNA expression in the TARPs γ3 and γ8 in the hippocampus 13 weeks after SE. Whole genome sequencing the GAERS and NEC rat strains. TThrough the proposed screen construct variants were identified in high-seizing F2 rats in the following genes; Cacna1h which codes for the CaV3.2 T-type Ca2+ channel, and F1LVI7_RAT and LOC300024. Similarly, non-seizing F2 rats are heterozygous for the mutations in RGD1308133, D3ZPQ1_RAT, Mkl and BiK. In the NEC and F2 non-seizing variants found in Abat, Cyp11b3 and Cyp11b2. Moreover, the flexibility of this method means that it can be applicable with other models of genetic and acquired epilepsies. Conclusions: Here is shown that blocking T-type Ca2+ channels with Z944 has encouraging positive preclinical evidence for disease-modifying in epileptogenesis and in epilepsy behavioural comorbidities that may be possible to translate to a clinical trial. Moreover, the analysis of T-type Ca2+ channels, AMPAR, TARPs and the discovery of novel potential molecular targets using whole genome sequencing uncovers overlap among genetic and acquired epilepsy. Importantly, the findings in this thesis could lead to discoveries that expand our knowledge of epileptogenesis, opening the door for the development of novel therapies.