Florey Department of Neuroscience and Mental Health - Theses
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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.