Florey Department of Neuroscience and Mental Health - Theses
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ItemChronic Upper Limb Pain in Stroke: Beliefs and Perceptions, Factors Impacting, and Clinical Implications( 2021)Chronic pain is commonly experienced following stroke, with post-stroke shoulder pain (22-47%) and complex regional pain syndrome of the hand (21-31%) being among the most frequently reported pains by stroke survivors. Individuals with chronic pain post-stroke experience higher rates of depression, fatigue and anxiety in addition to restricted mobility and cognitive functioning compared to those without pain. Pain is frequently identified as an unmet need by survivors of stroke, and there is a lack of evidence for effective interventions. Many interventions used in clinical practice are based on outdated models of thinking related to pain, and do not consider the multifactorial nature of chronic pain. However, there is currently a lack of knowledge informing the mixed nature of pain post-stroke, and as such, development of treatment strategies has been limited. The primary aim of this thesis was to characterise the nature of chronic pain for individuals with stroke, and identify factors that may impact on the pain experience. A further aim was to investigate the impact of a learning-based approach to sensory rehabilitation (SENSe Therapy) on pain outcomes in people with stroke who experience somatosensory loss and chronic pain. To achieve these aims, two main studies were conducted. The first (RECOGNISE) was an online observational study developed for this thesis. RECOGNISE utilised a survey questionnaire and a series of interactive tasks to characterise the nature and experience of chronic pain post-stroke from the perspective of the stroke survivor (N=533), and investigate if relationships existed between somatosensory ability, body perception and chronic pain. The second study was part of a larger randomised trial (CoNNECT) in which stroke survivors with somatosensory loss and chronic pain (N=29), undertook a program of SENSe Therapy over a six-week period, with assessments performed at pre-post intervention and follow up at six-months. Findings from RECOGNISE revealed that there is not a common chronic pain experience post-stroke, as individuals with stroke report a mix of symptoms (spontaneous, evoked and paroxysmal). Further, chronic pain post-stroke was often associated with negative pain beliefs, somatosensory impairment and altered body image. Review of existing literature showed that these identified factors are modifiable using targeted interventions in other complex pain conditions, resulting in a reduction in pain. Findings from the exploratory study targeting somatosensory function of the upper limb in stroke survivors with chronic pain found that somatosensory retraining is a viable treatment option that had a positive effect in reducing pain in stroke survivors. In conclusion, this thesis advances knowledge about the mixed nature of pain experienced by individuals with stroke, and identifies several opportunities for development of intervention strategies that can be tailored to the individual with stroke based on their symptoms and presenting features.
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ItemUsing structural and molecular approaches to investigate DLG2/PSD-93 dysfunction in neurodevelopmental disorders( 2021)The genetic basis of neurodevelopmental disorders such as autism spectrum disorder and intellectual disability is complex, involving rare and common variants in multiple genes. However, amidst this complexity, genomic studies have consistently revealed a convergence of mutations in synaptic genes. At excitatory synapses in the vertebrate brain, postsynaptic terminals contain highly conserved multiprotein complexes critical for signalling and behaviour. These postsynaptic complexes are organised by scaffold proteins such as PSD-93 (encoded by DLG2). PSD-93 binds glutamatergic receptors, anchoring them to the postsynaptic membrane and, in addition, assembles a downstream network of proteins to form the postsynaptic signalling machinery. In Chapter 2, we have identified a novel, missense DLG2/PSD-93R837N variant in a family where members have been diagnosed with intellectual disability and autistic features. Using structural and molecular techniques, we investigated the functional impact of this DLG2/PSD-93R837N variant. We show that the mouse DLG2/PSD-93R714N mutation (corresponding to human DLG2/PSD-93R837N) did not disrupt PSD-93 protein expression, nor the number of PSD-93-positive synapses in mouse hippocampal neurons in vitro. However, by examining the PSD-93-guanylate kinase (GK) protein domain where the mutation is located, we found that mutant DLG2/PSD-93-GKR714N exhibited decreased secondary structure folding which led to decreased protein stability and impaired binding to key postsynaptic interactors, SAPAP and MAP1A. From this, we propose that the PSD-93-GK protein exists in an equilibrium between two population states; a folded, binding-competent state and a partially unfolded, binding-incompetent state, and the DLG2/PSD-93R837N variant shifts this protein equilibrium to a more unfolded state. In Chapter 3, we further investigated the proposed PSD-93-GK protein equilibrium by utilising nuclear magnetic resonance spectroscopy. Initial analysis supported the presence of two conformational states in PSD-93-GKWT, however further optimisation and experiments are needed to confirm this finding. Additionally, we have identified conditions which produced PSD-93-GKWT protein crystals during sitting-drop vapour diffusion, allowing for future X-ray crystallography studies to determine the structure of the PSD-93-GK domain. In Chapter 4, we developed a lentivirus containing wild-type full-length PSD-93 which provides a valuable research tool to manipulate protein expression and investigate PSD-93 in vitro and in vivo. This thesis has utilised various structural and molecular approaches to investigate PSD-93 and how its function is impaired by the DLG2/PSD-93R837N variant identified in neurodevelopmental disorders. As a result, we have elucidated a novel mechanism of PSD-93 dysfunction and provided novel insights into the PSD-93 protein equilibrium. This has increased our understanding of how dysfunction in a synaptic gene can contribute to neurodevelopmental disorders which will provide valuable insights into the development of new targeted therapies in the future.
