Florey Department of Neuroscience and Mental Health - Theses
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ItemInvestigating mechanisms of mutant huntingtin toxicity by spatially mapping lipid metabolites in a mouse model of Huntington’s disease( 2022)Huntington’s disease (HD) features intraneuronal inclusion body formation by polyglutamine-containing fragments of the mutant huntingtin protein in many brain regions, including the hippocampus, neocortex and striatum. To better understand the molecular changes associated with inclusion body formation and associated pathogenesis, we examined the abundances and turnover rates of membrane lipids in the hippocampus, a region of pronounced inclusion formation associated with cognitive deficits, in a transgenic mouse model of HD (R6/1 line) using deuterium labelling in vivo. The R6/1 HD mice lacked inclusions in the hippocampus at six weeks of age, whereas inclusions were extensive by 16 weeks. We assessed one brain hemisphere collected at three timepoints (6, 12 and 16 weeks) by MALDI-mass spectrometry imaging (MALDI-MSI) and the other hemisphere for liquid-chromatography mass spectrometry (LC-MS) analysis. Hippocampal sub-fields (CA1, CA3 and DG) dense with inclusions showed a reduction in the relative abundance of neuronal-enriched lipids with roles in neurotransmission, synaptic plasticity, neurogenesis and ER-stress protection. Conversely, lipids in the phosphatidylinositol, phosphatidic acid and ganglioside class were increased in lipid synthesis in HD mice, relative to WT mice across all the age groups examined. The changes were also detectable in the HD mice at six weeks of age, indicating they arose prior to the formation of the inclusion bodies and disease symptoms. Since elevated synthesis of lipids in the PI, PA and ganglioside classes is a known adaptive response to Endoplasmic reticulum (ER) stress, our findings suggest this molecular mechanism serves as an early-stage adaptive response to ER stress in pre-symptomatic HD mice and may be targetable therapeutically. Additionally, our study has identified progressive changes in neuronal lipid abundances in the pre-symptomatic and symptomatic stages of HD that closely correlate with known hippocampal-dependent cognitive changes in HD, thus providing early lipid biomarkers that may be targeted therapeutically to slow down HD progression. Most importantly, we have spatially monitored disturbances in lipid metabolism at the primary site of inclusions in HD hippocampi, thus illuminating new insight into the cascade of molecular events in brain regions spanning the development of inclusions in HD mice. These findings required the development of a novel in-house bioinformatics software (KineticMSI), which is made available as an R package and will have broad neuroimaging applications.
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ItemInvestigating the potential role of the tau protein and metal ions in repetitive mild traumatic brain injury( 2022)Repetitive mild traumatic brain injury (r-mTBI) can lead to the development of chronic traumatic encephalopathy (CTE), a chronic and progressive neurodegenerative disorder characterised by cognitive impairment and hyperphosphorylated tau protein pathology (a neuronal microtubule-associated protein). The secondary events of a single TBI include increased tau hyperphosphorylation and metal dyshomeostasis which have been reported acutely following injury. These pathological processes are key players in the development of several neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, the development of tau pathology in animal models of r-mTBI is currently ambiguous, particularly in the chronic phase post-injury, bringing to question the relevance of the tau protein in driving neuropathology. Moreover, given that alterations in metal ions (such as iron, zinc and copper) contribute to behavioural impairments and are a prominent finding in several neurodegenerative disorders and following a single TBI, then the lack of metal ion research in the context of r-mTBI represents a surprising yet critical gap in the literature. Therefore, this project investigated the potential role of the tau protein and metal ions in a mouse model of r-mTBI. To explore this, we used the controlled cortical impact (CCI) model of TBI and assessed young-adult and aged mice at a sub-acute (1-month) time point, as well as young-adult mice at a chronic (12-month) time point following either single or r-mTBI. Firstly, we aimed to investigate the tau protein in young-adult mice sub-acutely following single or r-mTBI. Secondly, we aimed to characterise metal ions in young-adult mice sub-acutely following single or r-mTBI. Thirdly, we aimed to investigate the tau protein in aged mice sub-acutely following single or r-mTBI. Our fourth and final aim was to characterise the tau protein and metal ions in young-adult mice chronically following single or r-mTBI. We hypothesised that r-mTBI would lead to tau hyperphosphorylation and metal ion dyshomeostasis, initiating a neurodegenerative cascade of events sub-acutely which would be exacerbated chronically post-injury. We performed a battery of behavioural tests, immunohistochemistry and stereology, biochemical analyses via western