Acute cerebral ischaemic injury results in increased neurotransmitter release which can result in cell death via receptor and non-receptor mediated mechanisms. Furthermore a delayed apoptotic response has been described which may be attributed to the CD95 death receptor. Using the murine hippocampal HT22 cell line, this study has developed a novel FRET construct to investigate the biochemical pathways associated with two models of stroke induced neurotoxicity; CD95-induced apoptosis and glutamate-induced oxidative toxicity. CD95-induced apoptosis resulted in chromatin condensation and caspase activation which if inhibited rescued cells from death. Mitochondrial involvement was identified by cytochrome c release following CD95 stimulation. Furthermore the pro-apoptotic protein Bax was shown to be conformationally active and translocated to the mitochondrial following CD95 stimulation indicating Bax mediated cytochrome c release. The novel DsRed-EYFP FRET construct which showed caspase activity in living cells and further in vitro analysis confirmed loss of acceptor fluorescence was due to construct cleavage. Oxidative glutamate toxicity was shown to be mediated by calpains (calcium dependent proteases) and furthermore it was shown that the caspases contribute to the survival of the cell. Investigation into the role of the stress associated MAP kinase (SAPK/JNK) resulted in abnormal nuclear morphologies which were shown to be the result of cell cycle arrest. Inhibition of not only JNK but also glycogen synthase kinase (GSK) and calpains resulted in cell cycle arrest in the G2 or pre-mitotic cell cycle phase. In conclusion we have described a model where glutamate induced ROS production damages vital cellular components resulting in the activation of the cell cycle. Upon progression through the cell cycle the damage is identified and the cells enter a caspase independent, calpain mediated cell death programme.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:422912 |
| Date | January 2005 |
| Creators | Elphick, Lucy M. |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/842842/ |
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