Anatomy and Neuroscience - Theses
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ItemInvestigating the role of neuronal TrkB in remyelination( 2023-02)Demyelinating diseases, such as multiple sclerosis (MS), involve damage to the fatty, proteinaceous myelin sheaths surrounding nerve fibres and subsequent development of neurological symptoms related to the damaged area. Repair of the damaged myelin can completely or partially reverse the neurological deficits, however, over time with repetitive demyelinating events and incomplete remyelination this ultimately results in the degeneration of neurons and development of permanent disabilities. Currently, there is a lack of therapies effective in promoting myelin repair. A complete understanding of the factors that promote remyelination is critical for developing therapeutic strategies for myelin repair to prevent permanent neurological disability. Many studies have focused on the potential of oligodendrocytes (the myelin-generating cells in the central nervous system) to enhance the generation of myelin. However, neurons certainly have an intimate relationship with oligodendrocytes and are well-positioned to influence the process of myelination. Previously, neuronal expression of the tyrosine kinase receptor TrkB has been shown to promote myelination in early development and during ageing. However, whether neuronally expressed TrkB receptors also influences the extent of oligodendroglial and myelin damage post a demyelinating injury, and the subsequent process of remyelination has not been elucidated. In this thesis, I generated an inducible, neuron-specific TrkB receptor knockout mouse line to investigate the role of neuronal TrkB receptors in the extent of both demyelination and remyelination following the induction of a demyelinating insult with cuprizone administration. I found that following deletion of TrkB, these mice exhibit a normally myelinated nervous system, but do however develop a motor phenotype consistent with a decrease in precise motor control. Following administration with cuprizone, I revealed that neuronal expression of TrkB is important for the differentiation of OPCs (oligodendrocyte progenitor cells) that are generated in response to the demyelinating insult. Finally, although reduced expression of neuronal TrkB did not influence the overall extent of remyelination, I found that axons with small diameter required expression of neuronal TrkB to be efficiently remyelinated. This work is the first to identify that neuronally expressed TrkB receptors have a role in OPC differentiation following demyelination. This thesis suggests there is therapeutic potential in targeting neuronal TrkB signalling to promote myelin repair, albeit this may only be specific to smaller diameter neurons.
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ItemThe physiological relevance of ghrelin and dopamine D2 receptor (D2R) interactions( 2023-05)Disorders of colorectal function, especially constipation, are poorly responsive to drug therapy and are a substantial medical problem. It is estimated around 15-25% of Australians over the age of 65 suffer from constipation. This presents a burden for patients and society that must be addressed. Centrally penetrant ghrelin and monoamines (dopamine, serotonin) have pro colokinetic effects, enhancing colonic motility. Agonists likely act on the same neurons in the lumbosacral defecation centre, where their cognate receptors exist, although this has not been investigated. Intriguingly, the ghrelin receptor (GHSR) but not ghrelin can be identified in this region, suggesting either an orphan ligand or an apo-ghrelin receptor function. I hypothesised that dopamine, ghrelin, and serotonin act at parasympathetic preganglionic neurons (PGNs) in the lumbosacral defecation centre, where I predict GHSR, 5-hydroxytryptamine receptors (5-HTR) and dopamine D2 receptors (D2R) exist. Furthermore, I predict that GHSR and D2R functionally interact in these neurons, although this has not been examined. Using RNAscope I identified transcript for Ghsr