Florey Department of Neuroscience and Mental Health - Theses
Now showing items 1-12 of 124
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.
Developing peptidomimetic ligands for relaxin family peptide receptors
Relaxin family peptides that target G protein-coupled receptors (GPCRs), have important therapeutic applications (e.g., heart failure, fibrosis, cancer, and disorders related to psychiatry, drug addiction, eating, or colon motility). However, these family peptides have an insulin-like complex heterodimeric structure that is difficult to produce and modify to improve their pharmacokinetic properties (e.g., half-life). I have carried out structure-activity relationship (SAR) studies on these peptides leading to the generation of simplified agonists and/antagonists for the GPCRs, RXFP1, RXFP3, and RXFP4. These novel peptides are easier to prepare in larger yields and retain high affinity for their receptors. They are important research tools, and potential drug leads for the treatment of human diseases as mentioned above. Chapter 1 summarises the literature on the SAR studies of H2 relaxin, H3 relaxin and human INSL5 leading to the design and development of simplified agonist and antagonist. Chapter 2 describes all the materials and methods used in this study. Chapter 3 summarises the SAR studies on H2 relaxin. H2 relaxin is known to have strong anti-fibrotic, vasoprotective, angiogenic, and vasodilatory effects. The receptor for H2 relaxin, RXFP1, is a potential target for the treatment of heart failure, fibrosis and related disorders, including liver cirrhosis, and preeclampsia. Our laboratory has designed a B-chain-only analogue, B7-33, which was shown to be a potent RXFP1 agonist in cells endogenously expressing RXFP1 and in several animal models of fibrosis (mice vs rats). B7-33 is 27 residues long and has short circulation time in serum (t1/2= ~6 min). I have shown that B7-33 can be further truncated up to 4 residues, and it’s in vitro half-life in human serum can be increased from 6 minutes to 60 minutes. I also have developed a B7-33-based single-chain RXFP1 antagonist for the first time. These analogues are important research tools, and drug leads for the treatment of human diseases (e.g. fibrosis-related diseases, prostate, and other cancers). Chapter 4 summarises the SAR studies on the neuropeptide H3 relaxin. The receptor for the H3 relaxin hormone, RXFP3, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviours. This chapter has two parts. In the first part, I have developed four novel analogues based on two chains, and two disulfides bonded H3 relaxin analogue, also known as A2 analogue. I have introduced amide, alcohol, carbamate, and ester functionality at the C-terminus of the B-chain of A2 analogue. In terms of binding to RXFP3, the carboxylic acid remains to be the most suitable functionality. However, the cAMP response seems to be better with the C-terminal ester function. Such results highlight the possibility of generating drug candidates, as applied to H3 relaxin, to slow down the release or generating a long-acting analogue of H3 relaxin. In the second part, I have developed an alpha-helix stabilising chemical method. Using this method, I have produced a high yielding single-chain RXFP3 agonist and antagonist. The agonist peptide, H3B10-27-13/17alphaF, is 20-fold more stable in vitro in human serum compared with the control peptides (H3B10-27-13/17EA and H3B10-27-13/17FF). The novel RXFP3 antagonist, H3B10-22R-13/17alphaF, is only 14 amino acid long compared with the previously reported 23 residues antagonist. These analogues will facilitate the identification of physiological roles of RXFP3 and drug leads for the treatment of neurological disorders such as stress and anxiety. Chapter 5 summarises the SAR of insulin-like peptide 5 (INSL5), a gut hormone. Its receptor RXFP4 is a potential target for the treatment of anorexia, obesity, and colon motility disorders (e.g. constipation and diarrhoea). Our laboratory was the first to design and develop an RXFP4-specific agonist peptide known as analogue 13. However, there was no RXFP4-specific antagonist reported in the literature. The focus of this study was to utilise the non-specific RXFP3/RXFP4 antagonist, deltaR3/I5, as a template to design an RXFP4 specific antagonist rationally. Based on analogue deltaR3/I5, I have developed an analogue 17, which is RXFP4-selective. Analogue 17 is an ideal template for further development into a specific high-affinity RXFP4 antagonist as tool and drug leads. Chapter 6 summarises the findings in this study and provides future directions.
