Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 study and therefore identifying those genes and path- ways that define the dying neurons at this time point could add to our understanding of the molecular mechanisms involved in the neuronal death programme. In our data set, we identified 164 genes that were sig- nificantly up-regulated (FDR, p < 0.01) after NGF with- drawal and the expression of 48 of these genes increased by more than 2-fold. Conversely, 379 genes were down-regulated when the significance threshold was set at p = 0.01 and the expression of 86 of these genes decreased by 2-fold or more. We performed Gene Ontology (GO) analysis and functional enrichment ana- lysis to identify specific annotations that were enriched following NGF withdrawal. Whilst this type of analysis depends upon a controlled vocabulary and therefore has its limitations, it also represents a powerful method for extracting potentially useful biological information from our gene expression data. In an analysis of transcription-dependent neuronal apoptosis proceeding via the mitochondrial pathway, functional categories such as intracellular signaling cas- cades, transcription and mitochondrial changes might be expected to be enriched. Whilst these categories are indeed enriched after NGF withdrawal, other categories that contain genes which could suggest additional hypotheses about the mechanisms of neuronal death were also highlighted. The significance of the induction of ER stress-associated genes, for example, may offer new insights into the cell death process, especially since a similar response was observed in cerebellar granule neurons undergoing apoptosis [37] and experiments in other systems suggest a role for interactions between the mitochondria and the ER. On the other hand, the down-regulation of genes associated with cholesterol and fatty acid biosynthesis may be associated with an inhibition of cell growth since cholesterol and fatty acids are required for the synthesis of membranes. Cluster analysis allowed us to group the genes accord- ing to their pattern of expression, especially in the pre- sence of the MLK inhibitor, CEP-11004. The expression of many of the genes induced after NGF withdrawal is reduced by CEP-11004, suggesting that they may be tar- gets of the MLK-JNK-c-Jun pathway. This group includes c-jun, dp5 and mkp1 whose promoters contain ATF sites that bind c-Jun and which are important for their induction after NGF withdrawal (Figure 7A) [24,26,34]. The induction of a few genes, such as egln3, is not affected by CEP-11004, suggesting that the tran- scription of these genes may be regulated by other tran- scription factors that are activated after NGF withdrawal, but not regulated by the JNK pathway, for example, FOXO3a or Myb. Interestingly, CEP-11004 reverses the decrease in the level of expression of some of the genes that are down-regulated after NGF Page 12 of 17 withdrawal. Many of these genes encode proteins involved in fatty acid metabolism and cholesterol meta- bolism, e.g. insig1, sqle, hmgcr, and hmgcs1, and their transcription is activated by sterol regulatory element- binding proteins (SREBPs). In sympathetic neurons, the MLK-JNK-c-Jun pathway may negatively regulate the activity of a key transcription factor or signaling protein that is important for the transcription of this set of genes. The expression of only a small number of cell death genes changes after NGF withdrawal. Bim, dp5, and puma mRNA levels have been previously shown to increase after NGF deprivation and in this study we have confirmed this for bim and dp5. We also found that the bmf, caspase-12, caspase-3, and caspase-4 mRNAs increase in level whereas the expression of cyto- chrome c and prothymosin alpha (ptma; an inhibitor of the apoptosome; [39]) decreases after NGF withdrawal (Table 2). Thus in sympathetic neurons, as previously described for cerebellar granule neurons [37], the expression of the components of the intrinsic pathway (bax, cytochrome c, apaf-1, caspase-9), which are all essential for cell death, is not greatly altered by NGF withdrawal. However, what does change significantly is the level of expression of four genes that encode BH3- only proteins that activate the intrinsic pathway: dp5, bim, bmf (Tables 1 and 2) and puma [19]. NGF-deprived sympathetic neurons undergo several biochemical and morphological changes before commit- ting to the neuronal death programme and some of these are likely to play an important role in triggering apoptosis. Interestingly, levels of mitochondrial-pro- duced reactive oxygen species (ROS) are known to increase early after NGF withdrawal [40] and this causes a cellular pro-oxidant state which appears to be required for the release of cytochrome c [40]. The regulation of cellular redox balance is critically determined by the activity of several antioxidant systems [41,42] one of which is the thioredoxin system [42]. Thioredoxin itself is regulated by an endogenous inhibitor, Txnip [43,44] and a reduction in thioredoxin activity due to an increase in Txnip levels might lead to increased oxida- tion of thiol groups in cellular proteins and ultimately an increase in apoptosis. We found a 9-fold increase in the level of the txnip mRNA after NGF withdrawal and this was reduced to 1.73-fold in the presence of CEP- 11004 (Figure 5) which was confirmed in NGF-depen- dent differentiated PC6-3 cells (data not shown). Impor- tantly, the level of Txnip protein also increased significantly after NGF withdrawal and this increase was prevented by CEP-11004 (Figure 6). These data suggest that txnip is a potential target of the MLK-JNK-c-Jun pathway and may play an important role in triggering the apoptotic programme after NGF withdrawal.