Florey Department of Neuroscience and Mental Health - Theses
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Novel molecular tools for cellular- and subcellular-targeting of neuronal transgene expression
Among the questions addressed by modern neuroscience is how distinct cellular structures within the nervous system give rise to particular behaviours and cognitive functions. Our ability to address such questions has been greatly enhanced with the emergence of ‘transgenic’ technologies, which allow the introduction of foreign or engineered genes into biological systems of interest, such as neural circuits. This capacity to introduce and express transgenes in neurons has led to the development of a diverse catalogue of genetically encoded markers, sensors, and actuators, which provide powerful insights into the structure and function of the nervous system. In addition to their intrinsic utility, mechanisms exist by which expression of these transgenic tools can be selectively directed to cell-types and subcellular structures of interest. Studies described in this thesis sought to establish new molecular tools and approaches for localised transgene expression in neural structures of scientific and therapeutic importance. Transgene delivery to neural circuits of interest was mediated here in rats using viral vectors, particularly adeno-associated viral (AAV) vectors, from which targeted transduction of neurons expressing the highly conserved neuropeptide, relaxin-3, was achieved using a novel cell-type specific promoter. Relaxin-3 is most abundantly expressed by a subpopulation of neurons in the pontine nucleus incertus (NI) and has been identified as an important modulator of arousal and other interrelated cognitive functions and behaviours, making the relaxin-3 system a promising therapeutic target. To better understand this circuitry using transgenic approaches, I sought to identify a promoter sequence capable of regulating cell-type specific transgene expression in relaxin-3 neurons. In parallel to a relaxin-3 promoter sequence (1,736 bp), I also characterised transgene expression under an 880 bp tropomyosin receptor kinase A (TrkA) promoter, as TrkA is exclusively co-expressed with relaxin-3 in rat NI neurons. Eight weeks following stereotaxic injection of an AAV vector, expressing mCherry under the TrkA promoter, to rat NI, widespread non-specific transduction was observed. However, rats receiving injections of an equivalent AAV vector, where transgene expression was regulated by the relaxin-3 promoter, displayed almost exclusive mCherry production in relaxin-3-expressing neurons, demonstrating targeted, cell-type specific transgene delivery. Localisation of transgene expression at the subcellular level is also an important consideration. The need for rapid and efficient electrochemical communication between neurons has led these cells to evolve highly complex morphologies, with subcellular compartments each having distinct structural, biochemical, and functional properties. These differences can impact transgene function, as seen with the chloride-conducting opsin, GtACR2, where differing subcellular ion gradients cause GtACR2 to have hyper- and depolarising effects in either the somatodendritic or axonal membrane, respectively. To resolve these paradoxical effects, whilst simultaneously providing a mechanism for the functional dissection of closely interconnected circuitry, I developed fusion constructs between GtACR2 and trafficking motifs derived from either neurexin 1a (GtACR2nrxn) or the potassium channel, Kv2.1 (GtACR2Kv2.1), which endogenously localise to the axonal or somatic membrane, respectively. The preBotzinger complex (preBotC), a key driver of inspiration and respiratory rhythm, with close but poorly understood functional connections to neighbouring cardiovascular nuclei in the medulla, was selected as an ideal system for characterisation of these GtACR2 constructs. AAV vectors expressing GtACR2Kv2.1, GtACR2nrxn, or a control GtACR2-EYFP construct, under constitutively active promoters, were injected into the rat preBotC. Fusion of GtACR2 with the Kv2.1 motif was associated with enriched neuronal expression of this construct, but did not eliminate axonal expression, while subcellular localisation of GtACR2nrxn could not be determined histologically. However, a selective dissociation of respiratory and cardiac responses to optical manipulation, both of which were seen in rats expressing GtACR2-EYFP, was observed in rats expressing GtACR2Kv2.1 or GtACR2nrxn, respectively, supporting their differential trafficking in vivo. To further clarify these results and optimise the utility of GtACR2 fusion constructs in future experiments, a capacity for cell-type specific expression was required. I therefore developed an