Regulation of apoptosis in neural cells : two methods for overcoming asynchrony : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

Programmed cell death, or apoptosis, plays a major role in the development of the nervous system and in the pathogenesis of neurodegenerative diseases. Although many proteins that play a key role in apoptosis in other systems also appear to function in neurons, the mechanism that triggers apoptosis in neurons is unknown. Apoptosis occurs asynchronously in neural and differentiated neuronal cells, which makes biochemical studies difficult because a small number of cells are at a particular stage at any one time. Two strategies were devised to overcome asynchrony during neural cell death. The first strategy was to separate rat pheochromocytoma (PC12) cells at different stages of commitment to cell death on the basis of cell density using equilibrium density gradient centrifugation. Three populations were defined. Cells in population 1 were the most dense and committed to cell death. They showed extensive loss of mitochondria1 cytochrome c, DNA fragmentation, and chromatin condensation. Population 3 contained live cells that floated to the top of density gradients. Population 2 displayed some chromatin condensation, yet little DNA fragmentation and loss of cytochrome c. This population showed upregulation of the pro-death factor, c-Jun, and downregulation of pro-survival kinase, Akt. Importantly, these cells could be rescued from death by nerve growth factor (NGF) and thus represent an intermediate stage of apoptosis, upstream of irreversible commitment. The second strategy was to create a cell-free system to reconstitute apoptosis. The addition of cytochrome c to human neuroblastoma (SY5Y) cell extracts activated caspase-9 and -3, and nucleolytic events in PC12 nuclei. Using this system, requirements for ATP and phosphatase activity for caspase activation and nuclear apoptosis were characterised. In addition, pro-survival molecules Akt and Creb were identified as caspase substrates during apoptosis in vitro. To assess whether these events occurred in vivo, the kinase inhibitor staurosporine and the topoisomerase inhibitor camptothecin were used to induce apoptosis in intact SY5Y cells. The pro-survival signalling kinase Raf-1 was downregulated during both staurosporine- and camptothecin-induced apoptosis, but Akt was only downregulated by camptothecin. These studies illustrate the complex interactions of apoptosis and signalling mechanisms in neural cells.