Anatomy and Neuroscience - Theses
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ItemThe role of neurotrophic factors in osteoarthritis pain( 2023-10)Introduction: Osteoarthritis (OA) is a progressive disease of synovial joints and subchondral bone characterized by swelling, stiffness and pain. Brain-derived neurotrophic factor (BDNF) and artemin (ARTN) are neurotrophic factors that are important regulators of pain, and have recently been implicated in the pathogenesis of OA pain. This study aimed to explore roles for BDNF and ARTN in OA pain, by investigating whether the expression of BDNF and ARTN, and their receptors (TrkB and GFRa3), is altered in different tissues at different stages of OA, and whether blocking their signalling during late-stage OA can alleviate pain. Methods: The monoiodoacetate (MIA)-induced OA of the rat knee joint was used to explore roles for BDNF and ARTN signalling in OA pain. Pain behaviour was assessed using the dynamic weight-bearing apparatus to assay OA-induced changes in hindlimb weight bearing behaviour, at different stages of disease (early vs late). Histopathological alterations in the knee joint and surrounding bones were assayed using Haematoxylin and Eosin staining and scored using a modified OARSI scale. Changes in expression of BDNF/TrkB and ARTN/GFRa3 were explored using Western blot analysis of lysates from different tissues (joint, bone, and DRG), and at different timepoints of the disease (early vs late). The dynamic weight-bearing assay was used to determine if inhibiting BDNF signalling (with a peptide mimetic TrkB inhibitor) or ARTN signalling (with a sequestering antibody) could relieve pain at late-stage disease. Results: The results of this thesis highlight differential histopathological changes occurring in the early and late stages of OA, with joint involvement being prominent in early OA, and bone and cartilage involvement in late OA. BDNF expression was increased in the joint in early OA and in the bone in late OA. ARTN expression was also increased in the joint in both early and late OA and in the bone in late OA. Attempts to alleviate pain in MIA-injected animals by targeting the BDNF/TrkB and ARTN/GFRa3 signalling pathways did not yield pain relief outcomes with the therapeutic approach chosen in this study. Conclusion: Our findings suggest that altered pain behaviour in early MIA-induced OA is associated with changes in the joint not surrounding bones, while altered pain behaviour in late MIA-induced OA are attributable to the surrounding bones. Furthermore, BDNF and ARTN may contribute differentially to pain in early and late stages of MIA-induced OA through actions in joint versus bone. These findings support further investigations into the role of BDNF and ARTN signalling in OA pain and the development of novel targeted therapeutic approaches for managing OA pain.
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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 role of neuronal TrkB in central nervous system myelination( 2019)Myelin, the specialised membrane surrounding many axons in the nervous system, is vital for the normal sensory and motor function as well as high order function such as learning and memory. Despite the importance of myelin in neural development and functions, little is known whether neuronally expressed molecules can promote the central nervous system (CNS) myelinating process. Through selectively deleting the expression of neurotrophin receptor TrkB in neurons in vivo (TrKB cKO), I have found that neuronal TrkB is essential for oligodendroglial cell production and lineage progression in multiple CNS regions including the lumbar spinal cord white matter tracts, cerebral cortex and corpus callosum and importantly de novo myelination, independent of axonal number and calibre in vivo. I have performed ultrastructural analysis of myelinated axons and found that TrkB cKO mice have significantly fewer myelinated axons compared to control mice during early postnatal development, indicative of delayed initiation of myelination. The analysis of myelin thickness via G ratio has revealed thinner myelin membranes in TrkB cKO mice compared to littermate control mice during development, persisting to late adulthood (n=3 mice/genotype/timepoint). This hypomyelinating phenotype has resulted in impaired myelin function, as evident by reduced optomoter responses in TrkB cKO mice compared to control mice. Moreover, I have found that TrkB cKO mice have significantly fewer oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes in both white and grey matter regions throughout postnatal development and into adulthood compared to littermate controls. A 24-hour EdU labelling experiment has demonstrated that neuronal TrkB expression is required for oligodendroglial production. Interestingly, the overall number of astrocytes and microglia has remained unchanged after neuronal TrkB deletion, suggesting a specific effect upon oligodendroglial lineage cells and myelination. In addition, assessment of neuronal morphology revealed no significant difference in overall dendritic branching including the number of primary and secondary processes. Taken together, results of this PhD study identify that TrkB is a novel neuronal signal that instructs oligodendroglial lineage development and de novo myelination within different neural circuits, indicating a new mechanism that underpins nervous system plasticity.