and Drd2 in the same PGNs (identified by Chat transcript), in the lumbosacral defecation centre in rodents (both rats and mice). I also found retrogradely labelled PGNs in neonatal rats were excited by dopamine and the GHSR agonist (capromorelin) in the same neuron. In adult Drd2-tdTomato (D2R) reporter mice the postsynaptic excitatory effect of dopamine was masked by activation of inhibitory interneurons, an effect that was reversed using TTX. In addition to this, I showed using calcium imaging that dopamine increased calcium in D2R neurons in the lumbosacral defecation centre. The ability for dopamine to excite these neurons is unusual, as D2R coupling in midbrain neurons typically liberates Gbetagamma (Galphai/o), activating GIRK channels, leading to neuronal hyperpolarisation. Using whole cell electrophysiology and pharmacology, I uncovered that excitation of D2R by dopamine was dependent on GHSR, as antagonists and inverse agonists (YIL781/JMV2959) of GHSR reduced the excitatory effect of dopamine. Furthermore, I found that these GPCRs act synergistically through the downstream mediator PLCbeta, increasing [Ca2+]i and causing neuronal depolarisation, likely via a calcium sensitive non-specific cation channel. Work in vitro has complemented this work showing that PLCbeta activation is dependent on both Galphai/o and Galphaq signalling, as the inhibition of either of these G proteins or PLCbeta in stably transfected cells containing GHSR and D2R prevents [Ca2+]i release. Additionally, high resolution microscopy revealed that these receptors do not physically interact “heterodimerise” as many groups have hypothesised. Moving forward, these results provide the basis for promising therapeutic approaches to treat not only constipation but a wide range of other diseases. Additionally, it opens up the field of GPCR biology to start investigating other tissues where this altered receptor transducer signalling might be having a physiological effect.
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ItemCharacterisation of a novel liver progenitor cell marker in biliary atresia( 2022-06)The characterisation of liver progenitor cells (LPC) has proven to be difficult, likely due to plasticity in the regenerative/reparative processes of the liver based on the type and extent of injury. The recent development of an antibody to the GCTM-5 epitope has identified a putative liver progenitor cell population which was not previously described in the paediatric population. GCTM-5 was previously identified to strongly mark cells in the foetal ductal plate, early in the developmental pathway of both hepatocytes and cholangiocytes. This study describes the distribution of this antibody in patients with biliary atresia at time of paediatric liver transplant, and compares it to other known markers for cells with progenitor-like properties. We conducted a chart review of all patients with biliary atresia undergoing a liver transplant between 1995-2015 at the Royal Children’s Hospital, Melbourne. Multiple samples that had been stored from the explanted liver of patients with biliary atresia were stained for ENPRO-1, a second-generation antibody with greater affinity for the same epitope stained by GCTM-5. We found that the ENPRO-1 antibody most strongly marked ductular reaction cells in this human population, which is a histological description known to contain the LPC niche. It also identified undifferentiated EpCAM positive cells, most SOX9-positive cells, all NCAM-positive cells and some Lgr5-positive cells. As such, ENPRO-1 was found to mark a novel subset of liver cells of previously identified cells with progenitor properties. This is critical to validate future studies for a serum biomarker for activation of this LPC niche, as the epitope marked by the ENPRO-1 antibody is known to be secreted into blood. Unexpectedly, we found CD133/PROM1 to strongly mark almost all parenchymal hepatocytes in regenerative nodules, which has not previously been described, and may be unique to patients with end-stage liver failure due to biliary atresia.