additional AAV vector for recombinase-dependent GtACR2Kv2.1 expression in neurons. In characterising expression from this construct, I first validated a novel approach for cell-type specific transduction of an amygdaloid efferent population (CeA) projecting to the nucleus of the solitary tract (NST) in rats (referred to as CeA>NST neurons), using locally injected AAV vectors in the amygdala and a retrogradely transported, Cre-recombinase expressing canine adenovirus (CAV) vector delivered into the rostral NST. The CeA>NST pathway was chosen as a model system, given its empirically supported but poorly understood role in modulating autonomic function in response to emotional stimuli, which would benefit from cell-type specific transgenic investigations, and its long-range axonal projections, which provide a further opportunity to assess GtACR2Kv2.1 trafficking in vivo. While cell-type specific GtACR2Kv2.1 expression in CeA>NST circuitry was achieved, some axonal expression of GtACR2Kv2.1 was again present in these neurons. Overall, studies described in this thesis provide valuable contributions to the available toolset for targeted neuronal transgene expression. Changes in GtACR2 trafficking observed, following fusion with the Kv2.1 motif, are supported by independent publications which appeared in the literature concurrently with my studies, where a somatic enrichment, and significant, but not complete, reduction in axonal expression, was demonstrated for analogous GtACR2-Kv2.1 fusion constructs. As such, although effects arising from residual axonal expression must be considered and minimised, GtACR2Kv2.1 provides an improved and potent optogenetic construct for neuronal inhibition. Further studies may also establish GtACR2nrxn as a robust tool for manipulation of the axonal membrane. These, as well as a variety of other established and emerging transgenic actuators, sensors, and markers, can be selectively expressed in relaxin-3 NI or CeA>NST circuitry, among other precisely defined networks, using the strategies and resources developed here. In doing so, powerful insights into a range of neural systems of both scientific and therapeutic importance can be gained through future research.
Advancing the detection of short tandem repeats in health and disease
Short tandem repeats (STRs) are repetitive DNA sequences composed of repeat units with 2–6 base pairs in length. STRs make up around 3% of the human genome and have higher mutation rate than single nucleotide variants. Mutations in STRs loci have been linked to around 60 genetic disorders known today, the majority of them being neurological. Genotyping STRs has been difficult due to technical limitations but advances in DNA sequencing methods and the emergence of bioinformatic analysis tools have led to the discoveries of many new disease-causing loci. In this thesis, we focused on exploring different methods of STR analysis, their performance on high-throughput sequencing data and improving these existing methods. At first, we compared four genome-wide STR genotyping tools and determined their performance on whole exome sequencing data. We found that all tools have their merits and there is no clear winner. Difference between tools were observed when genotyping some repeat types with some tools having significantly higher error rates on two base-pair repeats. Next, we characterised STRs in whole genome sequencing data to determine genotyping accuracy of different types of repeats in various genomic settings and library preparation methods. We saw differences in accuracy between repeat unit sizes, repeat lengths and repeat composition. This demonstrates the need to use customised filtering strategies for different repeats that would improve quality of calls while retaining as much data as possible, which could be helpful detecting de novo mutations. Finally, we focused on known disease-causing STRs to improve and extend the current method of analysis. A comprehensive list of disease-causing STR loci was collated based on the literature. This was used to create a variant catalogue for a robust genotyping tool ExpansionHunter allowing genotyping of more than twice as many loci than it currently allows. In addition, it is now possible to genotype alleles longer than the fragment length in over forty loci. This catalogue was validated with an extensive series of simulations. We have used this updated catalogue on our sequencing dataset of individuals with rare disease to detect expansions in any of these loci. Moreover, we developed our own computational tool called STRipy, which has been made publicly available, to greatly simplify STR genotyping in sequencing data and enable researchers to genotype the highest number of known pathogenic STR loci compared to other existing methods.
