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ItemBone morphogenetic protein-4 signalling in glial cells of the central nervous systemGovier-Cole, Alistair Evan ( 2017)Multiple sclerosis (MS) affects over 20,000 Australians and over 2-3 million people globally. MS is a demyelinating disease of the central nervous system (CNS) in which the insulating myelin sheath of the neuron is degraded. This leads to severe impairment of neuronal signal transmission throughout the CNS. Oligodendrocytes are specialised cells of the CNS that form the myelin sheath. Oligodendrocyte progenitor cells (OPCs) are present in the adult brain and respond to demyelinating injury, but these cells often fail to differentiate from precursor cells into oligodendrocytes to replace damaged myelin in MS lesions. One group of factors linked to this ‘differentiation block’ is the bone morphogenetic protein (BMP) family. This thesis aims to clarify the role of BMP signaling in two major classes of CNS cells: oligodendrocytes, which are formed by OPCs, and astrocytes, which play a role in regulating oligodendrocyte differentiation during CNS injury. Firstly, to understand the influence exerted by BMP4 on oligodendrocytes during remyelination, BMP4 signalling was disrupted by infusing LDN-193189, a pharmacological inhibitor of BMP4 receptors BMPRIA and BMPRIB, following cuprizone-induced demyelination in mice. This resulted in a significantly higher number of mature oligodendrocytes present in the murine corpus callosum after one week of recovery from cuprizone treatment. Furthermore, this increase in oligodendrocyte number was coupled with a significant increase in the degree of remyelinated myelin sheaths in the murine corpus callosum. In vitro analysis demonstrated that LDN-193189 has a direct positive influence on OPC differentiation into mature oligodendrocytes, and reduces the astrogliogenic effect of BMP4. Inhibiting BMP4 signalling in OPCs in vitro also promoted myelination in a dorsal root ganglion co-culture experiment. Analysis of gene transcription in OPCs treated with BMP4 and LDN-193189 suggested that the positive effect of pharmacologically inhibiting BMP4 on oligodendrocyte differentiation and myelination was mediated by downregulation of a DNA binding protein, ID4. This protein has been previously shown to inhibit oligodendrocyte differentiation in response to BMP4 signalling activity. To further understand the signalling mechanisms by which BMP4 elicits its inhibitory effect on oligodendrocyte differentiation, a transgenic mouse with an inducible conditional deletion of Bmpr1a was used to disrupt BMP4 signalling through BMPRIA. Cultures of OPCs with a BMPRIA deletion recapitulated most of the positive effects observed on oligodendrocyte differentiation and myelination as seen in OPCs treated with LDN-193189. This suggested that BMPRIA may exert a critical influence in transmitting the inhibitory BMP4 signal in OPCs compared to BMPRIB. Secondly, the influence exerted by BMP4 signalling on astrocytes was assessed using in vitro and in vivo techniques. Mature astrocyte cultures responded to BMP4 by increasing their proliferation and transcription of Gfap, a key intermediate filament protein that is upregulated in astrocytes in response to CNS injury. Furthermore, BMP4 stimulates the secretion of factors that, when applied to OPC cultures, inhibit their differentiation. This effect is normalised when astrocytes are treated with LDN-193189, suggesting the influence of BMP4 signalling on astrocytic secreted factors is mediated through BMP4 Type I receptors. To understand the function of BMP4 signalling in astrocytes during demyelination, mice with an inducible, astrocyte-specific deletion of Bmpr1a underwent cuprizone administration to cause demyelination in the murine corpus callosum. However, mice with a Bmpr1a deletion in astrocytes did not show any improvement in remyelination, or a reduction in the number of GFAP+ astrocytes in the corpus callosum compared to vehicle-treated mice. This suggests that the influence exerted by BMP4 on astrocytes during demyelination is negligible, or alternatively, that the function of BMPRIA is compensated by BMPRIB in astrocytes in vivo. This thesis has further clarified the inhibitory influence exerted by BMP4 on oligodendrocyte differentiation in the CNS. Inhibiting BMP4 signalling through its Type I receptors BMPRIA and BMPRIB following demyelination significantly enhances subsequent remyelination in the murine corpus callosum. Additionally, this thesis has identified novel astrocytic responses to BMP4 signalling in vitro, including a potential influence on secreted factors that inhibit OPC differentiation. However, further investigation is required to fully elucidate this relationship. In a broader context, it is hoped that the research reported in this project may contribute to improved clinical options through identification of treatments for improving remyelination in demyelinating disease.
