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Exploring molecular and cellular strategies for neuroprotection and neuroregeneration in multiple sclerosis
thesisposted on 21.02.2017, 02:32 by Short, Martin
Multiple Sclerosis (MS) is an incurable neurological condition that affects close to 1 in 1000 Australians. Research into the complex aetiology and pathophysiology of MS is rapidly advancing. Many new treatments targeting the immune system are now available and can partially prevent damage caused by inflammation. However, all patients have a degree of axonal loss and neurodegeneration that can contribute to long-term disability. So far, no treatment has demonstrated an ability to directly protect the central nervous system (CNS) of MS patients from axonal injury or neurodegeneration. No treatment has shown to remyelinate or regenerate injured tissue in clinical trials. There is therefore a great demand for a therapy that can not only modulate the immune system but also protect the brain from axonal injury and facilitate repair processes. The aim of this thesis was to explore potential therapeutic approaches to neuroprotection and neuroregeneration in Multiple Sclerosis. Direct cell replacement was assessed using bone marrow derived cells in an animal model of Multiple Sclerosis, experimental autoimmune encephalomyelitis (EAE). The ability of mesenchymal stem cells (MSCs) and amnion epithelial cells (AECs) to protect human neural stem cells from oxidative stress injury and to differentiate through the production of neurotrophic factors was examined in vitro. Finally, several cytoskeletal proteins important for axonal growth were characterised. Their relationship with the Nogo receptor and changes due to inflammation from EAE were investigated. The ability of bone marrow derived cells to directly replace and regenerate cells within the uninjured or inflamed brain and spinal cord was assessed in the first part of the thesis. Despite the use of a highly sensitive multi-colour flow cytometry, an insignificant number of the bone marrow derived cells transplanted into mice were able to transform into neural cells. These findings were confirmed using immunofluorescence microscopy and virtual slide imaging. Although more bone marrow derived cells migrated to the brain and spinal cord in mice with EAE than controls, they retained hematopoietic cell markers, hence confirming the lack of transformation. While bone marrow predominantly contains hematopoietic cells and their precursors, there are other cell types including MSCs that have been investigated as a possible novel therapy for Multiple Sclerosis. As well as modulating the immune system, these cells have potential for neuroprotection and regeneration. Rather than direct cell replacement, these cells possibly have their effect through the production of soluble factors. We have demonstrated that MSC express RNA for many neurotrophic factors and in particular produce IL-6, BDNF and HGF. The quantity of these factors was increased following exposure to pro-inflammatory cytokines, particularly TNF-α. In the second part of the thesis, a novel culture system in which neural stem cells (NSCs) derived from a patient with MS using induced pluripotent stem cell techniques was used to assess the ability of human MSC to protect against oxidative stress and enhance differentiation through the production of neurotrophic factors. NSCs exposed to MSC conditioned media for 5 weeks reduced the expression of GFAP expression and a low molecular weight component within the conditioned media ameliorated oxidative stress. Although bone marrow derived MSC have many attractive attributes for use as an immunomodulatory and neuroregenerative therapy in MS, the need for cell culture and expansion may limit its use. AECs are an alternative cell type obtained in large numbers from otherwise discarded placentas that has promise for use in inflammatory conditions including MS. The neuroprotective and regenerative potential in MS has not previously been examined. The ability of AECs to produce neurotrophic factors was investigated and compared with MSCs. AECs expressed fewer neurotrophic factors and did not produce IL-6, BDNF or HGF. AEC conditioned media still reduced GFAP expression in NSCs over 5 weeks of culture but appeared to increase oxidative stress and cell death. Similar to MSCs, the AEC conditioned media appeared to ameliorate the effect of an exogenous oxidative stress. While cell therapies have great potential in regenerative medicine, there are a number of other promising avenues of research. In the final part of the thesis, possible mechanisms underlying Nogo receptor mediated restriction in axonal growth have been explored. Two proteins downstream of the Nogo Receptor, CRMP-2 and cofilin were examined. Phosphorylated CRMP-2 correlated well with axonal loss in both the animal model of MS (EAE) and biopsy sections from an MS patient. Levels of phosphorylated cofilin were higher in mice lacking the Nogo receptor than the controls. Since cofilin phosphorylation is linked to axonal growth, if substantiated, these findings could potentially lead to novel and targeted therapies for regeneration in MS. This thesis has explored a range of different strategies that show promise for neuroprotection and regeneration in MS from direct cell replacement by bone marrow stem cells, the production of neurotrophic factors by MSCs and AECs, and modulation of the Nogo receptor via CRMP-2 and cofilin. Due to the great demand for neuroprotective and regenerative therapies these and other therapies are already being translated into early clinical trials. Although current immunomodulatory therapies can significantly reduce relapses and lesion load over time, a combination of immunomodulatory and neuroprotective/regenerative therapies may have a greater impact on reducing long-term disability and improve the quality of life for patients with MS.