blotting, metal analyses using three different analytical techniques such as laser-ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS), size exclusion chromatography-ICP-MS (SEC-ICP-MS) and bulk ICP-MS, and finally differential gene expression analysis via RNA sequencing. Interestingly, the sub-acute period following r-mTBI in young-adult mice was characterised by a lack of overt tau pathology (despite trends towards increased phosphorylated tau at Ser396) but prominent changes in tau-regulatory proteins (such as decreased pGSK3-beta and Pin1 levels) and an increase in amyloid precursor protein (APP), indicative of axonal injury. Tau-regulatory protein changes occur upstream of tau phosphorylation, therefore the changes in tau-regulatory proteins observed herein suggested that tau pathology may require a longer time frame to develop following r-mTBI. The sub-acute period was also characterised by significant neuronal loss via stereology, and significant neurological impairment, gait deficits and anxiety-like behaviour, although no cognitive deficits, as assessed via behavioural testing. Importantly, we demonstrate for the first time that the sub-acute period following r-mTBI in mice is characterised by altered metal ion homeostasis (specifically iron, zinc and copper) using LA-ICP-MS. LA-ICP-MS analysis revealed increased iron levels with a concomitant increase in the iron storage protein ferritin (assessed via western blot) in the contralateral hemisphere. Although there were no changes in zinc levels in either hemisphere, by far the most prominent changes were observed for copper, with significantly increased copper levels in both hemispheres of the brain following r-mTBI. At 1-month post-injury the profile of metal-bound protein complexes assessed via SEC-ICP-MS revealed a significant decrease in iron-bound protein complexes associated with peak 4 (an uncharacterised protein peak) following a single injury but no changes following r-mTBI. Zinc-protein complexes were significantly decreased following r-mTBI (peak 5, uncharacterised protein peak) and significantly increased following a single injury (peak 6, uncharacterised protein peak). There were no changes in copper-protein complexes in either injury group. In aged mice (>18 months old), there was no overt tau pathology or changes in tau-regulatory proteins. While the aged mice with r-mTBI displayed a hyperactivity phenotype, gait deficits and anhedonia (a feature of depression), there were no cognitive impairments. Anhedonia was seen in both 5x Sham and 5x TBI groups, but not in 1x Sham or 1x TBI groups, suggesting a possible effect of repeated anaesthesia on the 5x Sham group. In these mice, we note the possibility that old age may have masked a true TBI effect (if any), and we also consider the possible negative effects of repeated anaesthesia on these pathological outcomes. The chronic period following r-mTBI in young-adult mice revealed a hyperactivity phenotype, gait deficits, mild anhedonia and mild learning and memory deficits without short-term memory impairment or anxiety-like behaviour. Despite the presence of mild cognitive impairment, there was no overt tau pathology or changes in tau-regulatory proteins at 12-months following r-mTBI. We also conducted an RNA sequencing experiment at 6-months following r-mTBI only (5x Sham and 5x TBI mice) to assess whether r-mTBI would lead to the differential expression of genes associated with neurodegenerative processes chronically post-injury. Surprisingly, we found no differentially expressed genes as a function of r-mTBI but pronounced sex differences, suggesting the possibility that the sex variable may have masked an injury effect, if any, and that this data may need to be re-analysed separately for male and female mice. However, metal analysis using bulk ICP-MS revealed a significant increase in iron, zinc and copper in the contralateral parietal cortex (the area opposite to the impact site), demonstrating for the first time that the chronic (12-month) period following r-mTBI results in significant changes in metal levels in an area distal to the impact site, representing the widespread nature of the injury. Moreover, we employed western blot analysis of iron-regulatory proteins to further interrogate iron metabolism in the brains of r-mTBI mice. While there were no changes in any iron-regulatory proteins in the contralateral hemisphere (where there was increased iron levels), there were significant changes in these proteins in the ipsilateral hemisphere (where there were no changes in iron levels). The regulatory protein changes observed in the ipsilateral parietal cortex were indicative of the molecular signatures of iron overload. Indeed, there was a reduction in iron transport into the cell (transferrin receptor), an increase in cytosolic iron import (via divalent metal transporter-1), an increase in intracellular iron storage in ferritin and a slight trend towards reduced iron export (via the ferroportin exporter) following five impacts. Based on these data, we suggest that iron levels may have been increased in the ipsilateral hemisphere prior to the 12-month time point post-injury, and the observed changes in iron-regulatory proteins may indicate the ability of this