Studies on the Bcl-2 Family of Apoptosis Regulators in the Nervous System

Abstract: Apoptosis is a type of cell death with a specific morphology and molecular program, which is essential for the development of the nervous system. However, inappropriate cell death has been implicated in several neurodegenerative diseases. The Bcl-2 protein family is a class of proteins, which can regulate the cell death program in either a positive (pro-apoptotic family members) or a negative (anti-apoptotic family members) way. This thesis further elucidates the role of Bcl-2 family members in the nervous system. Special focus has been put on the anti-apoptotic family member Bcl-w, whose function in the nervous system was previously unknown, and the pro-apoptotic family member Bad which serves as a link between growth factor signalling and apoptosis. Bcl-w mRNA was found to be upregulated during rat brain development suggesting increasing importance of Bcl-w with age in the nervous system. In contrast, mRNA levels encoding the anti-apoptotic protein Bcl-x were downregulated during development. Bcl-w was also found to have an anti-apoptotic function in neurons, rescuing sympathetic neurons from cell death after nerve growth factor deprivation. To further elucidate the mechanism by which Bcl-w exerts its function, we screened a yeast two-hybrid library for proteins interacting with Bcl-w. Two of the isolated positive clones encoded the pro-apoptotic protein Bad and a novel splice variant of Bad with a different carboxyterminal sequence. Both isoforms of Bad induced cell death in sympathetic neurons, which could be counteracted by Bcl-w, indicating that Bcl-w and Bad can interact both physically and functionally. Further studies on the genomic structure of the Bad gene suggested the presence of an additional splice variant, not expressing the first exon. Immunohistochemical analysis indicates that the isoform(s) not expressing the first exon is more widely expressed in adult rat brain than the known forms. Finally, we show that high cell density can enhance survival of cerebellar granule neurons and that bcl-2 and bcl-x mRNA levels are upregulated in high density cultures.