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ItemThe pathophysiology of major depressive disorder: an investigation of traditional neurochemical hypotheses and novel candidate genes in the anterior cingulate cortex( 2021)Major depressive disorder (MDD) is a debilitating psychiatric disorder which is often chronic and recurrent in nature, highlighting the need for novel therapeutics to improve patient outcomes. Central to this is extending our understanding of the underlying pathology of MDD. Part 1. Our laboratory has previously reported lower serotonin 2A (5-HT2A) receptors in the anterior cingulate cortex (Brodmann’s area (BA) 24) of MDD subjects. Given a pathological change in one receptor subtype has the potential to cause a breakdown in homeostasis within the serotonergic system, I investigated levels of the serotonin 1A (5-HT1A) receptor and serotonin transporter (SERT) in the same mood disorder cohort (MDD, n=20; bipolar disorder (BD), n=18; non-psychiatric control, n=20). In situ radioligand binding with autoradiography was performed to measure the density of [3H]8-OH-DPAT (5-HT1A receptors) and [3H]citalopram (SERT) binding. There was no variation in the levels of 5-HT1A receptors and SERT in BA24 of MDD subjects compared to BD and non-psychiatric controls. When taken with the previous finding of lower 5-HT2A receptors in the same mood disorder cohort, this could indicate aberrant functioning of the serotonergic system in MDD and a net functional deficiency in serotonergic neurotransmission. Furthermore, our laboratory has previously reported lower levels of M2 muscarinic acetylcholine receptors (M2 receptors) and higher levels of transmembrane tumour necrosis factor (tmTNF) in BA24 of the same mood disorder cohort. Thus, I investigated whether levels of nicotinic acetylcholine receptors (nAChRs) are also altered in MDD, as the assembly of nAChRs containing a4, b2, and b4 subunits is promoted by TNF. Using the same mood disorder cohort, in situ radioligand binding with autoradiography was performed to measure [3H]epibatidine binding in BA24. There was no variation in the levels of nAChRs between diagnoses. Additionally, quantitative polymerase chain reaction (qPCR) showed no variation in mRNA expression of the nAChR subunit CHRNB2 in these subjects. The data suggests that lower levels of M2 receptors in BA24 of mood disorders is not associated with a corresponding change in nAChR expression. However, serendipitous data showed a significant increase in the mRNA expression of cyclophilin A (PPIA; p <0.0001), alpha synuclein (SNCA; p <0.001), and mitochondrial dimethyladenosine transferase 1 (TFB1M; p <0.0001) in BA24 of MDD compared to BD and non-psychiatric controls. Part 2. I performed qPCR to measure mRNA expression of PPIA, SNCA, and TFB1M in two additional brain regions (BA9 and BA10) from subjects with MDD (n=14), BD (n=15), and non-psychiatric controls (n=15). Relative quantities of PPIA, SNCA, and TFB1M mRNA expression were normalized to the geometric mean quantities of two stably expressed reference genes. There was no significant variation in PPIA, SNCA, and TFB1M mRNA expression between diagnoses in BA9 or BA10. The data suggests that region-specific changes in PPIA, SNCA, and TFB1M could contribute to the pathophysiology of MDD and supports the notion that the cortical dysregulation in MDD is the product of small changes in many genes, across multiple brain regions. Furthermore, the diagnostic specificity of these findings has implications for understanding the biochemical differences underlying MDD and BD.
