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<p>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. </p><p> 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.</p><p> 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.</p><p> 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.</p><p> 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.</p><p> 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.</p>

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.

N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...