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ItemCommitment to Serotonergic Signalling: Evolution and Distribution of the Serotonin Receptors( 2022)Serotonin is a subclass of neurotransmitters and a relatively simple metabolite. The serotonin system underpins a multitude of biological functions by coupling with diverse serotonergic signalling. The effectors of serotonergic signalling are the serotonin receptors: a family of GPCRs (G protein-coupled receptors). These receptors are associated with diverse biological functions and are the primary sites of action for many psychotherapeutic drugs. We describe what commitment to serotonergic signalling entails by quantifying the diversity of this family in terms of receptor sequences, and expression patterns in the brain and systemic tissues. Sampling receptor sequences from the HTR (serotonin receptors) family, we constructed an updated phylogeny. We interpolated Bayesian priors to include key evolutionary events and to date the evolution of the family. Using RNA sequencing data, we provide the first systematic evaluation of the HTR distribution in the human body and organised the family based on their shared expression patterns. The phylogeny recapitulated early radiation of HTRs predating vertebrate evolution and demonstrated the three present clades of Galphas-coupled HTRs lack a singular ancestral node. The RNA-sequence analysis identified the systemic as well as brain-specific receptors, and reproducibly detected ten family members in the brain, which could be sub-classified by their co-expression patterns in cortical and subcortical regions. Together, the phylogenetic tree and the transcriptome map underscore the diversity of the HTR family, with multiple members evolved to activate all types of Galpha pathways specifically in the brain as well as systemically. Thus, through applying ‘omics data, the thesis outputs present the first systematic description of the family. The findings of this thesis reinforce the fact that multiple serotonin receptors are not evolutionary redundancies, but rather each receptor corresponds to specialised tissue distribution. An accurate understanding of the commonalities and contrasts among the subtypes would aid the development of subtype-specific drug targets.
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ItemPlasticity in the Enteric Nervous System( 2022)The gut is constantly exposed to fluctuating environments and stimuli. To maintain optimal digestion, absorption of nutrients and waste disposal, the gut must adapt to these changing conditions. Within the gut wall is a network of neurons and glia known as the enteric nervous system (ENS), which regulates the major functions of the gut, including secretion, absorption, and motility. However, the ENS circuitry that underlies plasticity in the gut is unclear. This thesis aimed to explore different aspects of plasticity in the myenteric plexus using the mouse as an animal model, to increase our understanding of how changes in gut motility is regulated. In Chapter 3, the role of Group I metabotropic glutamate receptors (mGluRs) in enteric neurotransmission was investigated. Group I mGluRs are involved in synaptic plasticity in the central nervous system (CNS), but their role in the ENS has been unclear. Live Ca2+ imaging was performed on ex vivo preparations of Wnt1-cre;GCaMP6f mouse colon, where all neurons and glial cells of the ENS express the genetically encoded calcium indicator, GCaMP6f. It was observed that some myenteric neurons were activated by the Group I mGluR agonist, DHPG, exhibiting increases in intracellular Ca2+ concentration ([Ca2+]i). Responses were recorded from calbindin and nNOS-immunoreactive neurons, which mostly represent the intrinsic sensory neurons and inhibitory motor neurons of the ENS, respectively. The role of the individual Group I mGluRs (mGluR1 and mGluR5) on endogenous synaptic transmission was examined by the application of specific antagonists following electrical stimulation of interganglionic fibre tracts. In addition, an ex vivo video imaging assay of colonic motor complexes (CMCs) revealed a role for mGluR1 in the initiation of CMCs, as but no role for mGluR5 in CMC generation. These data suggest a complex role for Group I mGluRs in the ENS. Chapter 4 investigates the effect of circadian rhythm on ENS function. Colonic motility fluctuates during the circadian cycle, and gut dysfunction can occur when the circadian cycle is disrupted. This chapter investigated whether ENS function is altered during the circadian cycle in mice. Using live Ca2+ imaging, responses to the application of different neurotransmitters and neurotransmitter receptor agonists were examined. Overall, myenteric neurons had increased [Ca2+]i transients in response to the application of agonists during the dark phase (when mice are active) compared to the light phase (when mice are asleep). A separate cohort of mice were fasted for 13 hours prior to live Ca2+ experiments, and showed increased [Ca2+]i following agonist application during the light phase compared to the dark phase. This suggests that circadian neuroplasticity in the ENS can occur within a short timeframe and depends on the availability of food and feeding time. Finally, Chapter 5, explores viral transduction as a technique for understanding neuronal connectivity. Abdominal surgery was performed on mice to inject AAV9-CB7-eGFP into the distal colon. Dense labelling of GFP was observed in the distal colon myenteric plexus, near the injection site, while sparse labelling of myenteric neurons was observed in the proximal colon. GFP was observed in neurons expressing Calbindin, Calretinin, and nNOS. Some colocalisation was observed between GFP+ varicosities and Enk+ varicosities, but there was no colocalisation between GFP+ and VIP+ or CGRP+ varicosities. These data confirm that AAV9 can be used to transduce different neurochemical subtypes in the myenteric plexus, which is important for future studies investigating neuronal connectivity. Together, this thesis provides the first evidence that Group I mGluRs are involved in neural control of gastrointestinal motility in mice, and that there is short-term plasticity of myenteric neurons during the circadian cycle. Combined with preliminary observations of using AAV9 to transduce myenteric neurons, these data lay the groundwork for future studies to explore neuronal connectivity and plasticity in the ENS.
