Signal transduction pathways involved in inhibition of axonal regeneration after CNS injury

2017-02-28T23:33:26Z (GMT) by Ozturk, Ezgi
SCI and MS are devastating conditions of the CNS that propagate neurological dysfunction with poor long term prognoses. In the past few decades, much effort has gone in to understanding mechanisms of neuroinflammation, demyelination and neurodegeneration that combined, drive the neurological deterioration observed in patients. However, currently available treatments are only effective in a small percentage of sufferers. Therefore there is an urgent need for the discovery of new and more effective therapies to dramatically improve patients quality of life. In recent years, a body of research has shifted towards discovering therapies that modulate mechanisms of neurodegeneration rather than immunomodulation. It is now evident that recurring axonal injury and eventual failure in regenerative compensatory mechanisms in the CNS are the major causes of irreversible neurological deficit observed in SCI and MS. The role of MAIFs and their potent capacity to drive neurodegeneration in damaged axons has been convincingly presented in the literature. Nogo-A, which signals through the NgR1 complex on axons, has been deemed the most potent inhibitor of regeneration. The overall aim of this thesis was to investigate Nogo-A/NgR1 signalling and their downstream mediators in the context of neurodegeneration in SCI and in the animal model for MS, EAE. Furthermore, we explored the potential of therapeutic agents in reversing the affects of Nogo-A/NgR1 signalling in an attempt to hinder neurodegeneration in these models. The first part of this thesis assessed the deposition of Nogo-A and the ROCKII-dependent phosphorylation of its downstream mediator CRMP-2 in a mouse model of SCI with and without the therapeutic intervention of daily LIF administration following injury. LIF administration resulted in substantial improvements in functional locomotor recovery in treated animals. At the molecular level LIF treatment reduced Nogo-A deposition at the lesion site. Similarly, RhoA-GTP and PCRMP-2 molecules which negatively regulate the cytoskeleton showed diminished activity. In contrast, factors indicative of growth and regeneration such as Rac-1 and GAP-43 were elevated in animals receiving LIF. Therefore factors like LIF that limit Nogo-A deposition in the injured CNS could have therapeutic potential in SCI. The second part of this thesis, a time-course evaluation of neurodegeneration in the MOG-induced EAE model was carried out, with a particular focus on the modulation of CRMP-2 in disease progression. CRMP-2 expression reached maximum levels at peak stage of EAE, however, its expression was evident as early as pre-onset stage of ii disease. In addition, levels of PCRMP-2 was associated with and only activated in neurodegenerative axons. Furthermore, molecular transport of tubulin heterodimers by CRMP-2 to promote microtubule assembly was impeded. Moreover, therapeutic administration of the function blocking Nogo-A antibody in EAE animals reduced clinical severity and in parallel decreased PCRMP-2 expression. These data indicated that Nogo-A signalling could mediate axonal degeneration through post-translational modification of CRMP-2 by negatively regulating microtubule assembly. Therefore, in the third part of this thesis, a direct relationship between NgR1 signalling and CRMP-2 phosphorylation and their role in mediating axonal degeneration in the progression of EAE was investigated. The clinical severity was significantly reduced in ngr1-/- mice with EAE. Correspondingly, ROCKII-dependent CRMP-2 phosphorylation was also diminished at all time points. Furthermore CRMP-2 association with tubulin heterodimers was restored, allowing for positive microtubule assembly and therefore regeneration. Moreover, the immune capacity of naive and EAE-induced ngr-/- mice remained unchanged, signifying that the observed alleviation in disease severity is due to alterations in mechanisms related to axonal injury and not mechanisms of immunomodulation. Collectively, research conducted in this thesis describe a common mechanism of neurodegeneration that can be activated in both SCI and EAE. It was demonstrated that growth inhibition can be initiated by MAIFs such as Nogo-A through the NgR1 complex in axons. A direct consequence of this is the ROCKII-dependent phosphorylation of CRMP-2 which impedes CRMP-2 transport of tubulin, negatively regulating microtubule assembly. Of particular relevance is the capacity of the CNS to regenerate when this mechanism is hindered by way of therapeutic intervention. Therefore, it can be concluded that targeting the components of MAIF/NgR1 signalling following CNS injury may provide novel therapeutic avenues by which neurological decline observed in patients can be diminished.