Centre for Neuroscience - Theses
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ItemIdentification of cellular responses to autoimmune injury induced in neurons( 2012)Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that is pathologically characterized by focal inflammatory lesions featuring demyelination, gliosis and axonal injury. Immune-mediated axonal damage and loss are accepted as major determinants of irreversible neurological disability in MS patients. However, it remains unclear how inflammatory cells damage axons in acute MS lesions, and whether axons or neurons respond to this injury to limit its extent. Here we used a common mouse model of MS pathology, murine MOG35-55 experimental autoimmune encephalitis (EAE) to characterize potential endogenous responses to autoimmune injury in neurons. The MOG-EAE model features marked inflammatory axonal injury in the spinal cord encompassing axons that originate from corticospinal motoneurons, located in the motor and sensory cortex of the brain. To reveal specific neuronal responses to axonal damage in the spinal cord, we compared gene expression in cortical regions of EAE and healthy control mice by microarray. Although inflammatory infiltrates were absent in the cortex, we detected gene expression alterations in EAE mice. Unexpectedly, many of these genes encoded for proteins that were functionally associated with the extracellular matrix (ECM). Expression of the ECM adaptor molecule, matrilin-2 (Matn2), correlated with EAE disease severity and was specifically increased in cortical areas projecting to the spinal cord. In addition, Matn2 protein was expressed by neurons in these regions. In the spinal cord of EAE mice, Matn2 was detected within acutely damaged axons in developing lesions, and with disease progression, was predominantly found extracellularly in and around the lesions. These observations suggest that Matn2 is regulated following neuronal damage and is subsequently released into the ECM. In addition, Matn2 markedly accumulated in the perivascular space of inflamed blood vessels. Importantly, in tissue samples of MS patients, Matn2 was also detected in neurons and Matn2 depositions were found in acute and chronic lesional parenchyma as well as in perivascular areas, suggesting a potential relevance of Matn2 to MS pathology. Functional analyses using Matn2 knockout (ko) mice in EAE showed an ameliorated disease severity at the acute disease stage. This coincided with smaller and fewer lesions, reduced axonal injury and decreased expression of genes encoding markers of microglial/macrophage activation (CD45 and CD11b) and pro-inflammatory molecules predominantly expressed by activated macrophages (Il-1b, Tnfa, Il-6, Cox-2 and iNos). Although, the number of immune cells in spinal lesions was unchanged in wt and ko EAE mice with equal (matched) EAE grades, expression of these genes was still reduced in ko mice. This led us to hypothesize that Matn2 can modulate microglial/macrophage activation and induce pro-inflammatory gene expression. In vitro studies using a neuron/macrophage co-culture model showed that Matn2 gene expression increased in cortical neurons following acute axonal injury by activated macrophages. Importantly, addition of recombinant Matn2 to neuron/macrophage co-cultures resulted in macrophage activation and induced pro-inflammatory cytokine expression as well as in acute axonal injury. These results support the described in vivo findings and give insight into a potential mechanism of Matn2-mediated axonal damage. Collectively, the present study introduces Matn2 as a novel mediator of neuroinflammatory disease and the innate immune response, in the EAE model. We show that Matn2 is an endogenous neuronal protein that is induced and potentially secreted/released following acute axonal injury. We found that Matn2 can modulate macrophage activation to promote pro-inflammatory activity, and consequently, axonal injury in acute inflammatory lesions. Strikingly, deletion of Matn2 significantly ameliorated disease severity and reduced axonal damage. Therefore, targeting Matn2 in the acute lesion environment presents a potential therapeutic strategy in MS pathology.
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ItemThe therapeutic effect of LIF in EAE-associated axonal injury( 2009)Axonal degeneration is a major pathological feature of the central nervous system (CNS) inflammatory demyelinating disease multiple sclerosis (MS). This axonal degeneration has major consequences, as functional axonal regeneration in the CNS is largely absent. Cumulative axonal degeneration is the likely cause of the majority of progressive MS-related disability, and therefore, the need for novel neuroprotective therapies for MS exists. Experimental autoimmune encephalomyelitis (EAE), an animal model of MS pathology, also produces axonal injury. In particular, the optic nerve and spinal cord are key sites of neuroinflammation in mouse EAE. By utilizing this model, the short term and long term effects of the putative neuroprotective cytokine, leukaemia inhibitory factor (LIF), were investigated in the optic nerve and spinal cord utilising a number of outcome measures of axonal dysfunction. These included MRI measures of water diffusivity along (ADC ||) and across (ADC┴) the optic nerves, serum levels of phosphorylated neurofilament heavy chain subunit (pNF-H) and histological morphometric measures. LIF treatment reduced EAE grade and pNF-H plasma levels, decreased ADC┴, but had no effect on ADC ||, axon counts or inflammatory infiltration. In contrast, genetic deletion of LIF and its sister cytokine ciliary neurotrophic factor (CNTF), not only increased EAE grade and pNF-H levels, but also decreased optic nerve ADC|| and optic nerve and spinal cord axon densities. After reviewing current literature, we hypothesize that the target cell for endogenously upregulated LIF in EAE may be the neuron or axon, whereas the target cell for exogenously administered therapeutic LIF may be another cell type, possibly infiltrating macrophages and activated microglial cells. LIF antagonist treatment did not have any affect on EAE grade, pNF-H levels or MRI parameters. This lack of effect may be due to the inability of the LIF antagonist to enter the CNS, supporting the hypothesis that endogenous LIF has a centrally acting mechanism.