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ItemTropomyosin related kinase B (TrkB) regulates neurite outgrowth via a novel interaction with suppressor of cytokine signalling 2 (SOCS2)( 2017)Suppressor of cytokine signalling 2 (SOCS2) negatively regulates cytokine signalling but it also has a positive effect on neurite outgrowth of cortical and dorsal root ganglion neurons. It has a high expression profile in the central nervous system in early development stages; hence any changes in the expression of SOCS2 results could result in alternations in the structure of neurites and dendritic morphology. SOCS2 has the highest expression in the hippocampus compared to other members of the SOCS family and this study aims to reveal the role of SOCS2 in the growth of hippocampal neurites in the presence of BDNF. Hippocampal neurons derived from neonatal mice are a great model of BDNF dependent study mediated by neurotrophin receptor BDNF. Neurons were derived from mice with overexpression or knock out of SOCS2 and the length of neurites were analysed in response to BDNF. While normal levels of SOCS2 did not result in any change in the length of neurites, neurons with overexpression of SOCS2 had increased length in response to BDNF. Knockout of SOCS2 resulted in shorter neurites although with BDNF treatment the length of neurites reached normal levels. The co-regulatory role of SOCS2 and BDNF in neurite outgrowth of hippocampal neurons in-vitro draws a possible link between SOCS2 and BDNF receptor, TrkB. The interaction of SOCS2 and TrkB was shown and studies in detail. Truncated isoforms and deleted mutants of TrkB and SOCS2 were employed to reveal the regions involved in the interaction. This study showed the complex interaction of SOCS2 and TrkB happens at two points. The first point is the protein-protein link between the kinase domain of TrkB and the SH2 region of SOCS2. The second point of interaction is through the juxtamembrane region of TrkB. Domains of SOCS2 that are involved in interacting with TrkB were assessed in their ability to regulate neurite outgrowth. Rat hippocampal neurons transfected with deleted mutants of SOCS2 shows that deletion in the BOX domain increases neurite length compared to SOCS2 full length indicating the importance of the interaction of the SH2 and N terminal domain in neurite growth. Intracellular events and downstream pathways stimulated by BDNF and mediated through TrkB play an important role in the outcome of TrkB signalling. SOCS2 through interacting with TrkB can influence such events. SOCS2 increases both phosphorylation and ubiquitination of TrkB, ubiquitination of TrkB being the result of the association of the juxtamembrane region of TrkB and the BOX domain. Bimolecular florescence complementation assay was employed to study the effect of SOCS2 on TrkB trafficking and it was determined that SOCS2 increases TrkB receptor turnover in the cell with an increase in trafficking of the receptor to late and recycling endosome. In summary, this study presents evidence of a novel role of SOCS2 in regulating TrkB neurotrophin signalling. The molecular and biochemical aspects of this regulation was analysed in detail in-vitro. These finding has potential implications in developing new therapeutic strategies in neurodegenerative diseases where BDNF signalling is involved.
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ItemMolecular mechanisms of BDNF signalling in central nervous system myelination( 2016)Myelination within the CNS allows the fast and efficient transmission of action potentials along axons. Both myelin and the oligodendrocytes that generate myelin and wrap the axons are crucial for the development and maintenance of an adaptive and healthy nervous system. Interestingly, the process of myelination is responsive to neural activity so myelination also contributes to a form of neuroplasticity called white matter plasticity. Disorders as varied as multiple sclerosis and schizophrenia result when myelination is compromised, and experiences in the world, from childhood neglect and maltreatment to learning a second language, leave their trace in the white matter of our brains. Much remains to be discovered about the factors that regulate myelination and the signalling networks these factors utilise. Our laboratory has taken an interest in a growth factor that has a recognised role in synaptic plasticity and is epigenetically regulated. This molecule is brain-derived neurotrophic factor (BDNF). Our studies reveal that BDNF exerts a pro-myelinating effect in vitro and utilises Erk1/2 to mediate this effect. Specifically, BDNF acts on oligodendroglial TrkB receptors to myelinate the axons of DRG neurons in co-culture with oligodendrocytes. Studies in vivo confirm a role for BDNF, TrkB and Erk1/2 in myelination. Previous work from our laboratory indicates that loss of TrkB from myelinating oligodendrocytes leads to a phenotype of thinner myelin in the CNS, and my results reveal that when TrkB is deleted from the time of oligodendrocyte specification, this phenotype is restricted to thinner myelin only around large diameter axons. This suggests that there compensation occurs for the loss of TrkB but the factors that contribute to this putative mechanism of compensation are not yet known. Surprisingly, despite TrkB null oligodendrocytes only yielding a relatively mild hypomyelinating phenotype in vivo, I discovered that in the context of a myelinating co-culture, TrkB null oligodendrocytes exhibit a severely reduced capacity to myelinate. This led me to pose the question: how were oligodendrocytes that had a very limited capacity to myelinate in vitro able to myelinate the majority of axons normally in vivo? The result suggested that a cell or factor that was present