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ItemThe interplay between clonal hematopoiesis and cardiometabolic diseases( 2022)INTRODUCTION: Clonal haematopoiesis of indeterminate potential (CHIP) is a blood disorder arising from somatic mutations in haematopoietic stem and progenitor cells (HSPCs), providing these cells with a competitive advantage. This results in an increased abundance of mutant cells (>2%) in the blood. CHIP correlates with an increased risk of atherosclerotic cardiovascular disease (ACVD), proportional to the clonal expansion rate. However, the factors that control clonal outgrowth are not well understood. The most frequently mutated genes in CHIP are the epigenetic regulators DNMT3A and TET2. Dnmt3a driven-CHIP has been shown to aggravate disease progression in CVD, however the role of Dnmt3a-CHIP in ACVD remains limited. Importantly, diabetic patients present with higher incidence of CHIP. Diabetes induces TET2 loss-of-function via dysregulation of the metabolic sensor AMPK in mice and humans. In CHIP, DNMT3A and TET2 are commonly affected but their combined deficiency results in a myeloproliferative disorder. Thus, the hypothesis of this PhD thesis herein was two-fold. Firstly, that Dnmt3a-driven CHIP is accelerated in diabetes due to AMPK-TET2 dysregulation and re-activating this pathway will reverse DNMT3A clonal expansion in the setting of diabetes. Secondly, we hypothesized that Dnmt3a-driven CHIP will play a causal role in atherogenesis. METHODS AND RESULTS: In isolated BM HSPCs we observed that high glucose levels impaired the AMPK-TET2 axis, resulting in a reduction in DNA hydroxymethylation (5hmC), which was restored by activating AMPK. In a murine model of type 1 diabetes, we observed monocytosis, which was associated with TET2 dysfunction in BM HSPCs and blood myeloid cells. We next investigated how diabetes induced TET2 dysfunction would affect Dnmt3a-driven-CHIP. Using competitive bone marrow transplants (cBMT), we mimicked human mutant Dnmt3a-driven CHIP in WT mice and noted that diabetes exacerbates mutant myeloid clonal expansion due to enhanced myelopoiesis. Notably, BM HSPCs from mutant Dnmt3a CHIP diabetic mice exhibited TET2 dysfunction. Diabetes-driven TET2 dysfunction in HSPCs and DNMT3A-mutant myeloid clonal expansion were reversed by AMPK activation. Another metabolic state associated with CHIP is obesity, where AMPK activity is also reduced. We hypothesised that obesity would also induce TET2-dysfuntion. Obese models of Dnmt3a-CHIP had TET2 dysfunction in BM HSPCs along with monocyte clonal expansion and an overall decline and whole-body metabolism. This was associated with mutant macrophage infiltration in white adipose tissue and liver. Finally, we explored how Dnmt3a-CHIP affects atherosclerosis, and if clonal expansion was associated with worsened atherosclerotic outcome. Indeed, in a plaque progression model we confirmed that Dnmt3a-CHIP was associated with myeloid clonal expansion and enhanced thrombopoiesis. The increased platelet counts were associated with mutant macrophage infiltration into the livers and higher levels of TPO in the plasma. Dnmt3a-CHIP mice presented increased mutant monocytes, neutrophils and macrophages in the aortic arch which was paralleled with increased lesion size and lipid content. CONCLUSION: Herein we show that diabetes dysregulates the AMPK-TET2 axis in BM HSPCs and promotes myeloid clonal expansion in Dnmt3a-CHIP. Importantly, activating AMPK restores TET2 function and reduced clonal expansion in diabetes. CHIP associated with Dnmt3a deficiency was shown to accelerate atherosclerosis progression due to pronounced myeloid clonal expansion and thrombopoiesis. These studies suggest that targeting AMPK-TET2 pathway may provide a novel approach to limit clonal outgrowth in diabetic patients with CHIP.
