Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 The endoplasmic reticulum (ER) plays a significant role in how cellular proteins are processed, folded, mod- ified and transported. In neurodegenerative diseases, these cellular processes may go wrong leading to various levels of ER stress that may contribute to neuronal death [45]. When sympathetic neurons are treated with the ER-stressor, tunicamycin, c-Jun becomes phosphory- lated but this can be prevented using CEP-11004 [46]. Bcl-2 and Bcl-xL have been found to associate with both the mitochondrial outer membrane and the ER mem- brane and it has been reported that crosstalk can occur between the ER and the mitochondria in cells under- going apoptosis [47,48]. We found that two of the most up-regulated genes after NGF withdrawal, trib3 and ddit3, are associated with the ER-unfolded protein response and CEP-11004 prevented their increase in expression suggesting that they are potential MLK-JNK- c-Jun targets (Figure 5). Furthermore, functional analysis revealed that the ER unfolded protein response annota- tion was the most overrepresented gene category after NGF withdrawal suggesting that an ER stress response occurs in sympathetic neurons under these conditions. The exact role of these genes in ER stress-induced apoptosis remains unclear, however, it has been shown that CHOP10, a known AP-1 target gene, is induced by both ER stress and oxidative stress [49]. A propapopto- tic role for CHOP10 has been reported since its overex- pression can lead to apoptosis [50], whilst MEFs derived from CHOP10-/- mice are resistant to ER stress-induced cell death [51]. However, the mechanism by which CHOP induces apoptosis still remains unclear. It has been shown that CHOP-induced cell death is associated with the translocation of Bax from the cytosol to the mitochondria and that CHOP-induced cell death can be prevented by the overexpression of Bcl-2 or the knock- down of Bax [52]. The link between CHOP and Bax translocation could involve a novel ER-stress inducible gene, trib3. It has been shown that trib3 is induced via the ATF4-CHOP pathway through the identification of a CHOP-binding site in the proximal portion of the pro- moter [53]. Also, ER stress can activate bim through CHOP-C/EBPa dependent transcriptional activation [54] and in other studies CHOP has been found to bind to the promoter of the proapoptotic Bcl-2 family mem- ber puma [55]. The relationship between ER stress, CHOP, Trib3 and BH3-only proteins may suggest an important role in the apoptotic pathway after NGF with- drawal. Furthermore, we also identified a conserved ATF site (TTACATCA) 14 bp upstream of Exon 1 in the rat trib3 gene (Figure 7C) which is identical to the reverse complement of the ATF site in the dp5 promoter. This potential c-Jun/ATF2 binding site in the promoter of the rat trib3 gene suggests that this gene might also be a direct target of the MLK-JNK-c-Jun pathway. We also Page 13 of 17 found evidence of an increase in Trib3 and Ddit3/ CHOP10 protein levels after NGF withdrawal and this increase was prevented by CEP-11004 (Figure 6). NGF withdrawal leads to a decrease in PI3K and Akt activity resulting in FOXO activation. FOXO3a translo- cates into the nucleus [56,57] and has been shown to trigger apoptosis by activating the transcription of genes necessary for cell death, such as bim. The mxi1 gene is also a target of FOXO3a, which binds to sites in the first intron downstream of the Mxi1-SRa promoter [58]. Mxi1 dimerises with Max and binds to E-boxes and represses c-Myc and MycN target genes by recruiting co-repressors to their promoters [59]. Interestingly, the mxi1 mRNA increases in level after NGF withdrawal (Figure 5) whereas c-myc and mycn mRNA levels decrease (Tables 1 and 2). Overexpression of MycN in sympathetic neurons has been shown to partially protect against NGF withdrawal-induced death [60], so by antagonising MycN, Mxi1 might have a proapoptotic role in this system. Furthermore, the expression of genes that are activated by c-Myc and MycN decreases after NGF deprivation, for example id2 and ptma (Table 2). Ptma can act as a repressor of the apoptosome [39] so it will be interesting to determine whether Ptma pro- tein levels also decrease after NGF withdrawal. Conver- sely, the expression of genes repressed by c-Myc and MycN increases after NGF deprivation, for example, ndrg1 (Figures 5 and 6 and Table 2). Conclusions The sympathetic neuron model is one of the best stu- died models of neuronal apoptosis. For the first time, we now have a global overview of the changes occurring at the transcriptional level in NGF-deprived sympathetic neurons. In the future, it will be interesting to determine how the regulated genes identified in this study contri- bute to the NGF withdrawal-induced death pathway. This may lead to the identification of new targets for the development of neuroprotective drugs that inhibit neuronal death following acute injuries to the nervous system or in neurodegenerative diseases. Methods Cell culture Animal experiments were performed according to the Animals (Scientific Procedures) Act 1986 under a license reviewed and approved by the Biological Services Unit at University College London. Sympathetic neurons were isolated from the superior cervical ganglia (SCG) of 1-day-old Sprague Dawley rats (Biological Services Unit, University College London, UK) and cultured in Dulbecco’s modified Eagle medium (DMEM; Life Tech- nologies, USA) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine (Invitrogen Ltd, UK) and