Towards a biobehavioral understanding of methamphetamine use disorder: Investigating psychiatric, cognitive, and genetic factors
Methamphetamine use is a major health concern globally, with ever-expanding market and an increasing number of users worldwide. In Australia, the number of people with methamphetamine use disorder has increased over the past 10 years, specifically amongst adolescents and young adults. This is a particular concern, as the age of onset of substance use predicts the severity of substance use disorder later in life. In addition, young people are more resistant to treatment. While it is still poorly understood why methamphetamine dependence is increasing in adolescents, literature suggests that methamphetamine use is associated with other psychiatric disorders, deficits in cognition, and certain genes involved in the neurocircuitry of addiction. Therefore, the aim of this thesis was to explore psychiatric, cognitive, and genetic factors associated with early onset of methamphetamine use to gain a holistic understanding of methamphetamine use disorder. To investigate these factors, I conducted a cross-sectional two-group study, recruiting people with a current diagnosis of stimulant use disorder, methamphetamine-type, and controls with no history of substance use disorder. All participants were administered a clinical interview to collect demographic and drug use characteristics. Psychiatric comorbidities and psychotic symptoms were also assessed. Following the interview, participants completed a cognitive task battery assessing attention, speed of processing, cognitive flexibility, working memory, and inhibitory control. Inhibitory control was also assessed using a cue reactivity task that I specifically developed for this project. It consisted of the pseudorandomized presentation of methamphetamine-related cues counterbalanced with control, food-related cues. Upon completion of the study session, whole blood was collected for single nucleotide polymorphism analysis in genes of interest. Genes were selected based on robust preclinical data and results from the meta-analysis presented in this thesis. Poor inhibitory control was identified as an age-dependent risk factor in this thesis, with an earlier age of onset associated with greater deficits. In addition, poor inhibition was associated with an increase in craving upon exposure to methamphetamine-related cues. This is critical as cue-induced craving may lead to relapse after abstinence, and therefore poor treatment outcome. Results from this thesis therefore suggest that pre-existing reduced inhibitory control in adolescence is a risk factor for developing methamphetamine use disorder when methamphetamine is first taken early in life, potentially by perpetuating methamphetamine use and inducing repeated relapses. This thesis also identified comorbid antisocial personality disorder as a strong predictor for age of onset of methamphetamine use. This highlights the need to treat cooccurring mental disorders in young people who use drugs to prevent them from transitioning into problematic use. Lastly, a mutation in the neuregulin-1 gene was associated with early onset methamphetamine use, suggesting that people carrying the gene variant are more likely to develop methamphetamine use disorder when exposed to methamphetamine earlier in life. Taken together, this thesis identified a range of psychiatric, cognitive, and genetic factors associated with early onset methamphetamine use. Findings from this work will contribute to the development of larger studies and clinical trials investigating new early interventions to prevent young people who casually use methamphetamine transitioning into a formal methamphetamine use disorder, thus alleviating the rising burden of disease
Protein engineering of arginine vasopressin receptor V1A for structural biology
Peptide hormone arginine vasopressin (AVP) and its cognate G protein-coupled receptor (GPCR), V1A, belong to the vasopressin-oxytocin signalling system – a highly complex and widespread endocrine system in the human body. AVP activation of V1A causes a stimulatory contractile effect on vascular and uterine smooth muscle, while, in the brain, AVP and V1A play an important role in anxiety, aggression and social behaviours. Due to these physiological mechanisms, AVP and V1A have been of therapeutic interest for decades; initially for the treatment of peripheral conditions such as heart failure and menstrual cramps, while recently, V1A antagonists have shown promise for treating aspects of autism spectrum disorder (ASD). Despite this longstanding interest, there are currently no approved drugs selectively targeting V1A. Subtype-selectivity is important among the vasopressin-oxytocin receptor family, as these receptors share a high degree of sequence similarity and structural homology, yet have differing, and sometimes opposing, effects. Furthermore, as there are currently no experimental 3D structures of V1A, there is a lack of understanding on exactly how AVP binds to V1A at the molecular level, which limits the design and optimisation of new, V1A-selective drugs. GPCR structural biology is a rapidly developing field; however, the low-expression level and instability of many GPCRs, including V1A, can be problematic for purification and subsequent structural study. This thesis aimed to apply various protein engineering techniques to V1A, in order to facilitate the purification and subsequent biochemical study of this important, but long unfulfilled, potential therapeutic target. Chapter 2 covers the first GPCR-engineering venture, which was to introduce expression and stability augmenting missense mutations to V1A via a well-characterised method – directed evolution. This method, which uses E. coli display of receptor mutants, has successfully produced high-expressing, stabilised receptor mutants for the structural study of other neuropeptide receptors, including NTS1 and NK1. However, upon application of this method to V1A, stabilised V1A mutants were not produced. Instead, a V1A truncate was selected, which contained a small section of the V1A N terminus. The reason this protein was selected was unclear – but can primarily be attributed to the issues that arise when using E. coli for GPCR expression. It was evident that further engineering of V1A would require the use a different cell type, such as insect or mammalian cells. Chapter 3 explores the development of a mammalian cell-based directed evolution method. This new method was developed to overcome the expression issues encountered using E. coli, and required optimisation and innovation to accommodate the new cell type, including the use of lentivirus as the gene-delivery method. The method was developed, optimised and applied to V1A – producing V1A mutants that exhibited improved expression levels compared to wild-type V1A. Furthermore, several mutations were identified that appeared in multiple V1A mutants, indicating that they were contributing to this expression-boosting effect. Chapter 4 covers the purification of V1A using a combination of protein engineering approaches – including the introduction of the expression-boosting point mutations discovered in Chapter 3. Ultimately, functional V1A was able to be purified with the aid of these mutations, which provided a substantial increase in functional yield of purified receptor. The two main outcomes of this thesis are: the development of a mammalian cell-based directed evolution method that improves the functional yield of purified receptor – and the successful purification of functional V1A. Firstly, the new, mammalian cell-based selection method presents as a generic, rapid and viable means of improving the heterologous expression of low-expressing GPCRs – and potentially other complex membrane proteins with expression issues. Secondly, the substantial increase the yield of purified, functional V1A is a significant breakthrough in the biochemical study of V1A, as it enables the application of structural biology techniques, such as cryo-electron microscopy (cryo-EM), as well as ligand screening methods for new, V1A-selective ligands.