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ItemSez6: a new potential target for neuropathic pain hypersensitivity( 2022)Seizure-related protein-6 (Sez6) is a neuronal protein that supports excitatory synapse development and maintenance. Sez6 has already been implicated in numerous psychiatric and neurodegenerative conditions, and new evidence from our lab has demonstrated that Sez6 may also be involved in neuropathic pain. Neuropathic pain is a severe form of chronic pain that affects up to 10% of the global population. Despite the high prevalence, a lack of efficacy and dangerous side effects common in current therapies means that new treatments are desperately needed. A growing body of research has demonstrated the essential role of the peripheral somatosensory nervous system in neuropathic pain. Targets within this system present an exciting opportunity for new therapeutics. This study aims to map out Sez6 expression in the somatosensory nervous system and determine if peripherally expressed Sez6 is involved in injury-induced neuropathic pain. To characterise Sez6 expression in the peripheral somatosensory nervous system, dorsal root ganglia (DRG) from multiple vertebral levels and sympathetic superior cervical ganglia (SCG) were dissected from WT mice and immunostained for Sez6 and various neuronal markers. This showed that Sez6 expression was more prominent in sensory neurons of the DRG than sympathetic neurons of the SCG and was mostly expressed in peptidergic C-fibres. To examine the contribution of peripheral Sez6 in a chronic constriction injury (CCI) model of neuropathic pain, Advillin-Cre(ERT2)xSez6(fl/fl) mice were used to delete sez6 in neural crest-derived peripheral neurons, including DRG neurons. Peripheral neuron Sez6 knockout (KO) ameliorated heat hyperalgesia in mice, and post-mortem examination of the injured sciatic nerve showed a reduced density of CD11b+ immune cells. The transmembrane isoforms of Sez6 can be cleaved into an active soluble protein by beta-site amyloid precursor protein cleaving enzyme-1 (BACE1). BACE inhibitors have been previously used in clinical trials for Alzheimer’s Disease. The final aim of this thesis was to investigate the possibility of repurposing these BACE1 inhibitors to treat neuropathic pain. Sez6 KO and wildtype (WT) mice were given either BACE inhibitor or vehicle control and subjected to CCI. Hargreaves and von Frey’s testing revealed that BACE inhibition in WT mice completely abolished mechanical allodynia and heat hyperalgesia. This thesis presents the first comprehensive study of Sez6 in the peripheral nervous system and provides evidence for both peripheral Sez6 and BACE1 as potential targets for new neuropathic pain treatments.