The Role of MERTK in Central Inflammatory Demyelination
Multiple sclerosis (MS) is, at least in part, an autoimmune demyelinating disease of the central nervous system. It is the most common cause of neurological dysfunction in young adults. In addition to demyelination, autoimmune destruction in MS results in oligodendrocyte loss, local inflammation and neurodegeneration. Later in the disease course, a pattern of neurodegeneration may emerge without obvious evidence of adaptive immune activation. Current evidence implicates dysregulation of the innate immune system as a major contributor to the neurodegeneration of progressive MS. It is well established that innate immunity influences central nervous system damage and modulates myelin repair. The TAMs (TYRO3, AXL and MERTK) are a family of receptor tyrosine kinases expressed by discrete innate immune cell types, including macrophages, microglia and dendritic cells. They are involved the homeostatic regulation of adult, fully differentiated tissues that are subject to constant intrinsic and environmental challenge. The primary aim of this thesis was to provide additional insight into the biology of the MERTK receptor by examining its role in commonly employed mouse models of MS. In addition, I sought to identify whether serum levels of MERTK correlated with disease activity in MS patients experiencing relapse. I established that heterozygote deletion of Mertk in CD11+ve cells worsens the clinical severity of EAE in male mice, demonstrating for the first time that changes in the expression of a TAM receptor can alter the outcomes of a model of MS. Conversely, homozygous deletion did not result in EAE exacerbation. Heterozygote deletion of Mertk in CD11+ve cells does not alter the outcomes of cuprizone-induced demyelination, suggesting that CNS-resident CD11c+ve cells (microglia) are not implicated in the clinical exacerbation observed in EAE cohorts. In human subjects, MERTK expression by monocytes and dendritic cells does not correlate with periods of increased inflammatory activity in MS. MERTK expression by circulating dendritic cells is very limited. Taken together, the data in this thesis offer compelling evidence that MERTK is an important regulator of the outcomes of central inflammatory demyelination. They justify further research efforts in this area to better understand the mechanism of these disease states and to develop therapeutic targets for the treatment of MS.
Sleep-wake dysfunction in human ischaemic stroke
Sleep-wake dysfunction is increasingly recognised as a key modifiable risk factor and consequence of stroke. Chronic sleep-wake abnormalities, characterised by excessively long sleep duration or sleep disorders, increase the risk of ischaemic stroke. Following stroke, de novo sleep-wake impairment is common and associated with poor recovery. However, the pathogenesis and evolution of sleep-wake disturbances in stroke have been poorly characterised thus far, largely due to methodological limitations. In this thesis, gold-standard sleep measurement tools and advanced MRI methodologies were applied to investigate the impact of chronic stroke on sleep-wake function. There were three primary research questions for this thesis, and each formed the conceptual framework for three major studies: (1) To what extent is sleep-wake dysfunction associated with both stroke risk and post-stroke evolution? (2) What are the neurodegenerative markers of sleep-wake after stroke? (3) What are the sleep architectural and sleep-respiratory characteristics of chronic stroke patients relative to healthy controls? To address the first question, a scoping systematic review of over 5,000 studies was conducted in order to assess the bidirectional relationship between sleep and circadian rhythm dysfunction in human ischaemic stroke. A qualitative synthesis of the extant literature showed that excessively long sleep duration and sleep disorders significantly increase the risk of ischaemic stroke. On the other hand, acute stroke patients exhibit fragmented sleep architecture in the weeks following the incident event – potentially driven by newfound sleep disorders which may also be associated with post-stroke topography and recovery. These findings support a bidirectional relationship between sleep-wake dysfunction and ischaemic stroke with important clinical implications. To expand on limitations of prior studies identified in the aforementioned systematic review, the associations between regional neurodegeneration and objectively measured sleep were investigated in a cohort of mild-to-moderate stroke patients and healthy controls from the Cognition and Neocortical Volume After Stroke (CANVAS) study. Stroke patients with excessively long sleep duration and poor sleep efficiency exhibited volumetric reductions to the thalamus and amygdala relative to healthy controls. Next, a novel method known as a whole brain fixel-based analysis was utilised to investigate fibre-specific white matter degeneration in stroke patients with poor sleep. Stroke patients with excessively long sleep duration exhibited tract-specific neurodegeneration to the cortico-ponto-cerebellar tract. These findings suggest that poor sleep efficiency or long sleep duration may contribute to neurodegeneration following stroke. The final study in this thesis aimed to characterise hemispheric sleep architecture and sleep-respiratory characteristics in stroke patients >4 years after their incident event using gold-standard polysomnography. In a subsample of patients from the CANVAS study, stroke patients and matched controls underwent overnight ambulatory polysomnography