Early life stress and the extinction of conditioned fear in the developing rat
Early life stress is a known antecedent to anxiety- and fear-related disorders. It may disrupt the ability to regulate fear and act to trigger the development of an anxiety- or fear-related disorder. Hence, in this thesis, I examined the link between early life stress and fear regulation across development. To do this, I used Pavlovian fear extinction as a model of fear regulation to assess the effects of infancy and peri-adolescent stress in rats. In Chapter 2, I examined whether a chronic infancy stressor altered fear extinction in juvenile rats. This series of experiments utilized the limited bedding and nesting model of chronic infancy stress. I found that chronic infancy stress resulted in the precocious emergence of relapse-prone fear in male juvenile rats. However, chronic infancy stress had little effect on extinction behavior in female juvenile rats, as they exhibited relapse-prone fear regardless of stress condition. In Chapter 3, I investigated whether chronic peri-adolescent stress altered fear extinction in adolescence and adulthood. This series of experiments utilized social isolation as a model of chronic peri-adolescent stress. Peri-adolescent stress had a sex-specific effect on fear extinction. In males, peri-adolescent stress resulted in impairments in extinction recall in adolescent and adult male rats. However, in females, peri-adolescent stress had no effect on fear extinction behavior. In Chapter 4, I explored the interplay between peri-adolescent stress and physical activity on fear extinction in adolescence. Peri-adolescent stress, as modelled by social isolation, impaired extinction recall in male adolescents, but this effect was prevented by increased physical activity. Extinction recall in female adolescents was again unaffected by peri-adolescent stress. Surprisingly, increased physical activity was disruptive to extinction recall in peri-adolescent stressed females. Pharmacological suppression of cellular proliferation in peri-adolescent stressed adolescents blocked the effect of physical activity on extinction recall in both sexes. This suggests that peri-adolescent born cells mediate the interplay between peri-adolescent stress and physical activity effects on extinction behavior. Together, these findings highlight sex-specific outcomes of peri-adolescent stress and physical activity on adolescent brain and behavior. In the final Chapter, I propose the Variable Speed Stress model to interpret the effects of stress in infancy and peri-adolescence. Further, I outline potential reasons for the sex effects and possible avenues for future research. Overall, the findings in this thesis contribute to our understanding of how early life stress affects fear extinction throughout development. It suggests that after early life adversity, stress-induced changes to extinction learning development in males may contribute to a reduction in treatment efficacy for exposure-based anxiety disorder therapies.