hemisphere to respond to changes in iron content and adequately restore iron metabolism. Because no changes in iron-regulatory proteins were observed in the contralateral hemisphere, despite a significant increase in iron content, then this may suggest that either these proteins may be altered beyond the 12-month time point studied herein, or this may demonstrate a failure of the iron-regulatory proteins in restoring iron homeostasis. Alternative rationales for these interesting observations exist and thus warrant further analysis in the future. In conclusion, the work conducted in this thesis has uncovered a potential novel secondary injury mechanism following r-mTBI in both the sub-acute and chronic stages post-injury. To the best of our knowledge, we are the first to extensively characterise metal ion changes in a mouse model of r-mTBI. This work has great implications for the future of the r-mTBI field, considering that there are currently no approved treatments for either single or r-mTBI. This work highlights the potential benefits of using metal targeting compounds such as metal chelating and chaperone agents, which have already yielded favorable outcomes in other neurodegenerative diseases such as AD and PD, as well as in single TBI animal studies. Future experiments should focus on investigating tau pathology using techniques other than western blots, as bulk digestion of proteins does not allow for more sophisticated morphological and microscopic examination of proteins, which may yield more insightful results. Moreover, future studies should conduct more in-depth behavioural testing to further examine anxiety and depressive-like behaviours as well as cognitive deficits. Lastly, it is also possible that the young-adult mice studied herein were not aged long enough to develop significant tau pathology. Indeed, metal ions are known to bind to and interact with tau, therefore given the extensive metal dyshomeostasis we report following r-mTBI, then it is possible that tau pathology may require a longer time frame to develop. Nonetheless, these novel findings provide a greater understanding of the pathological mechanisms underlying r-mTBI, and contribute crucial knowledge to the neurotrauma field.
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ItemNo Preview AvailableGene Regulatory Networks and MicroRNAs in Microglia( 2022)Microglia are important regulators of central nervous system (CNS) development, homeostasis and immunity. Microglia exhibit a significant level of cellular plasticity which is facilitated by a dynamic transcriptional landscape that is modulated by genetic and environmental factors. However, our understanding of these mechanisms and the complexity of microglial transcription remains incomplete. Dysregulation of microglial gene expression and cellular function is implicated in numerous neurodevelopmental and neurodegenerative diseases, including the autoimmune demyelinating disease multiple sclerosis (MS). Therefore, understanding the transcriptional control of microglia provides invaluable knowledge of these cells and their unique roles in the CNS. The central aim of this thesis was to further characterise microglial transcription by investigating two genetic mechanisms that are strongly implicated in microglial biology. The first genetic mechanism that was investigated was the influence of small non-coding miRNAs on microglial gene expression. Here, I have comprehensively sequenced and characterised miRNA expression in microglia derived from a developmental mouse cohort and human resected brain tissue, identifying distinct subsets of known and novel miRNAs that regulate key microglial processes. Integrated miRNA-mRNA expression network analysis revealed miRNAs involved in microglial developmental processes including neurogenesis, myelination, endocytosis as well as key immune signalling pathways. Evidence from both species indicated a significant influence of age, but not sex on miRNA expression in these cells. The second genetic mechanism that was investigated was the transcriptional regulation of MERTK. MERTK has been shown to regulate microglial/myeloid cell-mediated apoptotic clearance and the resolution of inflammation in the CNS. More specifically, genetic variation in MERTK is associated with MS susceptibility. In this study, I investigated the single nucleotide polymorphism (SNP), rs7422195, which is associated with both changing MERTK expression in CD14+ve monocytes and modifying MS risk, hypothesising that this SNP is a causal disease modifying variant. While in vitro evidence suggested that variation at this genomic site drives differential protein binding, I was not able to experimentally validate specific protein interactions occurring at this region in vivo in primary monocytes. Furthermore, to increase our understanding of the transcriptional regulation of MERTK, I characterised the promoter region of MERTK gene, identifying distinct regulatory elements that may be responsible for driving gene expression in microglial/myeloid cells. Overall, this thesis has contributed to our knowledge of two separate, but important mechanisms that regulate microglial phenotype with important consequences for their function in the healthy and diseased CNS.
