Anatomy and Neuroscience - Theses
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ItemBone morphogenetic protein-4 signalling in glial cells of the central nervous system( 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.