Investigating the role of Tyro3 in the central nervous system
One of the more remarkable aspects of myelination is the relationship between axon diameter and the correlating myelin thickness, whereby larger axons are ensheathed with more myelin. However, key regulators of myelin thickness in the CNS remain elusive. Previously, it was shown that that the tyrosine kinase receptor, Tyro3, is a key mechanistic component involved in controlling myelin thickness in the optic nerves of adult mice. Moreover, provision of the Tyro3 ligand, Gas6, dramatically increases clinical outcomes in a demyelinating mouse model. Tyro3 ligand provision also increased the corresponding total amount of myelin produced by myelinating oligodendrocytes in vitro. In addition, the promyelinating effects of Gas6 in vitro were lost when oligodendrocytes were devoid of Tyro3, suggesting the myelinating effect of Gas6 is up oligodendrocytes and is mediated by Tyro3. This thesis aims to further investigate the myelin regulatory role of Tyro3, firstly by assessing myelin thickness and oligodendrocyte maturation in a separate white matter tract in adult mice, the corpus callosum. Furthermore, this study aims to investigate the role of Tyro3 in demyelination and remyelination using the cuprizone induced, demyelinating mouse model. In addition, this study looks to identify the intracellular pathways in which Tyro3 and Gas6 regulate myelination in mouse oligodendrocytes. Finally, this study aims to understand how a nervous system devoid of Tyro3 functions by investigating action potential conduction in visual evoked potentials and compound action potentials. This research project shows that Tyro3 is a critical regulator of adult mouse myelin thickness, and that its expression is protective during demyelination and acts as a positive regulator of remyelination in the corpus callosum. Importantly, hypomyelination in the absence of Tyro3 occurred without alterations to oligodendrocyte maturation or mature oligodendrocyte density during the myelin establishment processes. In addition, although demyelination was more severe in the Tyro3 KO mice, microglial activation was normal. However, I was unable to identify an intracellular pathway in which Gas6 and Tyro3 may regulate myelin synthesis in mouse oligodendrocytes. Furthermore, the previously reported, promyelinating effects of Gas6 was not recapitulated when oligodendrocytes were grown on synthetic nanofibers rather than live neurons. These results suggest that the promyelinating effects of Gas6 could potentially be indirect and through neurons. Moreover, this study finds that in the absence of Tyro3, myelin loop arrangement at the node of Ranvier is significantly more disrupted than that of WT mice. However, the hypomyelinated and abnormal paranodal loop phenotype of the Tyro3 KO mice did not alter action potential conduction in the optic nerve or corpus callosum. To expand upon this, when the myelin structural parameters of Tyro3 deficient mice were placed within a synthetic action potential conduction model, a significant decrease in conduction velocities was observed in a single axon; however, it was lost when 400 axons were modelled as a myelinated tract. Finally, I show that in the absence of Tyro3, retinal function is significantly decreased, with fewer retinal ganglion cells present and a significant decrease in retinal ganglion cell dendritic density.
The Microbiome-Gut-Brain Axis in Huntington's Disease
Huntington’s disease (HD) is progressive neurodegenerative disorder caused by the abnormal trinucleotide repeat expansion in the Huntingtin gene that is expressed throughout the brain and the body. Despite being a monogenic disease, patients show a range of central and peripheral symptoms with complex pathogenesis. Whilst the focus of much research has been on the cognitive, psychiatric and motor symptoms of HD, the extent of peripheral pathologies and its potential impact on the central symptoms has been less intensely explored. The microbiome-gut-brain axis represents the bidirectional communication between the brain and the trillions of microorganisms in the gut, whose collective genome is known as the gut microbiome. The role of the gut microbiome in modulating brain health and behaviour has been widely documented. Importantly, recent evidences have highlighted its regulatory role on the small RNA expression in the brain. Gut dysbiosis (imbalance of the gut microbiome) has been reported in a number of neurological diseases, implicating altered communication between the gut microbiome and the brain in these diseases. Hence, this body of work aimed to investigate the microbiome-gut-brain axis and its role in the pathogenesis of HD using the R6/1 transgenic mouse model. This study, first, established a dysregulation of brain miRNAs expression in the R6/1 HD mice at 12 weeks of age, prior to overt motor symptoms. At that age, we also presented the first evidence of gut dysbiosis in Huntington’s disease, coinciding with impairment with weight gain despite higher food intake. We then extended those findings by profiling the R6/1 HD gut microbiome from 4 to 12 weeks of age, representing young and adult stage of life, to pinpoint the age at which gut dysbiosis occurs in these mice, as well as to determine the changes in the gut microbiome function. In conjunction with this, plasma metabolomics was performed at 12 weeks of age to ascertain potential effects of gut dysbiosis on the plasma metabolome profile given that microbial-derived metabolites could cross the blood-brain-barrier and may regulate brain miRNA expression. Overall, we found significant gut dysbiosis as well as perturbation of the gut microbiome function in R6/1 HD mice at 12 weeks of age and not at earlier time points. Profiling of the plasma metabolome revealed no major alterations in R6/1 HD mice when compared to their wild-type littermates at 12 weeks of age. Multi-omics integration analysis revealed some novel correlations between the gut microbiome composition and the plasma metabolome profile. However, given the lack of alterations in the microbial-derived short-chain fatty acids in the blood of R6/1 HD mice, there is no strong evidence to support the role of gut dysbiosis in partially regulating striatal miRNA expression in addition to the HD gene mutation effect at 12 weeks of age. Collectively, this thesis provided new insights into the microbiome-gut-brain axis in HD, especially during the period prior to the manifestations of overt central symptoms of HD. Importantly, the work presented here highlights the gut microbiome as a potential avenue for therapeutic interventions which may be beneficial in managing the disease in gene positive individuals.