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ItemTherapeutic targeting of necroptosis and ferroptosis cell death pathways in MND( 2021)Since its characterization in 1872, the mechanism that underlie motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS) remains unknown. The current study unravels the impact of two cell death pathways, necroptosis and ferroptosis, and highlights their significance in ALS. The impact of inhibiting necroptosis was assessed by genetically ablating the executioner of necroptosis, pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) in SOD1G93A mice. Our results demonstrate that MLKL is absent in the CNS of mice and humans. Further, inhibiting necroptosis does not change the disease course of SOD1G93A mice, implicating that necroptosis is dispensable in ALS. To study ferroptosis in ALS, key biochemicals markers of ferroptosis were assessed in postmortem ALS spinal cord in addition to 3 different in vivo models of ALS. Models included the SOD1G93A, TDP43Q331K and C9orf72500 mice. Critically, glutathione peroxidase 4 (GPX4), the critical inhibitor of ferroptosis was lost at disease onset in the spinal cords of all three in vivo models, implying that GPX4 loss precedes MN degeneration. The loss of GPX4 was also exacerbated in the spinal cord of SOD1G93A mice at endstage, suggesting that the loss of GPX4 drives ALS pathology. In line with our in vivo findings, GPX4 expression was also lost in the spinal cords of both familial and sporadic ALS patients. Immunohistochemistry revealed that loss of GPX4 is neuronal, suggesting that MNs are sensitive to ferroptosis in ALS. Due to the loss of GPX4, SOD1G93A mice overexpressing human GPX4 (hGPX4) were generated to assess the impact of inhibiting ferroptosis in ALS. Critical overexpression of hGPX4 in SOD1 mice delayed onset and improved SOD1G93A survival, showcasing the therapeutic potential of inhibiting ferroptosis in ALS in vivo. Altogether, we have shown that the loss of GPX4 is central to MND pathology and suggest that ferroptosis may play a pivotal role in driving MN loss in ALS.
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ItemNovel antidepressant-like properties of the iron chelator deferiprone in the 5-HTT knock-out mouse model of depression( 2021)Current therapeutics for major depressive disorder (MDD) have limited utility due to their extensive side effect profiles and limited efficacy. Additionally, there is an absence of extensive fast-acting antidepressant therapies. Serotonin is a neurotransmitter which is implicated in MDD and is responsible for various functions including mood regulation and stress responsiveness. Serotonin transporter knock-out (5-HTT KO) mice model the serotonergic dysfunction and behavioural endophenotypes intrinsic to a population of MDD patients. Iron is a metal which plays a role in various mechanisms relevant to MDD neuropathology and there is clinical evidence that brain iron levels may be elevated in MDD. Deferiprone is an iron chelator which has the capacity to rapidly cross the BBB. The aim of this thesis was to determine the behavioural effects of deferiprone on the 5-HTT KO mouse model of depression, relative to wild-type (WT) littermates. Furthermore, this thesis investigated brain regions involved in deferiprone treatment and its interaction with an acute stressor in the form of swim stress. Finally, deferiprone’s molecular mechanism of action was also investigated. The data presented indicates that deferiprone has acute antidepressant-like properties in both 5-HTT KO and WT mice. The antidepressant-like properties of deferiprone are more robust on the 5-HTT KO mouse model of depression, relative to WT littermate controls. Deferiprone also has pro-cognitive effects in the form of enhanced short-term spatial memory performance following chronic administration, irrespective of genotype. The acute antidepressant-like properties of deferiprone were attributed to regions of the lateral/basolateral amygdala, bed nucleus of the stria terminalis (BNST) and lateral septum. Graph theory-based analyses and hierarchical clustering was adopted to determine the effects of deferiprone and swim-stress exposure on the functional network of 5-HTT KO and WT mice. This revealed a divergence of brain region hubs of activity following deferiprone treatment in either genotype. This is likely to reflect neurodevelopmental effects on specific brain regions in the 5-HTT KO mice due to genetic ablation of 5-HTT protein function, although adult absence of 5-HTT may also play a role. The prefrontal cortex is a region which has extensive glutamatergic innervation and synaptic dysfunction in MDD and the 5-HTT KO mouse model. Deferiprone treatment in 5-HTT KO mice resulted in acute post-translational modification effects on proteins involved in synaptic transmission, cellular cytoskeletal proteins and glutamatergic signalling in the prefrontal cortex. 5-HTT KO mice did not have extensive iron dysregulation, and chronic deferiprone treatment increased levels of iron in the prefrontal cortex and dorsal hippocampus, indicating that deferiprone’s antidepressant-like actions may be iron independent. Investigating novel mechanisms involved in MDD neuropathology and therefore potential antidepressant therapies is critical for improving disease outcomes. This thesis provides evidence for deferiprone having acute antidepressant-like effects. Furthermore, the acute antidepressant-like activity of deferiprone was confirmed by specific changes to brain region activity and molecular mechanisms implicated in existing antidepressants. A further exploration of the novel antidepressant-like properties of deferiprone and its mechanism of action may reveal unexplored avenues of MDD pathogenesis and enhance antidepressant discovery for the treatment of this devastating disorder.