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ItemModelling sensory neuron plasticity in pulmonary disease with novel co-culture approaches( 2022)The mucosa of the respiratory tree is densely innervated by sensory nerve fibres that monitor the local environment and contribute to pulmonary function and defence. Hypersensitivity of this sensory circuitry accompanies mucosal dysfunction and excessive cough in a variety of pulmonary diseases, thus contributes significantly to patient morbidity. The local mucosal mechanisms driving hypersensitivity and the accompanying phenotypic effects in sensory neurons are not well described. One hypothesis is that the epithelium-nerve functional unit is altered, perhaps mediated by disease-induced changes in the epithelium. Indeed, there exists a large body of literature demonstrating injury and reprogramming of the epithelium in many pulmonary diseases. It is proposed that this dysfunctional epithelium changes the phenotypic and/or functional properties of innervating neurons. This has not been directly confirmed, however, as it is difficult to study nerve-epithelium interactions in vivo and there are currently limited in vitro models available to directly investigate disease-specific changes to sensory neurons and their interaction with epithelial cells. In this methodological-driven project, I set out to develop novel in vitro preparations consisting of stem cell-derived or primary vagal sensory neurons and human airway epithelial cells in order to further understanding of the epithelium-nerve functional unit and facilitate the future identification of novel targets for alleviating the sensory-associated symptoms of lung disease. In the first model, human epithelial cells and murine vagal sensory neurons were utilised. The epithelial cell line BCi-NS1.1 was grown at air-liquid interface (ALI) to generate tissue that expressed important proteins and genes that define epithelial cell subtypes; secretory cells (MUC5A and MUC5B), basal cells (TUBA1A, TP63) and ciliated cells (TUBB4A, FOXJ1). Vagal ganglia were dissected from wildtype C57BL/6 mice, enzymatically dissociated and cultured, with neurons identified and characterised via their expression of pan-neuronal markers (TUBB3, ACTB) and specialised markers (P2X2, PPT-A, TRPV1 and TRPA1). Media conditions were optimised to allow ALIs to be indirectly co-cultured with these sensory neurons (shared media but no direct cellular contact). Co-cultured neurons possessed significantly longer neurites than those grown without epithelial cells (neurons only = 640 + 226.6 cm; co-culture = 916 + 350.6 cm; P<0.05) but had molecular expression profiles different to acutely isolated cells, as percentage of neurons expressing PPT-A, P2X2, TRPV1 and TRPA1 was reduced. Altered growth of neurons in co-culture conditions was indicative of the existence of epithelial paracrine mediators in the co-culture system that evoke minimal phenotypic changes in neurons. The mechanism of communication between epithelial cells and neurons was not completely dependent on growth factors since blockade of Trk tyrosine kinases did not completely attenuate the increased growth in co-culture conditions. The indirect (paracrine) co-culture model has also enabled preliminary investigation into interaction of the epithelial cells and neurons under challenge conditions, specifically looking at the viral analogue poly(I:C). This challenge was chosen because respiratory viral infections have been linked to the development and exacerbation of respiratory symptoms in a range of pulmonary diseases, and evidence has accumulated suggesting that perturbed antiviral host defence processes may contribute to disease susceptibility. Treating the ALI epithelial tissue with 12 ug/ml of poly(I:C) significantly reduced the growth of co-cultured neurons (co-culture sham (PBS in apical compartment) = 764 + 22.34 cm; co-culture PIC = 468 + 11.79 cm; P<0.05), consistent with the challenge evoking damage to the neurons. Treating neurons with the selective TRPA1 antagonist HC-030031 prevented this epithelial poly(I:C) treatment-mediated reduction in neurite growth (co-culture PIC = 468 + 11.79 cm; co-culture PIC+HC = 938 + 11.50 cm; P<0.05). The second model under development utilised human cells only. Unlike epithelial cells, human sensory neurons could not be harvested for cell culture but were derived from human embryonic stem cells (hESCs) differentiated into sensory neurons by optimising a protocol using small inhibitors and growth factors. Neural crest induction was assessed using SOX10 expression. Young protocol cultures stained positive for SOX10 at day 5 (60% of all cells), day 6 (71%), day 7 (94%), day 8 (83%), day 9 (75%) and day 10 (73%) of differenation. To determine whether the derived cells resembled sensory neurons, morphological and functional endpoints were assessed. Electrical recordings from single cells demonstrated functional properties that are consistent with immature sensory neurons (membrane potential -44.67 + 10mV, action potential threshold -36.02 + 4.88mV). Calcium recordings demonstrated transient responses to TRPV1 agonist capsaicin (21%). In addition to staining positive for TRPV1 (24%), hESC-derived cultures stained positive other important proteins and genes that define sensory neuron subtypes; MAP2 (34%), CALB (4%), PGP9.5 (46%), PRPH (32%), P2X3 (40%), P2X2 (8%), SP (0%), NKR1 (34%), CGRP (24%) and VGLUTs (23%). RNA sequencing confirmed the presence of Tac1, Ntrk2, Prph, Trpv1, Scn9a, Piezo2, and Slc17a7 in Young cultures.