and completed an array of sleep and circadian questionnaires. Over half of all stroke patients in this sample exhibited undiagnosed moderate to severe obstructive sleep apnoea. Stroke patients had nearly 40% less restorative slow-wave sleep and potentially compensatory increases in lighter sleep stages relative to healthy controls. Sleep architectural disturbances were not attenuated by obstructive sleep apnoea. There were no sleep architectural differences in the stroke-affected versus healthy-hemisphere. These findings suggest that sleep impairment post-stroke is unlikely to be driven by comorbid obstructive sleep apnoea or the hemispheric distribution of stroke lesions. Furthermore, these results highlight the importance of formal sleep studies in stroke patients in order to identify undiagnosed obstructive sleep apnoea and fragmented sleep architecture. The overall findings from this thesis offer valuable insight into the potential in vivo pathogenesis of sleep-wake dysfunction after stroke and the evolution of sleep abnormalities in the chronic stages of stroke. The clinical-pathogenic implications of sleep-wake dysfunction in stroke are unravelled, and a research agenda for future studies in this emerging field of medicine is outlined.
The modulation of cortical dendrites during sensory perception and learning
Sensory perception arises in the cortex by integrating external information from the environment with internal representations and the current brain state. This process is supported by the structure of the neocortex and the organisation of excitatory inputs onto its core computational element: the pyramidal neuron. In layer 2/3 pyramidal neurons, external (feedforward) information mainly target the somatic region, while internal (feedback) information runs through layer 1 and lands onto distal tuft dendrites. Cortical dendrites have active properties that could be important for sensory processing and could provide a cellular mechanism to support the flexibility of sensory representations during learning. In this thesis, I addressed the role of L2/3 pyramidal neuron dendrites during sensory perception and learning in three parallel studies that investigated: 1) how inputs from another sensory area affect early stages of sensory processing; 2) the modulation of sensory processing in dendrites of the auditory cortex following fear conditioning; 3) changes in dendritic activity during perceptual learning of an auditory discrimination task. To tackle these questions, a combination of two photon Ca2+ imaging and whole cell patch clamp electrophysiology in vivo, together with behavioral testing and optogenetics manipulation, was used. The results presented here confirm that dendrites encode sensory information and show that they can undergo plastic changes during learning. The findings also illustrate the existence of compartmentalised activity in L2/3 pyramidal neurons and of a cellular mechanism for the control of action potential generation that involve dendritic integration. The results presented in this thesis highlight the importance of the upper layers of the cortex for flexible sensory representation through the integration of feedback and feedforward information.
Remyelination in the aging brain and the effects of growth factor supplementation on oligodendrogenesis
In multiple sclerosis (MS), chronic demyelination initiated by immune-mediated destruction of myelin, leads to axonal damage and neuronal cell death, resulting in a progressive decline in neurological function. The development of interventions that potentiate remyelination could hold promise as a novel treatment strategy for MS. Seminal work in the Merson group has demonstrated that neural precursor cells (NPCs) residing in the subventricular zone (SVZ) of the adult mouse brain contribute significantly to remyelination in response to CNS demyelination and can regenerate myelin of normal thickness. However, aging takes its toll on the regenerative potential of NPCs and reduces their contribution to remyelination. In this study, I investigated how aging could affect the NPCs contribution to oligodendrogenesis during the remyelination process and whether the delivery of growth factors into the brains of aged mice could potentiate the oligodendrogenic potential of NPCs. To map the fate of NPCs in response to demyelination induced at different postnatal ages, Nestin-CreERT2; Rosa26-LSL-eYFP mice were gavaged with tamoxifen at either 8 weeks, 30 weeks or one year of age before being challenged with cuprizone for a period of six weeks. Using osmotic minipumps, I infused epidermal growth factor (EGF) and/or heparin-binding EGF-like growth factor (HB-EGF) or artificial cerebrospinal fluid (vehicle) into the cisterna magna for a period of two weeks beginning at the peak of cuprizone-induced demyelination (n=6-8 mice per group). Mice were perfused six weeks after cuprizone withdrawal and the contribution of NPCs to oligodendrocyte regeneration in the corpus callosum was assessed. Our data reveal that although NPC-derived oligodendrocyte generation declined dramatically with aging, this decline was at least partially reversed by growth factor infusion. Notably, co-infusion of EGF and HB-EGF increased oligodendrocyte regeneration by two-fold in some regions of the corpus callosum. The results of this study demonstrate the beneficial effects of EGF and HB-EGF for increasing the contribution of NPCs to oligodendrogenesis during the remyelination process and indicate that modulation of progenitor responses could hold therapeutic potential for combating the negative effects of aging upon remyelination efficacy.