in vivo but absent in the myelinating co-culture system was enhancing the oligodendrocytes capacity to myelinate. Interestingly, in vitro studies demonstrate that Fyn kinase associates with and is activated by TrkB, and that Fyn translocates neuronal TrkB receptors to lipid rafts in response to BDNF treatment. Similarly to BDNF, in vivo studies on Fyn kinase reveal that it too has a role in synaptic plasticity and myelination and this potentially suggests they utilise a common signalling pathway. In this work I interrogated BDNF signalling in oligodendrocytes and identified that BDNF and Fyn kinase do share a common signalling pathway: Fyn kinase is a downstream mediator of the pro-myelinating effect of BDNF. I then hypothesised that Fyn kinase could form part of a mechanism that compensates for the loss of oligodendroglial TrkB in myelination in vivo. I demonstrated that forced over-expression of Fyn kinase by TrkB null oligodendrocytes enhanced myelination in vitro. However, I also found that in isolated, cultured TrkB null oligodendrocytes the expression of Fyn kinase protein was lower than control oligodendrocytes although proportionally more of it was active. Taken together, these results suggest that Fyn kinase could, in principle, contribute towards redundancy in myelination but requires factors or cells, absent from the in vitro myelination assay, but present in vivo that increase its activation or expression level. Further studies are required to identify these factors and to determine if this putative compensation mechanism occurs in vivo. BDNF and Fyn kinase both have roles in neural plasticity and it is consistent with a maxim of biological economy that they would have complementary roles in white matter plasticity. BDNF switches oligodendrocytes into an activity dependent mode of myelination, and the findings of this study indicate that Fyn kinase mediates BDNF driven myelination, suggesting a role for Fyn kinase in white matter plasticity as well. Due to the burgeoning burden of neurodegeneration and the recognition that white matter plasticity is affected in mental illness and by experiences of trauma, it is imperative that we interrogate the molecular mechanisms of white matter plasticity to better target treatments to alleviate the distress caused by these challenges.
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ItemA study of depression in Huntington's disease( 2008)Huntington’s disease (HD) is an inherited neurodegenerative disorder that is caused by a mutation of a single gene, huntingtin. The disease is more commonly known for the characteristic choreiform movements that develop in the later, more advanced stages of the disease. However, cognitive deficits and psychiatric symptoms are frequently observed prior to the onset of the motor symptoms. Little is known about the pathological bases for the neuropsychiatric features which include increased irritability and heightened aggression. Depression affects 30-50% of HD patients and is the most commonly diagnosed psychiatric symptom. This is proportionally higher than in the general population and it is possible that inherent pathological changes in the HD brain render a HD-gene positive individual more susceptible to depression. Using a variety of behavioural tests, the R6/1 transgenic mouse model of HD was found to display altered responses reflective of depression-related behaviour, indicating that the HD mutation confers a genetic susceptibility for developing depression. The behavioural alterations were more robust in female HD mice reflecting a possible sex-dependent manifestation of the depression symptoms in the human HD population that has yet to be investigated. The onset and rate of progression of HD is strongly influenced by the environment and the development of depression is similarly impacted upon by environmental factors (e.g. stress, negative life events). The experimental paradigms of environmental enrichment and wheel-running slow the development of motor and cognitive symptoms in R6/1 HD mice and the present study reports that both paradigms also correct the depression-related behavioural phenotype. This study also found that HD mice had muted responses to two common classes of antidepressant drugs, highlighting the need for a detailed examination of the efficacy of drug treatments in HD patients. Depression susceptibility is linked to genetic variance in the human population and studies of gene candidates in mutant mice report the detection of behavioural phenotypes similar to the present study. The depression-related behavioural phenotype of the R6/1 HD model was found to be associated with early down-regulations in mRNA levels of the ii serotonin (5-HT) 1A and 5-HT 1B receptors in the cortex and the hippocampus. Additionally, female HD mice had reduced cortical 5-HT transporter gene expression. Collectively, these findings indicate that a disruption of serotonergic signaling in the HD brain contributes to the development of depression in HD. Brain-derived neurotrophic factor (BDNF) gene expression is down-regulated in the HD brain, however the expression pattern of exon-specific splice variants was previously unknown. This study reports that BDNF mRNA levels are reduced in the hippocampus by an early age but also reports that individual exon-specific transcripts are differentially down-regulated in males and females, although the functional relevance of this remains to be investigated. Overall, this study has demonstrated that the R6/1 transgenic mouse model of HD is ideal for further investigating the occurrence of depression in pre-motor symptomatic HD. It has also identified alterations in gene expression of key components of neuronal signaling which might be linked to the molecular basis of depression.