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ItemIdentification of novel biomarkers and treatments for non-alcoholic fatty liver disease and associated co-morbidities( 2022)Non-alcoholic fatty liver disease (NAFLD) is characterised by impaired lipid metabolism, hepatic fibrosis and linked to systemic insulin resistance, which is partly mediated by remodelling of liver-secreted proteins or “hepatokines.” There are no effective screening tools or approved pharmacotherapies for NAFLD and NAFLD-associated fibrosis. Proteomic studies estimate that ~25% of circulating proteins are liver-derived, suggesting that hepatokines could be biomarkers and, due to well described impacts on energy homeostasis, effective treatments for NAFLD and NAFLD-associated fibrosis. This thesis aimed to discover novel biomarkers and treatments for NAFLD, non-alcoholic steatohepatitis (NASH), and NAFLD with significant fibrosis (NAFLD F2-3). For biomarker discovery, bariatric patients were prospectively recruited, plasma and a liver wedge biopsy were procured, livers were precision-cut to assess hepatokine secretion and were grouped based on histological confirmation of NAFLD, NASH, and NAFLD F2-3. Chapter 3 identified 3333 liver-secreted proteins, of which, 107 and 5 hepatokines were remodelled with NASH and NAFLD F2-3, respectively. EMILIN1 secretion was increased in NASH, NAFLD F2-3, and in the plasma of NASH patients. However, plasma EMILIN1 poorly stratified patients NASH. In Chapter 4, we speculated that hepatokine secretion may not correspond to changes in plasma levels. Notably, highly abundant plasma proteins corresponded to ~62% of the proteins detected in the human liver-secreted proteome. Plasma proteomics showed few changes in patients with NASH and NAFLD F2-3. Proteins APOF and FCN3 stratified patients with NASH and IGKV2-28 and APCS outcompeted existing non-invasive scores to stratify patients with NAFLD F2-3. Together, this provides a novel resource characterising human liver-secreted and highly abundant plasma proteins and identified IGKV2-28, APCS, APOF, and FCN3 as biomarkers for NAFLD-associated fibrosis and NASH. In Chapters 5 and 6, we exploited hepatokine remodelling to uncover novel treatments for NAFLD and hepatic fibrosis. In Chapter 3, we detected EMILIN1, which was previously reported to reduce TGF signalling. Hepatocyte-specific overexpression of EMILIN1 with severe NASH and advanced fibrosis reduced TGF signalling and hepatic fibrosis, while overexpression of EMILIN1 mice in a mouse model of mild NASH did not impact fibrosis or metabolic co-morbidities, suggesting that EMILIN1 could be a therapeutic target for advanced, but not mild, fibrosis. Physical activity can resolve NAFLD and improve its metabolic co-morbidities, however, the effects of exercise-training on hepatokine secretion and the metabolic impact of exercise-regulated hepatokines in NAFLD remain unresolved. Chapter 6 assessed whether exercise-training could remodel hepatokine secretion to mediate improvements in NAFLD and associated co-morbidities. Mass spectrometry proteomics analysis detected 2657 intracellular and 1593 secreted proteins from isolated mouse hepatocytes. Exercise-training remodelled the hepatocyte proteome, with differences in 137 intracellular and 35 secreted proteins. Hepatocyte-secreted factors from exercise-trained mice improved insulin action in skeletal muscle and increased hepatic fatty acid oxidation. Hepatocyte-specific overexpression of SDC4 reduced hepatic steatosis, which was associated with reduced fatty acid uptake, and blunted pro-inflammatory and pro-fibrotic gene expression. Treating hepatocytes with recombinant SDC4 recapitulated these effects. Together, the work in this thesis addresses an unmet clinical need to discover novel biomarkers and therapeutics to treat NAFLD and co-morbidities.