Age related dopamine 1 and dopamine 2 receptor expression in addiction-related behaviours
Methamphetamine (meth) is a significant social and public health concern worldwide, and a growing problem in Australia. One factor contributing to meth use disorder is the lasting memory of its rewarding experience, which can lead to persistent use in vulnerable individuals. Epidemiological data show that adolescence is a period of heightened vulnerability for developing meth use disorder. Furthermore, sex differences exist in numerous aspects of meth use motivations, behaviours, and consequences. Despite this, few studies have investigated age and sex effects of meth on brain and behaviour. A unified neural mechanism by which substances of abuse, including meth, produce their addictive properties is by increasing dopaminergic transmission throughout the mesocorticolimbic dopamine system. Although dopamine binds to and activates several subclasses of receptor in brain regions implicated in reward processing, the dopamine receptors 1 (D1) and 2 (D2) have been reported to be particularly important for drug-affected behaviour. Importantly, levels of D1 and D2 have been shown to fluctuate throughout development. D1 and D2 signalling may therefore be important mediators of adolescent vulnerability to substance use. However, there are numerous inconsistencies in the literature that describe developmental changes in D1 and D2 expression. Systematic characterisation of these changes is therefore critical for a full understanding of how changes to the dopamine system may affect susceptibility to meth (and other substance) use disorder. As such, the first aim of my thesis was to investigate the postnatal developmental trajectory of D1 and D2 in addiction-related brain regions. This was achieved using D1- and D2-green fluorescent protein (GFP) transgenic mice, starting from the juvenile period through to adulthood. The results showed region-specific changes in D1 and D2 expression occur across development, with the insular cortex (insula) showing the most dramatic changes. In particular, the density of D1 compared to D2 expressing neurons (D1:D2 ratio) in the insula substantially increased from adolescence to adulthood in males. Since substance use disorders are male dominant disorders which often have an adolescent onset, this reduced D1:D2 ratio may be relevant in understanding the neurobiological basis of substance use disorders. The second aim of my thesis was to investigate the role of insula D1 and D2 in potential age and sex differences in meth conditioned place preference (CPP) and aversion (CPA). Although no age or sex differences were observed in CPP to 0.1 or 3 mg/kg meth at a group level, analysis of individual data revealed females were less likely to form a place aversion compared to males, and adolescents formed more of a place preference to 0.1 mg/kg meth compared to adults. Conditioning with 3 mg/kg meth led to age differences in insula D1:D2 ratio in males, reduced age differences in insula D1:D2 ratio in females, and increased the activation of insula D2 expressing cells in adults compared to adolescents. Insula activity and expression of D1 and D2 did not correlate with place preference behaviour. Taken together, these findings suggest distinct sex differences in D1 and D2 expression across development in addiction-related cortical and striatal brain regions may underly age- and sex-associated vulnerability to meth-related behaviours.
