Apoptosis occurs extensively during the development of the mammalian nervous system, during which time neurons depend on neurotrophic factors, such as nerve growth factor (NGF), for survival. This process is tightly controlled and the BCL-2 family of proteins are key regulators of neuronal apoptosis. DP5, a member of the proapoptotic BH3-only subfamily with highly restricted expression in the nervous system, is upregulated in sympathetic neurons undergoing NGF deprivation-induced apoptosis. Sympathetic neurons from dp5-/- knockout mice die more slowly after NGF deprivation and dp5-/- motor neurons have increased resistance to axotomy-induced death. DP5 is regulated at the transcriptional level and the c-Jun N-terminal kinase (JNK) pathway contributes to the induction of dp5 after NGF withdrawal. The goal of my project was to determine the mechanism by which NGF deprivation induces dp5 transcription in sympathetic neurons and to identify the regions of the gene and signalling pathways involved. Firefly luciferase reporter constructs containing different regions of the dp5 gene were tested in a microinjection assay using sympathetic neurons cultured in vitro. Levels of luciferase activity after NGF deprivation or following treatment with chemical inhibitors were measured using immunofluorescence or a dual luciferase assay. Three regions important for the induction of dp5 after NGF withdrawal were identified: 1 kb of promoter, a 400 bp region of the intron conserved between the rat, mouse and human genes, and ~5 kb of 3'UTR. Sequences within the dp5 promoter and intron were shown to be regulated by the MLK/JNK pathway. In addition, a functionally important binding site for the transcription factor E4BP4 was identified in the dp5 promoter region. Overexpression of E4BP4 strongly repressed dp5 promoter activity, whereas mutation of this site reduced basal promoter activity suggesting that an activator, such as a related PAR family transcription factor, might bind to this site in sympathetic neurons.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:428072 |
| Date | January 2005 |
| Creators | Towers, Emily Rachel |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1446620/ |
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