Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Table 1 Genes known to be regulated by NGF withdrawal Gene symbols Gene name Page 5 of 17 -NGF CEP v v +NGF +NGF Pathway References Bid3/Dp5/Hrk BH3-interacting domain-containing protein 3; Dp5; harakiri Trib3 tribbles homologue 3 (Drosophila) Dusp1/Mkp1 dual specificity phosphatase 1;MAP kinase phosphatase 1 Bcl2l11/Bim/Bod BCL2-like 11 (apoptosis facilitator) Egln3/SM-20 EGL nine homologue 3 (C. elegans) Gadd45g growth arrest and DNA-damage- inducible protein 45 gamma Jun Jun oncogene; c-Jun Ccnd1 cyclin D1 Myc myelocytomatosis viral oncogene homologue (avian); c-Myc Ngfr/p75NTR nerve growth factor receptor (TNFR superfamily, member 16); p75NTR 9.78 5.21 4.69 4.35 3.41 3.16 2.62 1.46 -1.76 -1.73 5.28 1.81 1.60 1.95 3.53 1.31 1.55 1.04 -1.32 -1.44 cell death 32, 34 ER stress 35 MAPK pathways 13, 24 cell death 16, 17 cell death 15 DNA damage inducible 33 AP1 family 13, 14 cell cycle 31 Myc network 31 neurotrophin signalling 31 The fold increase in RNA level after NGF withdrawal or after NGF withdrawal in the presence of CEP-11004 (average of 3 independent experiments) from the microarray is shown for genes that have been previously reported to be transcriptionally regulated in this system (see references above). Functional categories such as the ER unfolded protein response and negative regulation of protein kinase activity were significantly over-represented in the up-regulated genes whilst in the down-regulated genes, categories such as cholesterol biosynthetic process and the electron trans- port chain were significantly enriched (Figure 3). Notably, the proportion of up-regulated genes related to amino acid transport and positive regulation of developmental process was also significantly higher than expected by chance. Finally, a significant proportion of genes asso- ciated with the cellular components plasma membrane, mitochondria and the ER was strikingly different between the up- and down-regulated genes (Figure 3). The 50 most significantly up- and down-regulated genes were subjected to hierarchical clustering analysis (Figure 4). Both samples and genes were clustered according to their expression profiles using the Euclidean distance metric to calculate dendrograms. Genes with similar expression profiles may be regulated by common pathways and be involved in related functions. We observed four major patterns of gene expression (Figure 4): 1) genes induced after NGF withdrawal that require the MLK-JNK pathway, for example dusp1 and hamp (their induction was reduced by CEP-11004); 2) genes induced after NGF withdrawal that may not depend on the MLK-JNK pathway, e.g. egln3 and prg1; 3) genes that are down-regulated after NGF withdrawal that are res- cued by CEP-11004, e.g. hmgcr and insig1, and 4) genes that are down-regulated after NGF withdrawal whose decrease in expression is not affected by CEP-11004, e.g. dusp6 and prl6a1. Genes belonging to some of the most common networks are listed in Table 2. Validation of gene induction after NGF withdrawal by real time PCR Functional enrichment analysis revealed that the annota- tion ER-unfolded protein response was the most enriched term after NGF withdrawal suggesting that an ER stress response occurs in sympathetic neurons deprived of NGF. We therefore initially selected two regulated genes from this category for further validation. Trib3 (up-regulated 5.2-fold after NGF withdrawal) and ddit3/CHOP10 (up- regulated 4.11-fold) were the third and ninth most up- regulated genes respectively after NGF withdrawal (Figure 5A). The trib3 mRNA was previously shown to increase in level after NGF withdrawal in PC12 cells [35] but nothing is known about its role in sympathetic neurons. CHOP10 has not been studied before in sympathetic neurons. The increase in the level of the trib3 and ddit3/chop10 mRNAs was reduced by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway. To validate these exon array results, we cultured sympathetic neurons for 6 days in the presence of NGF and then for a further 16 hours in the presence or absence of NGF ± CEP-11004. The levels of trib3 and ddit3 mRNA were then measured by quantitative real time PCR (Figures 5B- C). After NGF withdrawal, the levels of trib3 mRNA and ddit3 mRNA increased by 3.33-fold and 3.68-fold respec- tively but this was reduced to 0.79-fold and 1.1-fold in the presence of CEP-11004 when normalised to gapdh (Figure 5B). A similar increase was seen in trib3 and ddit3 mRNA levels after NGF withdrawal when normalised to hprt1 (Figure 5C). We also found that the txnip gene was signifi- cantly up-regulated after NGF withdrawal. Txnip binds to and inhibits thioredoxin, a major antioxidant protein in neurons. Any perturbation of the redox system in neurons could lead to a cellular pro-oxidant state that is a neces- sary component of apoptotic death [36]. We found that the txnip mRNA levels mirrored the patterns from micro- array analysis (Figure 5). Interestingly, txnip mRNA levels increased significantly after NGF withdrawal (up-regulated 9.03-fold) and this was reduced to 1.73-fold in the pre- sence of CEP-11004 when measured by qPCR and nor- malised to either gapdh or hprt1. Two other genes were