Space for recovery after stroke: Exploring the role of the physical environment in inpatient rehabilitation facilities
The aim of this thesis was to explore the role of the physical environment of inpatient rehabilitation facilities in stroke recovery. The purpose of rehabilitation is to help stroke survivors to re-learn skills and abilities lost as a result of stroke, or to learn new skills to adapt to their changed condition. Research in other healthcare environments suggests that hospital design can impact patient outcomes, but there is little evidence specific to rehabilitation. This thesis embraces the complexity of rehabilitation environments and explores the physical environment as an essential and integrated component of this complex system. This exploration began with a scoping survey to identify and describe all inpatient rehabilitation facilities in Victoria, Australia. This survey revealed 64 facilities, most of which had not been purpose-built for rehabilitation. Rehabilitation facility design appears to be influenced by evidence from acute medical settings and current design trends, rather than reflecting the unique purpose of rehabilitation. A series of expert elicitation workshops were then conducted to define – for the first time – what is important in the physical environment of inpatient stroke rehabilitation facilities. Thirty experts participated, including policy makers, researchers and designers in learning and healthcare environments, clinical staff, and patients. A Value-Focused Thinking methodology was used to facilitate the workshops. The experts defined 16 criteria thought to be fundamentally important (including efficiency, patient practice, activity and rest, emotional well-being, and safety), and 14 criteria that could be a means to achieving these fundamentally important things. Together, these criteria comprise a framework which can be used to guide research and design in this complex area. This framework informed a multiple-case study in two stroke inpatient rehabilitation facilities. Convergent mixed-methods were used to produce a rich and thorough exploration of the cases. Twenty inpatients from Case 1 participated, and 16 from Case 2. The physical environment was described using field notes, photographs, floor plans, and checklists. Walk-through semi-structured interviews were used to explore patients’ experience of the physical environment. Systematic observation (behavioural mapping) and questionnaires were used to investigate patients’ behaviour and emotional well-being in the environment, and a retrospective audit of patient falls was conducted to investigate patient safety. Four interrelated themes described the patient experience: 1) entrapment and escape; 2) power, dependency, and identity in an institutional environment; 3) the rehabilitation facility is a shared space; and 4) the environment should be legible and patient-centred. Quantitative data revealed that patients spent over 75% of their time in their bedrooms. Comparison between cases suggested that the physical environment played a role in patients’ behaviour, emotional well-being, and safety. Qualitative and quantitative findings were then merged using joint display tables and narrative integration. This robust analytic process produced a new conceptual model of the role of the physical environment in stroke patients’ behaviour, emotional well-being, and safety in rehabilitation, emphasising the importance of variety and interest in the environment, privacy without isolation, and patient-centred design. The findings from this study provide meaningful direction for rethinking rehabilitation facilities and guiding real-world health design practice.
Sex differences in fear learning in juvenile rats
The present thesis examined the sex differences in fear learning in the developing rat. Traditionally, it has been widely assumed that pre-pubertal sex differences are negligible in developmental studies. However, epidemiological studies and sexual dimorphism in the brain prior to puberty, indicate that sex differences in fear learning may emerge early in life. In chapter 2, I assessed renewal, reinstatement and spontaneous recovery of extinguished fear (i.e. relapse of extinguished fear) in juvenile male and female rats. I found that P18 females showed all three fear relapse behaviors while P18 males did not. This finding implied that P18 female rats were able to form the contextual memories while P18 males could not. However, whether the ability to form a context-shock association contributes to fear extinction in juvenile rats, has not been explicitly tested. Therefore in chapter 3a and 3b, I directly compared renewal with context fear, using identical fear conditioning parameters in the developing rats. In chapter 3a, I found that P18 male rats did not show renewal while P25 male rats did. P18 and P25 male rats displayed comparable contextual freezing immediately after conditioning and after 24 hours delay. In chapter 3b, both P18 and P25 female rats displayed renewal. I also found that P18 female rats displayed a developmental deficit in context fear learning compared to P25 female rats. Together, findings from chapter 2, 3a and 3b strongly suggested that the emergence of context memory may be sex-dependent, where context-specific extinction emerges earlier in females while context fear learning emerges first in males. Given the critical role of the hippocampus in context-dependent learning, I thought the sex differences in the hippocampal function may underlie the observed sex differences in fear learning. In chapter 4, I examined the role of dHPC and vHPC in fear extinction in P18 male and female rats. I showed that temporary inactivation of the dHPC prior to extinction accelerated extinction acquisition in both male and female rats. I also showed that pre extinction inactivation of the vHPC reduced freezing during extinction and impaired extinction recall, regardless of sex. Collectively, these findings strongly suggest sexually dimorphism in fear learning emerges early in life and emphasize the importance of sex as a factor in the field of developmental learning and memory.
