Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 cycloheximide suggesting that new gene expression is required for cell death to occur [12]. The key prediction of this hypothesis is that the transcription of specific genes increases after NGF withdrawal and that the pro- teins encoded by these induced genes trigger cell death. To date only a limited number of induced genes that promote apoptosis have been identified, either by study- ing the expression of candidate genes (c-jun, bim, tp63, puma) or in mRNA differential display experiments (egln3). In the case of each of these genes the mRNA and protein increases in level after NGF withdrawal and experiments with knockout mice have demonstrated that the gene is required for NGF withdrawal-induced death [13-23]. However, the intracellular signalling path- ways that are altered by NGF withdrawal - the MLK- JNK-c-Jun pathway is activated and the PI3K-Akt and Raf-MEK-ERK pathways are inactivated - are likely to regulate the expression of a much larger number of genes. Some of these genes, like bim and puma, will directly regulate the intrinsic pathway of caspase activa- tion. However, other genes induced after NGF withdra- wal may be involved in other aspects of NGF withdrawal-induced death, e.g. alterations in signalling pathways, changes in cell shape, the decrease in the rate of protein synthesis or neurite fragmentation. No pre- vious study has comprehensively addressed these issues in sympathetic neurons. Recent advances in gene micro- array technology have allowed us to investigate the expression of all known genes in sympathetic neurons for the first time. The Affymetrix Rat Exon 1.0ST microarray allows more accurate measurement of gene expression at the whole gene level because there are several oligonucleotide probes for each exon of a gene. In addition, exon arrays can be used to measure the expression of individual exons, which provides informa- tion about alternative splicing. Microarray analysis represents an unbiased approach to the investigation of NGF-withdrawal induced apoptosis and will identify the majority of the genes that are up- or down-regulated after NGF withdrawal. Using developing sympathetic neurons as a model sys- tem, we have carried out a genome-wide analysis of gene expression at 16 hours following NGF withdrawal. Furthermore we have analysed gene expression in NGF- deprived sympathetic neurons in the presence or absence of the MLK inhibitor, CEP-11004 [24]. By including CEP-11004 in our experimental design we were able to identify which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK- c-Jun signalling pathway, which is activated after NGF withdrawal and required for NGF deprivation-induced death [13,14,25-30]. To provide further insight into the molecular mechanisms that underlie NGF withdrawal- induced apoptosis in sympathetic neurons we also Page 2 of 17 performed functional analysis that identified highly enriched genetic pathways. Our data provides a compre- hensive overview of how NGF withdrawal alters signal- ling pathways and global gene expression. This will increase our understanding of the basic mechanisms of neuronal apoptosis and may also identify new targets for the development of neuroprotective drugs. Results Temporal analysis of NGF withdrawal induced apoptosis in sympathetic neurons To comprehensively study the expression of all known genes in rat sympathetic neurons we used Affymetrix Exon arrays to profile gene expression at 16 hours after NGF withdrawal compared to +NGF as a control. We selected 16 hours because this was previously defined as the transcriptional commitment point [5] and induced genes known to be required for NGF withdrawal- induced death, e.g. c-jun, bim, egln3, are expressed at a high level at this time. To be able to relate any changes in gene expression that we might observe to the morphological and biochemical changes that are known to occur after NGF withdrawal we carried out a temporal analysis of NGF withdrawal- induced apoptosis using several well-defined markers (Fig- ure 1). The morphological changes that occur in sympa- thetic neurons following NGF withdrawal are apparent after 8-12 hours of NGF deprivation (Figure 1). During this time, the smooth appearance of the plasma membrane is lost and the cell becomes irregular in structure. This is accompanied by beading of the neurites. At later time- points (16-24 h), membrane blebbing and extensive neur- ite degeneration occur shortly before the neuron starts to lose its structural integrity. Nuclear changes such as chro- matin condensation and nuclear shrinkage were visualised by staining with Hoechst dye and DNA fragmentation was detected by TUNEL labelling. These changes occur rapidly after NGF withdrawal but become much more apparent from 12-16 hours (Figure 1). Other key apoptotic events such as cytochrome c release from the mitochondria and the activation of caspase 3 were also measured (Figure 1). Cytochrome c is released from the mitochondria following NGF withdrawal and eventually decreases in level. Simi- larly, caspase 3 becomes activated and is clearly detected in sympathetic neurons deprived of NGF from 8 hours. We also detected an increase in c-Jun phosphorylation at serine 63 following NGF withdrawal (Figure 1). This site is phosphorylated by JNKs, which are activated after NGF deprivation. Importantly, the level of c-Jun phosphoryla- tion increases before and peaks at 16 hours. Therefore at 16 hours, the timepoint chosen for our Exon microarray analysis, the MLK-JNK-c-Jun pathway has been activated in many neurons, and some cells in the population are already undergoing apoptosis.