Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 R E S E A R C H A R T I C L E Open Access Global analysis of gene expression in NGF- deprived sympathetic neurons identifies molecular pathways associated with cell death Mark Kristiansen, Francesca Menghi, Rosie Hughes, Mike Hubank and Jonathan Ham* Abstract Background: Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF. Results: We have used Affymetrix Exon arrays to study the pattern of expression of all known genes in NGF- deprived sympathetic neurons. We identified 415 up- and 813 down-regulated genes, including most of the genes previously known to be regulated in this system. NGF withdrawal activates the mixed lineage kinase (MLK)-c-Jun N- terminal kinase (JNK)-c-Jun pathway which is required for NGF deprivation-induced death. By including a mixed lineage kinase (MLK) inhibitor, CEP-11004, in our experimental design we identified which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun pathway. A detailed Gene Ontology and functional enrichment analysis also identified genetic pathways that are highly enriched and overrepresented amongst the genes expressed after NGF withdrawal. Five genes not previously studied in sympathetic neurons - trib3, ddit3, txnip, ndrg1 and mxi1 - were validated by real time-PCR. The proteins encoded by these genes also increased in level after NGF withdrawal and this increase was prevented by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway. Conclusions: The sympathetic neuron model is one of the best studied models of neuronal apoptosis. Overall, our microarray data gives a comprehensive overview of, and provides new information about, signalling pathways and transcription factors that are regulated by NGF withdrawal. Background During normal nervous system development, neurons depend on growth factors secreted by their target tissues for survival [1]. These neurotrophic factors bind to cell surface receptors on developing neurons and activate intracellular signalling pathways that inhibit pro- grammed cell death and promote neuronal growth. The regulation of programmed cell death by survival factors plays an integral part in ensuring that neuronal popula- tions of the correct size are established [1-3]. In * Correspondence: J.Ham@ich.ucl.ac.uk Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK addition, increasing evidence suggests that apoptosis contributes to the neuronal loss seen after acute injuries to the nervous system, such as stroke or trauma, or in cell culture and animal models of neurodegenerative dis- orders, such as Parkinson’s disease and Alzheimer’s dis- ease [4]. Developing sympathetic neurons have proved to be a valuable model for studying the molecular mechanisms of apoptosis and the signalling pathways that regulate neuronal death [5-11]. These cells require nerve growth factor (NGF) for survival during late embryonic and early postnatal development. When deprived of NGF, sympathetic neurons die by apoptosis and this death is inhibited by actinomycin D and © 2011 Kristiansen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.