Improving the safety and function of pluripotent stem cell-derived neural transplants for the treatment of Parkinson's disease
Clinical trials using fetal tissue have provided the necessary proof-of-principle evidence that transplanted dopamine neurons can appropriately integrate into the brain and alleviate motor symptoms in Parkinson’s disease patients for > 20 years. Pluripotent stem cells (PSCs), are now being pursued as an alternative cell source, circumventing ethical and availability issues, given their competent self-renewal and differentiation capabilities. Despite rapid progress in the field over the past decade, and recent advancement of these cells into clinical trials (Japan October 2018), we recognise a number of short-comings that require further attention. Despite the existence of efficient protocols for the directed differentiation of human PSC into ventral midbrain (VM) progenitors (i.e. cells capable of maturing into dopamine neurons and amenable to transplantation), a small proportion (10%) of cells in culture remain incorrectly specified. These cells are capable of significant expansion after transplantation, such that grafts commonly contain <5% dopamine neurons when examined after many months. These highly proliferative cells not only present an evident risk of neural overgrowth, but there also remains little knowledge of the identity these cells become and the impact they may have on the graft and patient. For these reasons, strategies have been pursued to improve the purity of donor tissue for grafting. However, to date, cell sorting approaches, to select for correctly patterned human PSC-derived cells prior to implantation have been suboptimal. In chapter 3 of the thesis we employ two human PSC reporter lines to demonstrate that the isolation of vm progenitors, but not vm precursors, results in viable cell grafts that are more predictable in their composition, retain integration and functional capacity to restore motor deficits in Parkinsonian rats, and importantly eliminate highly proliferative cells and populations known to contribute to graft-induced dyskinesias. An alternative safeguarding approach for transplantation studies is to employ suicide gene therapy – targeted at eliminating unwanted cells after transplantation. In Chapter 4, we employ a human PSC line carrying a suicide gene (thymidine kinase) that can be activated by administration of a prodrug (ganciclovir), to enable remote killing of proliferating cells within the transplant. We demonstrate that the timely activation of this ‘suicide switch’ can prevent excessive expansion of undesirable cells in the graft whilst preserving DA integrity - yet surprisingly, a small proportion of persistent dividing cells remained. We speculate that this was a result of suboptimal delivery of ganciclovir caused by insufficient vascularisation of dopamine grafts. In a third approach to improve graft outcomes, we address the impact of human PSC-derived VM progenitor donor age (Chapter 5). Despite historical efforts to understand the impact of different aged VM fetal tissue on graft survival, dopamine contribution and functional integration, there remains no comparable assessment of human PSC-derived vm progenitor age. While the current clinical trial in Japan has elected to use late stage human PSC-derived VM progenitors, in Chapter 5 we surprisingly show that VM-specified early-stage progenitors generated the most homogeneous DA grafts, containing a considerably lower component of unwanted, off-target cells than grafts derived from older progenitors. Importantly we demonstrated that while this effect was extremely consistent within human PSC lines, considerable inter-line variability was evident, highlighting the need for rigorous assessment on a line to line basis, prior to translation. In summary, the work provided within this thesis provides significant new insight into strategies to standardize human PSC-derived transplantation for Parkinson’s disease – importantly addressing the need to consider graft survival, proportion of dopamine neurons and their function, as well as the critical requirement to eliminate unwanted cell types to ensure maximal safety.