ItemThe role of the TAM receptors in CNS myelination and demyelinationAkkermann, Rainer ( 2015)Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The TAM family of receptor-tyrosine kinases, comprising Tyro3, Axl and Mertk, is widely expressed in the CNS and has been shown to be critical in the outcome of both toxin-induced, as well as inflammatory demyelination. In addition, previous work has demonstrated a direct impact of Gas6-mediated TAM signalling in the regulation of myelination. However, our understanding of the contributions of each individual receptor in these processes remains poor. In order to potentially utilise TAM receptor functions in the development of new therapeutics, it is of crucial importance to dissect these contributions as pan activation of all three receptors may have undesired off-target effects. The aim of this thesis was therefore to provide further insight into which TAM receptor transduces the pro-myelinating effects of Gas6 and which receptors may be important in limiting demyelination. Using electron microscopy, I found that while deletion of Gas6 only results in a mild, non-statistically significant reduction in developmental myelination, Tyro3 deficiency significantly impairs initiation of this process. In vitro data suggest that Tyro3 expressed on oligodendrocytes is required for normal myelination and that this receptor is required for Gas6-mediated enhancement of myelination. Oligodendrocytes deficient in Tyro3 display a reduction in the activation of Erk1, a signalling molecule involved in the induction of myelin gene expression, suggesting that the effects of Tyro3 upon myelination may be mediated at least in part by Erk1. I also could demonstrate that Tyro3 deficiency alone is not sufficient to significantly alter cuprizone-induced demyelination. This is also true for heterozygous microglia-specific Mertk deletion, indicating that homozygous deletion may be required to unravel potential effects of this receptor in experimental demyelination. Finally, injection of Axl or Mertk activating antibodies did not alter EAE disease course which may have been due to detrimental effects probably caused by antibody-mediated hyperactivation of the immune system. In summary, the data presented in this thesis describe for the first time that Tyro3 is a regulator of CNS myelination and that this is regulated by its expression on oligodendrocytes, possibly to an extent through Erk1 activation. Neither Tyro3 deletion nor partial deletion of Mertk in microglia alone affected cuprizone-induced demyelination. Finally, my findings suggest that TAM activating antibodies may not be ideal for therapeutic activation of these receptors in inflammatory conditions.
ItemTAM signalling in CNS demyelination and multiple sclerosisMA, ZHI-MING ( 2015)Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Involvement of the immune system in the pathogenesis of MS is a key feature of the disease, and an understanding of the mechanisms underlying how immune responses are shaped during CNS demyelination will provide insight into the development of new therapeutic strategies. The TAM (Tyro3, Axl, Mertk) family of receptor tyrosine kinases and their ligands Growth Arrest-Specific 6 (Gas6) and Protein S (ProS) have been shown to modulate many immunological processes important during central demyelination. The major aim of this thesis is to provide further insight into TAM biology in the context of both an animal model of inflammatory demyelination and human MS. By conducting a study examining MS patients and common genetic variations within TAM genes, I identified the MERTK gene as a novel MS susceptibility gene. Examination of plasma from MS patients revealed that levels of the TAM ligand PROS are decreased in MS and that low PROS levels are associated with increased MS disease severity. To interrogate the role of TAM signalling in modulating disease severity during inflammatory demyelination, I used the experimental autoimmune encephalomyelitis (EAE) animal model and observed major changes in TAM gene expression within the CNS and peripheral immune cells during EAE. Examination of Gas6-/- mice during EAE showed that absence of the TAM receptor ligand Gas6 results in both attenuated microglial/macrophage responses and disease severity during the effector phase of EAE. Conditional deletion of Mertk from dendritic cells (DC) resulted in worse disease during the effector phase of EAE. Stratification by sex revealed sexual dimorphism in TAM gene expression and also in the outcome of EAE in both Gas6-/- mice and mice with DC-specific deletion of Mertk. In summary, the data presented in this thesis suggest that the TAM family plays key roles in MS susceptibility and modulating innate immune responses during inflammatory demyelination, providing evidence for members of the TAM family as either markers of disease severity and/or therapeutic targets for the treatment of MS.