Investigation of molecular and cellular mechanisms underpinning the neurotoxicity of homocysteine and its metabolites in models of neurodegeneration

Abstract

Elevated levels of homocysteine (HCy) are a known risk factor in several disease states (1). HCy has several other metabolites, homocysteine thiolactone (HCy-T) and homocysteic acid (HCA). Whilst HCy-mediated neurotoxicity has been extensively studied, the underlying mechanisms of HCy-T and HCA mediated neuronal damage remain largely unknown. This thesis aims to explore the underlying mechanisms, triggered by HCy and metabolites which result in neuronal cell death, and may be appropriate targets for future research on disease-modifying interventions in neurodegenerative disorders. As ageing is the greatest risk factor for neurodegeneration, a novel model of human neuronal ageing was established, permitting investigation of the pathways triggered by HCy in ageing. Using SH-SY5Y cells, a novel differentiation protocol was established and categorised, once fully differentiated, these cells were shown to be fully functional neurons and could be maintained for a month in culture. Using a range of concentrations of HCy and HCy-T, the concentration cell death occurs at was determined using crystal violet and lactate dehydrogenase assays. Mechanisms of toxicity were determined using pharmacological intervention at the NMDA receptor, nitric oxide scavengers and antioxidants. Using a combination of immunocytochemistry, live cell imaging and ELISA, alterations in markers of cell damage could be examined. The results showed HCy and HCy-T have distinct mechanisms of toxicity. Whilst both are neurotoxic, HCy directly acts via the NMDA receptor, however HCy-T appears to be less potent. Additionally, HCy-T caused a greater increase in reactive oxygen species generation than HCy, and each metabolite also displayed distinct mitochondrial network abnormalities. Finally, using the long-term culture methods, the chronic effects of HCy, HCy-T and HCA were examined. However, extensive cell death was apparent at low doses in all metabolites therefore no definitive mechanisms could be determined. This culture method was deemed not appropriate for toxicity experiments.