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Title: Bcl-2 family proteins and cell death in cortical neurons
Author: Fricker, M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2008
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This thesis explores the function and regulation of a subset of Bcl-2 proteins, the pro-apoptotic BH3-only proteins (BOPs), in cortical neuron apoptosis. Sodium arsenite (NA) is used as a model toxin in murine cortical murine cultures. Three BOPs were upregulated, Puma, Noxa and Bim. Primary cortical neuron cultures derived from mice lacking Puma, Noxa or Bim expressions were used to demonstrate that Puma, but not Bim or Noxa, was required for execution of NA-induced apoptosis. Adenovirus-mediated expression of exogenous Puma in cortical neurons was sufficient to induce cytochrome c release form mitochondria and apoptosis in a Bax-dependent manner, implicating Puma as an important upstream activator of Bax in cortical neurons. Furthermore, Puma knockout afforded cortical neurons substantial protection against many apoptotic insults, establishing Puma as an important apoptotic mediator of a variety of disease-relevant apoptotic signalling pathways. Whilst some insults induced Puma expression and apoptosis in a p53-dependent manner, others, including NA and ER stress, were predominantly p53-independent. NA treatment resulted in an accumulation of TA-p73α and a p53-independent increase in a number of p53/p63/p73 target genes, indicating that p73 may be involved in Puma induction. Expression of the inhibitory ΔNp73α and –β isoforms largely prevented NA-mediated induction of Puma and other p53/p63/p73 target genes, as well as protecting neurons from NA-induced apoptosis. In addition, luciferase assays using Puma promoter fragments demonstrated that the promoter region encompassing the p53 response elements was required for arsenite-mediated activation of the Puma motor. Finally, a novel post-translational modification of Puma was investigated using cell lines. Puma-α is phosphorylated at several sites in cycling cells, the major site being serine 10.  Initial experiments have failed to identify a functional consequence of Puma phosphorylation, although the identification if casein-kinase I as a candidate Puma kinase merits further investigation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available