Genetic and pharmacological targeting of Heat shock protein 72 (Hsp72) in the 5xFAD*Tg30 mouse model of Alzheimer’s disease
The impact of Alzheimer’s disease (AD) is profound. In Australia, around 460,000 patients have dementia, AD being the most common form. An estimated 1.2 million people are involved in their care and it is the second leading cause of death. It is difficult to provide statistics purely on AD due to the difficulty in diagnosis, but an estimated 70% of dementia cases are AD (322,000 patients) (Dementia Australia 2018). Overexpression of the cytoprotective Heat Shock Protein 72 (Hsp72) is currently under investigation as a potential therapeutic option for prevention and treatment of AD due to its many protective mechanisms. The series of projects in this thesis aim to better understand the effects of upregulating Hsp72 in relation to behaviour, cognition and metabolism in a mouse model of AD. The aim of the first study was to replicate and extend on the characterisation of a recently developed mouse model of AD. The mutant 5xFAD (overexpressing human APP and Presenilin-1) was crossed with Tg30 (overexpressing tau) to produce 5xFAD*Tg30 mice and investigate the effects of tau accumulation in the presence of amyloid pathology, which closely resembles human AD. A comprehensive battery of behavioural and physiological tests were performed to observe cognitive and physical decline over time, between 3-8 months of age. In agreement with one previous report (Heraud et.al 2014), we observed a decline in Rotarod performance from 6 months onwards compared with wildtype control (WT). While we noted a decrease in spatial awareness and memory (Morris water maze) and significant genotype differences in anxiety-like behaviour (large open field) by 8 months, memory function (Y-maze) and novel object recognition were not significantly affected. The 5xFAD*Tg30 mice had a 40% decrease in survival by 10 months of age. Additionally, 5xFAD*Tg30 mice were smaller, with a significant difference in body weight, tibialis anterior skeletal muscle weight and tibia length. Our results successfully reproduced aspects of the previously published description of this model (Heraud et.al 2014), while adding further additional characterisation of the model. This initial characterisation study demonstrated that the model develops many aspects associated with AD including frailty, early death and initiation of cognitive decline to complement the previously described AD-like brain pathology, specifically the presence of amyloid-beta pathology with tau accumulation and neurofibrillary tangle (NFT) development, found in this model (Heraud et.al 2014). Hsp72 has been shown to play a cytoprotective role in AD- related research by inhibiting amyloid-beta oligomerization, enhancing its clearance, restoring tau homeostasis and inhibiting neuronal apoptosis. Hence, the aim of the second study was to genetically overexpress Hsp72 and study its effects on the 5xFAD*Tg30 mouse model of AD. As in the first study, we crossed 5xFAD and Tg30 mice, to create the double transgenic, 5xFAD*Tg30, then crossed the double transgenic mice with Hsp72 overexpressing transgenic mice (Hsp72 Tg) to create the triple transgenic, 5xFAD*Tg30*Hsp72Tg. BGP -15 is a compound described to be a co-inducer of Hsp72, and therefore, our third study hypothesized that pharmacologically activating Hsp72 with this compound may be advantageous in preventing or delaying AD progression and associated pathology. 5xFAD*Tg30 mice were treated with BGP-15 or vehicle in their drinking water in a randomised and blinded study in both male and female mice from the ages of 2-10months. A comprehensive battery of behavioural and metabolic tests was conducted for both the second and third studies, and results compared to littermate WT mice. We observed significant declines in Rotarod, Y-Maze and Novel Object performance and increased seizure activity in the 5xFAD*Tg30 mice compared to wildtype, however neither Hsp72 overexpression (study 2) nor BGP-15 treatment (study 3) rescued this decline. Hsp72 overexpression was partially effective in maintaining lean mass in male mice and improved performance on the elevated plus maze. In males there was a 38% decrease in survival rates by 10months of age in the 5xFAD*Tg30 mice, which was improved by BGP-15 treatment to only a 14% loss (p=0.07). In conclusion, BGP-15 may improve survival rates in a gender specific manner (male), while overexpression of Hsp72 leads to maintenance of lean mass in male 5xFAD*Tg30 mice. Both avenues used to target Hsp72 were insufficient, however, to protect against the observed cognitive deficits in the model.