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ItemAndrogen receptor dysregulation in Amyotrophic Lateral Sclerosis( 2021)Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder, resulting in the death of motor neurons. Sexual dimorphism is clear in ALS patients and mouse models. Capable of modifying disease onset and progression, sex differences can also manifest in the responsiveness and outcomes to drug therapies and interventions. Understanding the biological components contributing to the clinical heterogeneity in ALS is of great interest. The androgen receptor (AR), a nuclear steroid hormone receptor which mediates the biological actions of androgens, has strong links to motor neuron function and survival. Androgens are neuroprotective to cultured motor neurons and in response to nerve injury in vivo. An expansion mutation in the AR gene gives rise to lower motor neuron degeneration in the disorder known as spinal and bulbar muscular atrophy (SBMA). This thesis explores a potential role for AR in mediating the sex-differences in ALS using the transgenic SOD1G93A mouse model of ALS. Firstly, changes to the level and cellular localisation of AR, estrogen receptor alpha (ERa) and beta (ERb), and progesterone receptor (PR), were analysed within the lumbar spinal cord of male and female SOD1G93A mice relative to wildtype control mice, over the disease course. A robust decrease in spinal cord AR level occurred in symptomatic male SOD1G93A mice. This occurred in parallel with decreases in transcript levels of androgen metabolising enzyme, 5a-reductase type II. AR level in females was half that of males, whereas levels and localisation of ER and PR were comparable between sexes. ERa showed a robust localisation to astrocytic processes in symptomatic SOD1G93A mice; ERb was upregulated, possibly contributing to multiple glial cell responses. Secondly, a closer inspection of AR expression within the various motor neurons (MN) throughout the central nervous system (CNS) neuraxis was conducted. It was strikingly apparent that by endstage disease, the majority of MNs remaining in SOD1G93A male mice had reduced nuclear AR. A clear correlation between AR expression and vulnerability in ALS was not immediately evident, although unique MN clusters with high AR content were all preserved in endstage disease. In the vulnerable lumbar spinal cord MNs, AR levels were reduced in presymptomatic stages of disease. This was paralleled by a downregulation in AR within target skeletal muscle. The consequences of these perturbations to AR level remains unclear, although their early disruption could suggest their contribution to disease pathogenesis. Finally, this thesis explored the impact of disrupted AR signalling and expression levels, to SOD1G93A disease course. Firstly, chronic administration of AR antagonist, flutamide, worsened muscle pathology and exacerbated onset without affecting neurodegeneration and survival. Secondly, the Cre/LoxP system was used to conditionally delete AR from neuronal and glial cells of SOD1G93A male mice using the Nestin-Cre transgenic. Central AR deletion was not observed to have an impact on disease course, however, the metabolic phenotype of the Nestin-Cre mouse had a noticeable, and confounding, effect the on SOD1G93A lifespan. Thirdly, global overexpression of AR in SOD1G93A mice, showed subtle effects on disease onset, although did not have an appreciable impact on ALS disease course. In summary, the work of this thesis provides a robust characterisation of sex steroid hormone receptors in the SOD1G93A mouse model of ALS. Evidence of reduced AR level in MNs through disrupted local androgen metabolism may present a target for future intervention studies. Peripheral AR was identified as a modulator of disease onset, through chronic AR antagonism. Genetic manipulation studies did not identify AR as a modifier of disease outcome in the SOD1G93A.