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Title: On the neuroprotective actions of FK506
Author: Macleod, Malcolm
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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To examine the role of FK506 in neuronal apoptosis I have examined apoptotic cell death in the SHSY-5Y neuroblastoma cell line. Initial experiments used differentiated SHSY-5Y cells at high passage. Neither their morphology nor their response to various toxins was constant. I therefore identified a source of low passage SHSY-5Y cells and compared their behaviour with high passage cells under differentiating conditions. Treatment of low passage cells with retinoic acid and serum reduction for 7 days resulted in the development of neuron- like morphology, with stabilisation of cell number and the development of immunopositivity for the neuronal marker MAP2 and the cell cycle G0 marker p21waf. In terminally differentiated SHSY-5Y cells the calcium ionophore ionomycin caused rapid and simultaneous loss of mitochondrial respiratory activity and membrane integrity (determined using the MTS and LDH assays respectively) and had no effect on endogenous (PARP) or synthetic (Ac-DEVD-amc) Caspase 3 substrate cleavage. FK506 had no effect on ionomycin induced death. The broad spectrum protein kinase C inhibitor staurosporine also caused death in SHSY-5Y cells in a concentration dependent fashion, but the onset of the death was slower than with ionomycin. This was associated with increased cleavage of the synthetic caspase 3 substrate Ac-DEVD-amc. The decline in viability determined using the MTS assay, an index of mitochondrial function, was more pronounced than that seen using the LDH assay, a measure of membrane integrity. FK506 had no effect on staurosporine- induced death. Serum withdrawal caused the apoptosis of a proportion of SHSY-5Y cells, as evidenced by characteristic nuclear changes, caspase activation and cleavage of caspase substrates. FK506 partially reduced the decline in viability seen following serum withdrawal, but was without discernible effect on caspase activation. In conclusion, I have demonstrated that FK506 can be without effect on NOS activity in brain tissue. Secondly, I describe the terminal differentiation of SHSY-5Y cells. In these cells both serum withdrawal and staurosporine cause a decline in viability that has some of the feature of apoptosis; the effect of serum withdrawal, but not staurosporine, is partially reversed by FK506.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available