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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ItemDifferential responses to neural injury and disease in EphA4 knockout mice( 2011)Promoting repair and regeneration of the injured and diseased central nervous system is among the most critical fields in research today, and would improve the quality of life of people suffering from traumatic brain and spinal cord injury, multiple sclerosis and many other conditions. Mice lacking the developmental axon guidance molecule EphA4 demonstrate extensive axonal outgrowth and functional recovery following spinal cord injury (Goldshmit et al., 2004), and a greater understanding of the mechanisms orchestrating this regenerative response is the focus of this thesis. To identify differentially expressed genes which may contribute to axonal regeneration, RNA collected from adult EphA4 knockout and wild-type mice 4 days following lumbar spinal cord hemisection or laminectomy only was hybridised to Affymetrix All-Exon Array 1.0 GeneChipsTM. Microarray analysis indicated attenuated or otherwise altered expression of a number of inflammatory genes in injured EphA4 knockout spinal cords. These included inflammatory phospholipid-related genes lysophosphatidic acid receptor 1 (LPAR1, p=0.002), a receptor for lysophosphatidic acid, and alkaline ceramidase 2 (ACER2, p=0.006), a regulator of sphingosine production. In both genotypes at 4 days post-injury, LPAR1 was localised to reactive astrocytes surrounding the spinal cord lesion and ACER2 was predominantly localised to macrophages / activated microglia within the lesion. Microarray results also indicated arginase 1 (ARG1) was lower in injured EphA4 knockout compared to wild-type mice (p=0.014 and fold difference=3.17 between injured groups) and a lower proportion of ARG1-immunoreactive macrophages / activated microglia in EphA4 knockout spinal cords was confirmed histologically at 4 days post-injury. However there was no difference in the overall number or spread of macrophages / activated microglia in injured EphA4 knockout compared to wild-type mice at 2, 4 or 14 days post-injury. To further investigate whether a subtly altered neuroinflammatory response contributes to the regenerative phenotype of injured EphA4 knockout mice, the response of EphA4 knockout compared to wild-type mice was examined using a model of neural injury caused by inflammation, experimental autoimmune encephalomyelitis. In this animal model of multiple sclerosis, EphA4 knockout mice had less severe neurological symptoms compared to wild-type mice, indicating less neural injury. The proportion of ARG1-immunoreactive macrophages / microglia was not altered in experimental autoimmune encephalomyelitis-affected EphA4 knockout mice at 20 days post-immunisation, and whether the attenuated response to disease symptoms seen in EphA4 knockout mice is primarily due to differences in immune or central nervous system parenchymal cells remains to be determined. In summary, EphA4 knockout mice not only display regeneration following spinal cord injury but also have a less severe response to experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Alterations in the neuroinflammatory response of injured EphA4 knockout mice may contribute to their improved outcomes to models of CNS disease and injury. Investigations in this thesis have also resulted in the localisation of two inflammatory phospholipid-related genes, LPAR1 and ACER2, previously uncharacterised in the injured CNS.