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ItemMechanisms Underlying Excitability in an HCN1 Developmental and Epileptic Encephalopathy( 2021)Epilepsy is a neurological disorder characterized by seizures, which occur due to excessive and hypersynchronous neuronal activity. The Hyperpolarisation-activated Cyclic Nucleotide-gated channels (HCN channels) are a family of ion channels encoded by the genes HCN1, HCN2, HCN3 and HCN4, which are widely expressed throughout the brain and play key roles in regulating neuronal excitability and synchrony. Dysfunction and dysregulation of HCN channels has been closely linked to epilepsy. In particular, an increasing number of pathogenic variants in HCN1, which encodes the HCN1 channel isoform, have been identified and shown to give rise to epilepsy. Many of these variants cause Developmental and Epileptic Encephalopathy (DEE), a severe condition characterised by early-onset, pharmacoresistant seizures as well as developmental delays. The work described in this thesis aimed to identify the mechanisms underlying how HCN1 channel dysfunction can cause hyperexcitability and subsequent epilepsy, and to explore the best ways of treating this condition. To do so, we generated the first mouse model of HCN1 epilepsy, the Hcn1M294L heterozygous knock-in mouse. This mouse carries the murine homologue of the human HCN1 M305L variant, which has been identified in two unrelated patients with HCN1 DEE. The Hcn1M294L mouse accurately recapitulates several of the major phenotypic features of human HCN1 DEE, including having spontaneous seizures, epileptiform activity on electroencephalography (EEG), susceptibility to heat-induced seizures, and a learning deficit. Electrophysiological studies in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue from Hcn1M294L mice revealed that the disease variant causes a loss of voltage dependence, resulting in a constitutively open HCN1 channel that allows cation ‘leak’ at depolarised membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarised at rest and fired action potentials more readily, contributing to the hyperexcitability underlying the epilepsy. Pharmacological studies revealed the Hcn1M294L mouse to have similar pharmacoresponsiveness to the anti-epileptic drugs (AEDs) sodium valproate and lamotrigine as a human HCN1 DEE patient. These results positioned this mouse as a strong preclinical model with good face validity, on which potential treatments for HCN1 epilepsy could be trialled. A broad screen of ten currently available AEDs tested in Hcn1M294L mice revealed four drugs which significantly improved and three which significantly worsened neuronal epileptiform activity, providing a potential framework for the clinical treatment of HCN1 epilepsy. Finally, experiments exploring potential precision medicine treatments for HCN1 epilepsy demonstrated that the blood-brain barrier penetrant, broad-spectrum HCN channel blocking drug PTX-002 significantly reduced neuronal epileptiform activity in Hcn1M294L mice, providing an initial proof-of-concept that HCN channel block may be an effective treatment for HCN1 epilepsies caused by cation ‘leak’. Together, these results provide novel insights into the mechanisms underlying hyperexcitability in HCN1 epilepsies, and offer promising directions for future research and for the development of improved treatments for patients who live with these conditions.