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ItemIron homeostasis in models of skeletal muscle atrophy( 2022)Skeletal muscle is one of the most active, adaptive, and resilient tissues in the human body, with an innate capacity to regenerate after injury. Dysregulation of protein synthesis and protein breakdown can lead to skeletal muscle wasting which is associated with a wide range of conditions, including genetic mutations (e.g., Duchenne muscular dystrophy), age-related wasting (sarcopenia) and different forms of injury (including ischemic reperfusion damage). Despite differences in their causality, all of these chronic and acute illnesses/injuries are linked by mechanisms that include abnormal ROS generation and inflammation. Iron, one of the most abundant trace metals, plays a crucial role in oxidative metabolism but it can also generate ROS. Due to the high energetic demand, skeletal muscle contains a significant iron load, but few studies have investigated the detrimental consequences of excess iron. To this end, my thesis research investigated the contribution of iron dysregulation to muscle atrophy. We discovered and characterised skeletal muscle iron overload using novel laser ablation-inductively coupled-mass spectrometry (LA-ICP-MS) technology in multiple models of muscle atrophy, including genetic murine models of Duchenne muscular dystrophy (mdx and dko), aged mice relative to adult controls, and in injured muscles using a model of ischemia-reperfusion injury. We subsequently investigated the therapeutic potential of reducing iron levels via iron chelation (deferiprone; DFP) and by overexpressing myoglobin, the muscle specific iron binding protein. In Chapter 3, we showed that DFP treatment (4 weeks; 100 mg/kg/day) could improve aspects of the dystrophic pathology: fibrosis and ROS generation (DHE) were reduced in diaphragm muscles of DFP treated mdx mice. However, the reduction in iron decreased the abundance of haemoproteins (myoglobin and cytochrome c) and compromised mitochondrial function (reduced citrate synthase activity). To overcome this paradox, in Chapter 4 we demonstrated that overexpression of myoglobin in dko mice maintained haemoprotein expression while decreasing fibrosis. In Chapter 5 we review the literature regarding iron chemistry in skeletal muscle and discuss the emerging field of iron homeostasis in sarcopenia. In Chapter 6 we identified an exacerbated iron overload in an aged mouse model of haemochromatosis (a common genetic disorder). The increased skeletal muscle iron was associated with decreased haemoproteins, and proteins involved in oxidative metabolism. In Chapter 7 we found chronic (12-week) DFP treatment in aged mice reduced iron and ferritin in the liver but not in skeletal muscle. We found that iron overload was associated with increased lipid peroxidation (4HNE) and ischemia-reperfusion injury exacerbated the accumulation of iron, ferritin and lipid peroxidation in muscles of aged mice compared to adult controls. However, an attempt to reduce iron pre- or post-injury in aged mice exacerbated the already impaired regeneration. The lack of efficacy of DFP prompted a shift from iron to ferritin. In Chapter 8 we conducted preliminary experiments regarding the administration of ferritin to skeletal muscle and found it impeded muscle regeneration (after ischemia-reperfusion injury), which was associated with an exacerbated inflammatory response and dysregulation of haem metabolism. In conclusion, my thesis research characterised an underlying iron dyshomeostasis (increase in iron and ferritin) in multiple models of muscle atrophy associated with chronic inflammation and elevated ROS (including DMD, sarcopenia and IRI). We identified that iron chelation/reduction in skeletal muscle is largely ineffective and propose that future studies should alter iron distribution via modulating haem synthesis, increasing myoglobin and/or ferritin breakdown/clearance.