Investigation of the pattern of expression, regulation and function of the Bcl-2 family of proteins during neuronal development and apoptosis

The Bcl-2 and Bcl-xL proteins suppress programmed cell death, whereas Bax promotes apoptosis. In this thesis, the pattern of expression of Bcl-2, Bax and Bcl-x was investigated during neuronal differentiation and development. Using specific antibodies against Bax, Bcl-x, and Bcl-2 we found that all three proteins were widely expressed in the cortex and cerebellum of neonate rats. However, following the period of developmental cell death, Bax levels decreased dramatically and were 20 to 140 fold lower in the adult brain. In contrast, Bcl-x protein levels remained constant throughout development. Immunohistochemical analysis revealed that in the cerebellum, the Bax decrease in the adult was confined to the granule neurons. Purified cerebellar granule cell cultures, prepared from 8- day-old rats, and maintained in medium with serum and high extracellular potassium for up to 5 weeks, also exhibited a similar decrease in their Bax protein levels with time in vitro. Interestingly, the Bax downregulation that occurred in cultured cerebellar granule cells closely correlated with the appearence of the GABAA receptor a6 subunit, a specific marker for mature granule neurons. The balance between the level of Bax and the levels of other anti- apoptotic members of the Bcl-2 family such as Bcl-xL is one factor that affects the way cells respond to death signals. We found that overexpression of Bax in developing sympathetic neurons induced apoptosis in the presence of their trophic factor (NGF), and increased the rate of cell death after NGF withdrawal. This effect could be blocked by co-expression of Bcl-xL or the baculovirus p35 protein, a broad inhibitor of the caspases. These results suggest that during development of the brain the ratio of Bax to Bcl-x changes dramatically and is higher during the period when cells normally undergo programmed cell death. Furthermore, Bax-induced death requires the activity of the caspase family