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ItemInvestigating new therapeutic leads for Huntington’s disease( 2021)Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. The disease, characterized by motor, cognitive and psychiatric impairments, is caused by the expansion of a CAG repeat in the huntingtin gene. Despite the discovery of the mutation in 1993, no disease-modifying treatments are yet available. Understanding the molecular and cellular mechanisms involved in HD is therefore crucial for the development of novel treatments. Protein phosphorylation consists of the addition of a phosphate group on serine, threonine, and tyrosine residues on proteins. This process, catalysed by kinases, regulates numerous cellular mechanisms and has been shown to be dysregulated in other neurodegenerative disorders, including Alzheimer’s disease. Its study in such disorders has led to the discovery of novel therapeutics and biomarkers. However, a potential dysregulation in the phosphorylation machinery had yet to be studied in HD. By using mass spectrometry-based phosphoproteomics on brain samples from a mouse model of HD, we aimed to improve our understanding of the pathogenesis, gene-environment interactions, as well as find potential drug targets. The results presented in this thesis evidence that the protein phosphorylation machinery is dysregulated in the early stages of HD, primarily in the cortex and striatum. Further experiments investigated the involvement of the microtubule-associated protein tau, found hyperphosphorylated, in the development of HD symptoms. Our results indicate that tau, although found altered in HD, is not a suitable drug target to modify the progression of the disease. We also report the effects of an early environmental intervention on the protein phosphorylation dysregulation observed in HD. Environmental enrichment was able to rescue the changes observed in protein phosphorylation in the striatum of HD mice. Altogether, these discoveries provide new insights into the pathogenesis of HD and identify novel therapeutic targets.
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ItemInvestigating ligand- and cell-specific signal transduction at relaxin family peptide receptor 1( 2021)Relaxin, a peptide hormone and the endogenous agonist of the relaxin family peptide receptor 1 (RXFP1), has shown substantial promise for the therapeutic treatment of cardiovascular disorders and fibrosis. RXFP1 has received considerable therapeutic interest as a drug target over the years, and a lot of effort has been put into the development of novel ligands targeting this receptor. However, we do not understand how novel RXFP1 agonists work because we do not understand RXFP1 signalling in enough detail, and do not fully understand which pathways are important for the therapeutic actions, where they are activated in the signal transduction cascade, and in what cell types. Furthermore, development of novel ligands raises the question of biased signalling, which is the ability of different ligands for the same receptor to preferentially activate certain signal transduction pathways relative to one another. It may be possible to utilise bias to create effective drugs that have fewer side effects, but this requires us to first understand which effectors and signal transduction pathways are important for the therapeutic versus harmful actions. Furthermore, recent research has highlighted the importance of measuring the temporal aspects of signalling in order to understand bias, as signalling and bias can change over time, and using end point assays can produce a misleading picture of efficacy and bias depending on which time point was chosen. Additionally, validating findings from recombinant cells in physiologically-relevant cells is also important to understand how ligands signal across cell types, and to distinguish biased signalling from cell-specific signalling. The development of sensitive, real-time assays that can be used across cell types will aid our understanding of the molecular mechanisms underlying the actions of relaxin and other ligands targeting this promising receptor. The general aim of this thesis was to apply and develop bioluminescence resonance energy transfer (BRET)-based methods in conjunction with human native and primary cells in order to examine real-time signalling and bias at RXFP1. First, the functionality and versatility of the CAMYEL (cAMP sensor using YFP-Epac-Rluc) real-time BRET-based cAMP biosensor was demonstrated for RXFP1 and the related GPCRs RXFP2, RXFP3, and RXFP4. CAMYEL was a sensitive alternative to end point assays, as it detected concentration-dependent changes in cAMP activity at all receptors in recombinant cell lines, was dynamic and reversible, detected kinetic differences between different ligands for the same receptor, and showed potencies comparable to those seen in end point assays. CAMYEL was cloned into a lentiviral vector, and lentivirus was used to transduce THP-1 cells, which endogenously express low levels of RXFP1. THP-1 CAMYEL cells showed robust cAMP activation after relaxin stimulation and will therefore streamline the process of screening novel RXFP1 ligands. The lentiviral vector will also allow for the transduction of many mammalian cell types for real-time analysis of cAMP activity at various GPCRs, including in primary cells. However, it appeared that the CAMYEL assay was unable to detect a delayed, Gi3-mediated phase of RXFP1 cAMP activity that has been demonstrated using other assays, suggesting that CAMYEL might not detect cAMP generated in specific compartments of the cell. Second, we developed, validated, and characterised a BRET-based biosensor for cGMP activity, known as CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), based on the Forster/fluorescence resonance energy transfer (FRET) biosensor cGES-DE5. CYGYEL was cloned into a lentiviral vector, enabling its use across different mammalian cell types. CYGYEL was initially characterised in HEK293T cells, where it was shown to be sensitive, dynamic, reversible, and also very selective for the detection of cGMP