Identification of genes regulated during neuronal apoptosis using oligonucleotide microarrays
Neuronal apoptosis occurs extensively during the normal development of the mammalian nervous system and ensures that appropriate connections are made between neurons and their target cells. Developing sympathetic neurons and the PC6-3 cell line, a PC 12 sub-clone, are useful in vitro systems for studying neuronal apoptosis. Both sympathetic neurons and neuronally differentiated PC6-3 cells depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal in a transcription and translation-dependent manner. The c-Jun protein is known to be required for neuronal cell death following survival factor withdrawal, however the direct targets of this basic/leucine zipper transcription factor are unknown. The aim of this thesis was to identify and study c-Jun target genes involved in neuronal apoptosis. In experiments using Affymetrix GeneChip oligonucleotide microarrays, 78 genes were identified that had an altered pattern of expression after NGF withdrawal from neuronal PC6-3 cells. One of the induced genes, ATF-3, a basic/leucine zipper transcription factor that can dimerise with c-Jun, was confirmed as an up-regulated transcript during PC6-3 cell apoptosis and this occurred after the induction of c-Jun. In sympathetic neurons, atf-3 RNA and protein levels increase between 8 and 16 hours after NGF withdrawal and remain elevated at 24 hours. This protein induction occurs after that of c-Jun and is inhibited by the c-Jun N-terminal kinase inhibitor SP600125 and the mixed-lineage kinase (MLK) inhibitor CEP-11004. This suggests that induction of ATF-3 may require JNK activity in neurons and that the atf-3 gene might be a target of c-Jun. The function of ATF-3 in sympathetic neurons was investigated in microinjection experiments. Injection of expression vectors for c-Jun and ATF-3 decreased the survival of sympathetic neurons in the presence of NGF, as measured at 72 hours after injection. In contrast, overexpression of ATF-3 or the ATF-3 basic/leucine zipper domain in the absence of NGF increased the survival of sympathetic neurons. In addition, when ATF-3 expression was knocked down, using a pSUPER ATF-3 RNAi expression vector, the amount of cell death observed after 48 hours of NGF deprivation in neurons was increased.