Whitfield, Jonathan Richard; (2000) An investigation of the regulation and role of c-Jun during neuronal apoptosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Apoptotic cell death plays a key role during the development of the nervous system. Sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It is known that expression of a c-Jun dominant negative mutant promotes sympathetic neuron survival following NGF withdrawal, whereas overexpression of c- Jun induces apoptosis in the presence of NGF. The aims of this thesis were to investigate how c-Jun expression is regulated in sympathetic neurons and to determine the mechanism by which c-Jun activates the cell death programme.In microinjection experiments, a c-jun promoter/CAT reporter gene was activated following NGF withdrawal and this required c-Jun/ATF-2 binding sites within the c-jun promoter. Expression of dominant negative c-Jun blocked the NGF withdrawal-induced increase in endogenous c-Jun protein, suggesting that c-Jun positively regulates its own synthesis. Expression of activated MEK kinase 1 (MEKK1), an upstream component of the c-Jun N-terminal kinase (JNK) pathway, increased the level of N-terminally phosphorylated (activated) c-Jun and induced apoptosis in the presence of NGF. Therefore, stimulation of the c-Jun pathway increases the level of phosphorylated c-Jun and triggers apoptosis. To investigate how c-Jun promotes neuronal apoptosis, an adenoviral gene delivery system was developed. Infection of sympathetic neurons with a virus that expressed dominant negative c-Jun prevented cytochrome c release from mitochondria, a key step in neuronal apoptosis. Expression of MEKK1 caused cytochrome c to be released in the presence of NGF. The pattern of expression of several potential regulators of cytochrome c efflux was studied. A large increase in the level of bim mRNA and protein was observed and this was reduced by expression of dominant negative c-Jun. Overexpression of Bim in microinjection experiments induced both cytochrome c release and apoptosis in the presence of NGF. Thus, the effect of c-Jun on cytochrome c release is mediated, at least in part, by Bim.

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