over cAMP. CYGYEL was then used to detect cGMP after transduction of human primary vascular cells, namely endothelial and smooth muscle cells. CYGYEL detected differences in cGMP signalling kinetics both between cell types, and also between ligands that increased cGMP production via soluble versus membrane guanylate cyclases. So far we have been unsuccessful at detecting GPCR-mediated cGMP using CYGYEL, but further work is required in this area. Regardless, CYGYEL still has many uses for drug discovery. Finally, we used a variety of BRET-based assays for G protein association, second messenger activity, and ERK1/2 activity, as well as physiologically-relevant primary cells, in order to understand the mechanisms of action underlying the beneficial actions of the relaxin peptide analogue B7-33. According to previous work, B7-33 appeared to show cell-specific signalling and biological responses, whereby it had weak activity in recombinant and cancer cells, but potent activity in fibroblasts and vascular cells, as well as in vivo. Our results across several cell types indicated that B7-33 is a biased agonist that favours signalling via Gi3/cGMP over Gs/cAMP, relative to relaxin which signals potently via both pathways. These findings are consistent with B7-33’s actions as a potent vasodilator and anti-fibrotic, which depend on cGMP rather than cAMP. Relatedly, we demonstrated that B7-33 shows transient cAMP activity relative to relaxin in all cell types tested, and that in a real-time cAMP assay involving ligand washout, the cAMP response from B7-33 dropped drastically relative to relaxin, suggesting that B7-33 dissociated from RXFP1 far more readily than relaxin. We thus hypothesised that the bias shown by B7-33 is related to kinetics, whereby the relaxin/RXFP1 complex catalyses more cycles of Gs activation due to the sustained duration of the active receptor conformation, relative to B7-33 which has a faster off-rate associated with its weaker activation of Gs. However, both agonists equally activate Gi3 suggesting that the relative rates of activation and deactivation of the different G proteins may also be important. Finally, it was also observed that ERK1/2 is activated by its upstream effectors in a cell type-specific manner. Specifically, whereas previous findings have shown that ERK1/2 is primarily downstream of Gi/o in native cells, our findings show that ERK1/2 is activated downstream of Gs in HEK-RXFP1 cells, which explains B7-33’s weak ERK1/2 activation in HEK-RXFP1 cells but potent activation in native cells. These findings have implications for the development of novel biased drugs targeting RXFP1, as it is believed that the negative actions of exogenously-administered relaxin, including for example its ability to promote tumour growth in mouse models in vivo, are related to its potent cAMP activity. Conversely, equi-molar doses of B7-33 do not promote tumour growth but do retain the beneficial actions of relaxin which occur via Gi. Thus, we could potentially aim to retain the kinetic bias to maintain potent cGMP signalling, while minimising cAMP activity, and at the same time aim to develop compounds that are more drug-like with longer half-lives.
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ItemInvestigation of the neural circuits underlying stress eating( 2021)The incidence of eating disorders and obesity has reached epidemic levels/proportions worldwide and this has been further exacerbated by the COVID19 pandemic. Binge eating is a hallmark feature of most eating disorders and some subtypes of obesity. Negative affect, such as stress, can strongly influence eating behaviour and drive bingeing episodes and overconsumption of palatable food independent of homeostatic need. This is often followed by feelings of guilt and shame, resulting in greater distress and further perpetuating the stress binge cycle. Notably, stress related binge eating disproportionately affects females more than males, yet the precise biological mechanisms underscoring behavioural sex differences remain underexplored. This has in part been due to the lack of robust animal models that faithfully recapitulate the human condition. In this regard, during my PhD, I developed a highly reproducible model of stress-induced binge eating in female mice that is independent of caloric restriction. Cyclic caloric restriction is commonly applied in animal models of binge eating but is suboptimal for examining reward based feeding and binge eating driven by hedonic processes independent of the metabolic memory of negative energy balance. Having this model established, I then aimed to investigate 1) possible endocrine and centrally mediated processes involved with stress-induced binge eating in female mice; 2) changes in neuronal activation across multiple brain regions following aberrant eating behaviours precipitated by stress; 3) neuronal circuits involved in driving stress induced binge eating. To address these questions, I used a combination of behavioural testing with anatomical tracing and chemogenetic manipulation. The results presented in this thesis demonstrate the reproducibility and applicability of the animal model here developed to investigate multiple aspects of stress eating. Crucially, the stress induce binge eating seen in this model occurred in ovariectomised female mice, suggesting factors other than ovarian hormones must be involved, and that centrally mediated processes instigate this behaviour. Indeed, additional results here reported show the possible involvement of key regions from the mesocorticolimbic, thalamic, and hypothalamic systems in stress induced binge eating and provide insight into a possible neural circuit driving this behaviour. Finally, I show that chemogenetic inhibition of a discrete projection from the paraventricular nucleus of the thalamus to the insular cortex suggested a causal role for this pathway in the emergence of overeating following stress in female mice. The results presented in this thesis provide important insights into underlying biological factors involved in stress-induced binge eating and overeating in female mice.