Functional and structural brain networks during epileptic spikes in human brain
Accurate localisation of the brain region responsible for the seizure onset is a critical step for refractory epilepsy patients to gain access to surgical treatment. In this work, EEG, functional MRI and diffusion-weighted MRI were used to enhance the current understanding of functional and structural properties of brain networks involved in epileptic activity in patients with refractory focal epilepsy. The findings of the studies included in this thesis support the view that epilepsy is a network disorder, and epileptic activity is organised in a complex network. This work showed that results from simultaneous EEG-fMRI studies can have a critical impact on patient management, including influencing the decision for targeted surgical resection, with good subsequent seizure outcomes. Preforming EEG-fMRI studies in patients with frequent spikes during inpatient video-EEG monitoring, while on reduced or withdrawn antiepileptic medications, may lead to a much higher proportion of successful cases. Similarly, automatic spike detection method can improve detection of epileptic spikes in scalp EEG recordings, significantly shorten the analysis time, and enhance the statistical power of EEG-fMRI analysis, revealing additional elements of the patient’s epileptic networks. This work proved that the addition of spike variability information into standard fMRI analysis of the EEG-fMRI data allows for better localisation of the origin of epileptic activity and its spread across the network. In addition, investigating the structural connections that may underlie functional networks can enhance the current understanding of the propagation pathways used by epileptic activity. This work demonstrated the utility of advanced analysis methods in the investigation of refractory epilepsy. Advanced neuroimaging techniques can better localise the epileptic onset and propagation of epileptic activity. Combination of information from different diagnostic modalities bridges the gap between neurophysiology (EEG) and imaging (MRI) of epilepsy and may be a useful adjunct to the presurgical assessment of refractory epilepsy.
Targeting HCN4 channels in epilepsy
Epilepsy is a prevalent neurological disorder that affects around 65 million people worldwide. Despite optimal treatment with modern antiepileptic drugs, about one third of patients will continue to have seizures, along with undesirable side effects. Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4). HCN channels have four isoforms (HCN1-4) and produce HCN-mediated currents (Ih) that exhibit pacemaker properties critical for regulating the hyperexcitable neuronal activity seen during seizures. This thesis explores the impact of both pharmacological and molecular HCN channel block on seizure susceptibility and neuronal excitability. Broad-spectrum HCN channel block using ivabradine significantly reduced the seizure susceptibility of wildtype and Scn1a Dravet mice in two proconvulsant assays. Testing isoform-selective HCN channel blockers, the HCN2/1-preferring channel blocker, MEL55A, increased seizure susceptibility. Whereas, the HCN1-preferring channel blocker, MEL57A, had no effect on seizure susceptibility. The HCN4-preferring channel blocker, EC18, significantly reduced seizure susceptibility in two proconvulsant assays in vivo while displaying a safe drug profile. Furthermore, the conditional knockout of HCN4 channels in adult mice was also sufficient to significantly reduce seizure susceptibility in proconvulsant tests with minimal behavioural effects. Interestingly, EC18 showed no effect on seizure susceptibility when administered intraperitoneally to the conditional HCN4 knockout mouse model indicating seizure protection is HCN4-dependant. Moreover, electrophysiological as well as multi-electrode array (MEA) recordings indicated a significant reduction in parameters relating to neuronal excitability after treatment with the HCN4-preferring channel blocker, EC18. Together these results indicate that HCN4 channels are important mediators of neuronal network excitability suggesting they are promising anti-seizure drug targets with minimal adverse effects.