Studies on the mechanism of binding and activation of relaxin family peptide receptors
The peptide hormone relaxin is involved in reproductive processes but has also been investigated for several decades as a treatment for a range of disease states such as scleroderma, acute heart failure, and fibrotic conditions. The receptor for relaxin, RXFP1, is an integral membrane protein belonging to the G protein coupled receptor (GPCR) family. RXFP1 is therefore a therapeutically tractable target for which a thorough understanding of its mechanism of binding and activation is required to develop better relaxin-like drugs. The aims of these studies are to investigate the mechanism by which relaxin binds and activates RXFP1 using a variety of molecular pharmacology approaches in a HEK293T cell model system recombinantly expressing RXFP1 in various forms. Specifically, a hypothesis was tested that a homodimer of RXFP1 might be the minimal functional unit required for receptor activation. GPCR dimers are postulated to interact via their transmembrane helices, so initial investigations aimed to disrupt RXFP1 homodimerisation by incorporation of peptides representing single transmembrane segments of RXFP1 as well as recombinant expression of RXFP1 transmembrane domains. There was no evidence that RXFP1 homodimerisation is required for receptor activation. Following this, the evidence for RXFP1 homodimerisation was re-evaluated in the development of two methods which utilise principles of Bioluminescence Resonance Energy Transfer (BRET). Firstly, split Nanoluciferase was used to tag cell surface localised RXFP1 receptors in combination with mCitrine-tagged RXFP1 and BRET was measured to assess relative receptor proximity. This indicated that RXFP1 is unlikely to be a stable homodimer, intracellularly localised receptors predominate, and there is no change in receptor:receptor proximity upon relaxin stimulation. Secondly, Nanoluciferase-tagged RXFP1 receptors were used in combination with fluorescently labelled relaxin and BRET was measured to track relaxin:RXFP1 binding interactions. This allowed sensitive, real time measurements of the relaxin:RXFP1 binding interactions, demonstrating a multi-step mechanism of relaxin binding in which the linker domain of RXFP1 is critical for high-affinity interactions. Furthermore, there was no evidence of negative co-operativity of relaxin binding, contrary to previous reports which were used as evidence of RXFP1 homodimerisation. Overall, these studies indicate that relaxin does not activate RXFP1 via a mechanism involving a receptor homodimer. Several molecular tools were developed which will be useful for future investigations into RXFP1 pharmacology. This work adds incremental detail to the understanding of how relaxin activates RXFP1, hopefully leading to the development of novel therapeutically useful relaxin-like molecules in future.
Early gene expression profiling of motor neurons in the SOD1G93A mouse model of amyotrophic lateral sclerosis: insights into earliest pathogenesis
Although amyotrophic lateral sclerosis (ALS) typically presents in mid to late life, there is increasing evidence in patients and mouse models for a protracted preclinical period of motor neuron vulnerability and damage before clinical onset. We hypothesized that the seeds for the development of ALS may be sown shortly after conception and motor neuron vulnerability is induced in the perinatal period of life, leading to subsequent neuronal dysfunction and degeneration. The goal of this study was to identify the earliest gene expression patterns in the vulnerable lower motor neurons at very early and key developmental ages in the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We have implemented and fully characterised HB9:GFP reporter mouse to unambiguously identify and isolate the spinal alpha motor neurons for transcriptomic profiling using RNA sequencing, pathway analysis and target validation. Isolation of HB9:GFP+ spinal motor neurons using FACS was employed at embryonic day 12.5 (E12.5), E17.5, postnatal day 3 (P3) and P8 in SOD1G93A mice and control littermates. Purification of enriched motor neurons was only successful from E12.5 mice. At E17.5, P3 and P8 samples were enriched in glial cells. Gene expression profile of HB9:GFP+ spinal motor neurons from E12.5 SOD1G93A mice revealed significant dysregulation of RNA processing genes, consistent with key pathological pathways in ALS. Dysregulation of Gria2, an AMPA receptor subunit gene, implicated in Ca2+ mediated excitotoxicity was confirmed by real-time qPCR and immunohistochemical analyses in motor neurons from embryonic SOD1G93A mice and induced pluripotent stem cell lines of ALS patient carrying SOD1 mutations. We propose that dysregulation of the AMPA receptor may lead to mitochondrial pathology induced by increased Ca2+ influx and the intracellular increase of free radicals which confers an early susceptibility of spinal motor neurons to excitotoxicity in ALS. In summary, this study provides the first insights into gene expression profiles of motor neurons from SOD1G93A mice in utero. Importantly, our data identified dysregulation of RNA processing and the AMPA-mediated excitotoxicity receptor subunit as early as E12.5 which may account for an early, and likely a causal event in triggering selective motor neuron vulnerability in ALS, arguing for administration of early interventions using anti-excitotoxic therapeutic approaches in ALS.