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ItemToward the structural characterisation of the relaxin receptor, RXFP1( 2021)Relaxin is a peptide hormone that is involved in several physiological processes such as pregnancy, collagen breakdown, fibrosis inhibition and vasodilation. It has been investigated for the use of several disease states such as scleroderma, fibrosis, cancer and most recently acute heart failure. Relaxin’s cognate receptor is the relaxin family peptide receptor 1 (RXFP1), an integral membrane protein belonging to the G protein-coupled receptor (GPCR) family with a complex, multistep activation mechanism which is still not well understood. Given the physiological roles of relaxin, RXFP1 is a promising target for the treatment of abovementioned conditions. However, there is currently a lack of a detailed mechanism in which relaxin mediated activation of RXFP1 occurs and this makes the design of relaxin-like compounds such as long active peptide mimetics, small molecules or biologics targeting RXFP1, or understanding and optimizing existing compounds that act at RXFP1 difficult. The lack of a detailed mechanism of RXFP1 activation can be attributed to the lack of full-length RXFP1 structures. While there are proposed models of this activation mechanism, these models were derived from studies on isolated domains of RXFP1 and thus it cannot be assumed that the findings are similar to that of a full-length RXFP1. Thus, the aim of this thesis was to work toward active and inactive state structures of full-length RXFP1 using cryo-electron microscopy (EM). By solving active and inactive state structures, we can overlay these structures to determine key conformational changes and key residues that interact with relaxin to determine a complete mode of relaxin mediated activation of RXFP1. However, these studies are hampered by the limitations of cryo-EM to study inactive state GPCRs and the low recombinant expression of WT RXFP1 which makes producing sufficient amounts of purified RXFP1 for these studies very difficult. In this thesis we optimised the expression and purification of RXFP1 for the purposes of cryo-EM studies. We also developed and optimised a novel tool, monomeric ultra-stable GFP (muGFP) as an intracellular loop 3 (ICL3) fusion partner to overcome the limitations of inactive state cryo-EM studies. We applied this to a thermostabilised variant of the alpha1A-adrenoceptor and demonstrated its utility for cryo-EM studies before applying it to RXFP1. Next, we applied an established workflow for the production of active state GPCR-G protein complexes in insect cells for cryo-EM studies to WT RXFP1 for the active state studies of the receptor. We also experimented with the expression and formation of an RXFP1-G protein complex in a mammalian expression systems. However, we were unable to proceed to cryo-EM studies of either inactive or active state RXFP1 due to inability to produce sufficient quantities of protein To overcome the limitation of poor protein yield, we developed a novel mammalian cell-based method of directed evolution. Existing methods of GPCR directed evolution are primarily E. coli based, and as RXFP1 is unable to be expressed in E. coli due to requiring post-translational modification, a mammalian system was required. We applied this novel method to RXFP1 and were able to evolve mutant #35, which demonstrated an ~9x increase in recombinant RXFP1 expression. Additionally, we also identified 2 mutants that demonstrated interesting pharmacological changes from WT. This includes a mutant that demonstrated an increase in basal signalling, and another mutant that demonstrates a decreased pEC50 for relaxin, that is a higher concentration of relaxin is required to produce an equivalent response in WT. By evolving high expressing mutant #35, we could potentially overcome the bottleneck of insufficient purified protein yield for cryo-EM studies. By applying mutant #35 to the workflows developed in this thesis, we can potentially enable downstream cryo-EM studies of RXFP1 through the ability to produce ~9x more protein than WT. Through enabling these studies, we may be able to elucidate the mechanism in which relaxin triggers RXFP1 activation in a full-length receptor. Understanding this mechanism in atomic resolution detail through cryo-EM studies could then facilitate rational drug design of novel relaxin-like mimetics for the treatment of acute heart failure or fibrosis or antagonists for the treatment of certain cancers.