Early Mobilisation After Stroke: Barriers and Enablers for Effective Use of Population-Level Evidence to Inform Individualised Clinical Decision-Making
Early mobilisation, defined as sitting out of bed, standing or walking early after stroke, is an important constituent of acute stroke unit care. However, the multifaceted and complex nature of early mobilisation evidence and interventions, clinical practice guideline development and reporting, and the characteristics of stroke patients and their outcomes challenges individualised decision-making. The overall objective of this thesis was to investigate the barriers and enablers in the effective use of population-level evidence to inform individual patient-level clinical decision-making for early mobilisation post-stroke. The objective was achieved by conducting five studies: (1) a review of early mobilisation clinical practice guidelines as decision-support tools for individual patient-level decision-making; (2) a meta-analysis of individual participant data from EM trials, (3) an investigation of factors guiding early mobilisation decision-making by expert stroke clinicians, and investigation of how well (4) the utility-weighted modified Rankin scale, and (5) modified Rankin scale reflect post-stroke burden for an individual patient. (1) Based on the findings from the review of clinical practice guidelines, the decision-support requirements were met to a varying degree by early mobilisation clinical practice guidelines. Four key recommendations were formed for the future development of clinical practice guidelines. These included more granular descriptions of patient and stroke characteristics to allow tailoring of decisions to individual patients; clarity about when clinical flexibility is appropriate; a detailed description of the intervention dose, and physical assessment criteria including safety parameters. (2) The individual participant meta-analysis allowed the inclusion of individual patient-specific information and further strengthened earlier evidence from conventional meta-analyses that commencement of early mobilisation should only be considered after 24 hours post-stroke. It also reinforced the importance of adequate reporting of early mobilisation interventions to ensure the applicability of evidence to individual patients. (3) Interviews with expert stroke clinicians revealed that more than 80 percent of stroke experts considered stroke type and severity, medical stability to be the most important factors contributing to decision-making about early mobilisation. Inadequate staffing, equipment and low level of staff expertise were barriers for early mobilisation. (4) Based on the investigations of the utility-weighted modified Rankin scale, high variability in individual patient-centred utility values between and within mRS categories, over time post-stroke, and using different derivation methods was found. This variability is not adequately reflected in the utility-weighted modified Rankin scale. (5) Quality of life and activities of daily living domains demonstrated patient-specific patterns of post-stroke burden across the mRS and UW-mRS. From this research, gaps in the early mobilisation evidence base were identified that require future exploration of existing data and the development of new clinical trials to better support evidence-based clinical decision-making. Detailed areas of improvement for clinical practice guidelines as decision support tools were also identified to effectively translate population-level evidence to inform complex clinical decision-making at an individual patient-level. Finally, the identified multifaceted patterns of post-stroke burden across the utility-weighted modified Rankin scale and modified Rankin scale may facilitate appropriate assessment, articulation and interpretation of the outcomes for individual patient decision-making. The collective work has substantially contributed to systematically identifying the barriers and enablers in using population-level evidence to inform patient-level clinical decision-making in this challenging clinical context.
Characterization of white matter asymmetries in the healthy human brain using Diffusion MRI fixel-based analysis
Magnetic resonance imaging (MRI) has revolutionized the way to investigate brain structural connectivity non-invasively. Diffusion MRI can be used to obtain local estimates of the white matter fibre orientations in the brain, which in turn can be used to study changes in the local fibre specific properties and/or in conjunction with fiber-tracking algorithm to reconstruct a representation of the white matter pathways in the brain. In recent years, the Diffusion Tensor model has played an important role in modelling the diffusion of water within white matter bundles. Diffusion tensor derived metrics such as fractional anisotropy (FA) have been used extensively for investigating white matter using approaches such as voxel-based analysis. One of the limitations of the diffusion tensor model is that it is not capable of appropriately modelling regions that have complex fibre architecture (such as crossing fibres). This makes tensor-derived measures unreliable measures to assess the white matter. Recent contributions toward the study of brain asymmetry have suggested asymmetry of brain anatomy and function are observed in the temporal, frontal, and parietal lobes. Several studies have used diffusion tensor model to study asymmetry in various regions of the human brain white matter. However, given the limitations of the tensor model, the nature of any underlying asymmetries remains uncertain. This research aims to provide to provide a more robust characterization of structural white matter asymmetries than those previously derived using the tensor model, by using quantitative measures derived from the spherical deconvolution model, and a whole-brain data-driven statistical inference framework such as Fixel-Based Analysis, that is both sensitive and specific to crossing fibres; we furthermore apply this approach to a state-of-the-art publicly available diffusion MRI dataset.