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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.
ItemTesting paraclinical outcome markers in optic neuritis and a study of the phenomenology and treatment of tremor in multiple sclerosisVAN DER WALT, ANNEKE ( 2012)Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterised by multi-focal inflammatory demyelinating plaques and significant neuroaxonal degeneration. The clinical presentation and disease course of MS is heterogeneous and ranges from clinically isolated syndromes such as optic neuritis (ON), to disabling progressive disease at which stage tremor can be a feature. This heterogeneity reflects the multi-focal nature of the CNS injuries and suggests that the relationships between neuronal injury and clinical presentations are complex. Focused clinical and paraclinical assessments of defined neural pathways associated with discrete clinical presentations can help improve our understanding of complex clinico-pathological relationships. This thesis examined clinical manifestations and outcomes in two models at the opposite ends of the MS clinical spectrum namely acute isolated ON and MS-related tremor. The aims of section one was to comprehensively study changes in clinical and paraclinical markers after ON and more specifically to evaluate the ability of these markers to serve as predictive biomarkers of clinical and axonal outcomes after ON. The second part of this thesis aimed to perform a detailed clinical phenomenology study in MS tremor patients to help define possible underlying neuroanatomical networks and broaden understanding of injury in secondary progressive MS in general. In addition, the efficacy and safety of onabotulinumtoxin-a in the treatment of MS tremor was evaluated. In Section one, forty patients with acute, idiopathic, unilateral optic neuritis with early MS or at high risk of MS were recruited. Patients were studied prospectively over 12 months at multiple time-points. Ten healthy age and sex-matched controls were tested twice to inspect inter-scan and inter-subject variability. All participants had a detailed neurological assessment, visual acuity testing (high and low-contrast and colour vision), optic nerve diffusion tensor imaging (DTI), multi-focal visual evoked potential (mfVEP), and optical coherence tomography (OCT). The main outcome measures were 2.5% low contrast visual acuity, mfVEP amplitude and retinal nerve fibre layer thickness (RNFL) at 12 months after ON. Significant changes in clinical and paraclinical measures after ON, particularly in the first 3 months were demonstrated. Despite some initial recovery, persistent visual, functional and neuroaxonal deficits persisted at 12 months. Multivariate models identified decreased high contrast visual acuity (logMAR notation) at 1 month after ON to be the earliest predictor of persistent visual loss at 12 months. Decreased 3-month colour vision and RNFL thickness also both significantly predicted poor visual recovery. The earliest predictors of axonal outcomes at 12 months after ON were decreased 1-month mfVEP amplitude, low contrast acuity and optic nerve axial diffusivity. Decreased optic nerve axial diffusivity was the best predictor of severe combined (persistent mfVEP amplitude loss and/or RNFL thinning of greater than 30%) axonal loss. Both mfVEP amplitude and clinical assessments are limited in the hyper- acute setting due to inflammatory oedema, conduction block and variable patient performance. Therefore, the finding of an early structural marker, optic nerve axial diffusivity, that is able to predict axonal outcome after ON is important as this measure could serve as a biomarker in future studies of putative neuroprotective therapies by identifying those patients at most risk of permanent axonal loss. In Section 2 of the thesis, a cross-sectional study of 54 MS patients with and without tremor was undertaken. Of the 27 tremor cases, 25 were included in a double-blind randomized placebo-controlled crossover trial (RCT) to evaluate the efficacy of onabotulinumtoxin-a. A detailed comparison between MS patients with and without tremor was completed. Patients underwent a detailed history and neurological assessment that included the Expanded Disability Severity Scale (EDSS) as well assessments of cerebellar ataxia, dystonia and tremor-related quality of life. All patients were assessed for the presence of tremor and tremor severity was rated using the Bain score, a validated MS tremor severity score to rate tremor severity, writing and drawing an Archimedes spiral. Functional tasks such as writing, drawing, drinking from a cup and pouring water from one cup to another were completed. A blinded, independent observer, rated standardized video assessments. In the RCT, patients were assessed 6 weekly for 6 months with baseline and 3 months injections of either onabotulinumtoxin- a or placebo. MS tremor was found to be predominantly an action tremor of the upper limb with prominent cerebellar and dystonic features. Dystonic features (including geste antagonistes, mirror movements, dystonic posturing and writer’s cramp) were more prominent and more severe in patients with tremor than those without. A further novel finding was the impact of dystonia severity in the affected limb on tremor severity. This result highlights the complexity of MS tremor as a movement disorder with injury that is not localized to the cerebellum alone, but rather to the wider cerebello-thalamo-cortical, pallido-thalamic as well as cortico-cerebellar network. In a RCT of the efficacy of onabotulinumtoxin-a in MS tremor, the drug was found to reduce MS tremor severity by an average of 40%, with writing and drawing severity improving by an average of 30%. Injections were well tolerated with transient focal weakness being the predominant side effect. The efficacy of onabotulinumtoxin-a likely reflects modulation of the central tremor-generating networks by modification of peripheral stretch reflexes and muscle- spindle excitability. In addition, the efficacy of a drug used predominantly for focal dystonia underscores the importance of recognizing dystonia as a feature of MS tremor.