Apoptosis is a form of programmed cell death that has important functions during normal mammalian development and is important for tissue homeostasis and defence against pathogens. Apoptosis occurs extensively during the development of the nervous system and is important for establishing neuronal populations of the correct size. In addition, there is strong evidence that apoptosis is one of the major mechanisms of neuronal death following stroke or traumatic brain injury, and neurons often die by apoptosis in cell culture and animal models of chronic human neurodegenerative disorders. To study the molecular mechanisms of neuronal apoptosis we have used developing sympathetic neurons as a model system (Kristiansen and Ham, 2014). These cells require nerve growth factor (NGF) for survival and die by apoptosis in its absence. This death depends on de novo gene expression. The aim of our research is to identify genes that are induced by NGF withdrawal and study their function and regulation in sympathetic neurons.
Using the sympathetic neuron model, we were one of the first groups to show that the basic-leucine zipper transcription factor c-Jun, an AP-1 protein, increases in level as soon as 4 hours after NGF withdrawal and is required for NGF withdrawal-induced death (Ham et al., 1995). We found that c-Jun protein levels increase in sympathetic neurons after NGF withdrawal because the c-jun promoter is activated and that c-Jun N-terminal phosphorylation increases, which increases the ability of c-Jun to activate the transcription of target genes (Eilers et al., 1998). Increased N-terminal phosphorylation of c-Jun was preceded by an increase in c-Jun N-terminal kinase (JNK) activity, which is required for NGF withdrawal-induced death (Ham et al., 1995; Eilers et al., 1998; Eilers et al., 2001).
Some of the known transcriptional targets of the MLK-JNK-c-Jun pathway in sympathetic neurons.
One of the major goals of our research is to determine the mechanisms by which c-Jun promotes apoptosis. We found that the activity of c-Jun is required for the release of mitochondrial cytochrome c in sympathetic neurons deprived of NGF, a key event required for the activation of caspases, the effectors of cell death (Whitfield et al., 2001). Members of the Bcl-2 family of proteins regulate the release of mitochondrial cytochrome c. We found that the proapoptotic BH3-only protein BimEL increases in level after NGF deprivation and that c-Jun contributes to the induction of Bim after NGF withdrawal (Whitfield et al., 2001). Overexpression of BimEL induced cytochrome c release and apoptosis in the presence of NGF and sympathetic neurons injected with bim antisense oligonucleotides or isolated from bim (-/-) knockout mice died more slowly after NGF withdrawal (Whitfield et al., 2001). These results indicate that Bim plays an important role in the NGF withdrawal-induced death pathway. We have also studied the function and regulation of Dp5, another BH3-only protein that increases in level after NGF deprivation (Towers et al., 2009).
To understand how the intracellular signalling pathways that are altered by NGF withdrawal regulate the expression of bim we cloned the 5' end of the bim gene and studied how its transcription in sympathetic neurons is regulated by c-Jun as well as FOXO3a, NF-Y and CBP/p300 (Gilley et al., 2003; Hughes et al., 2011). We showed that the expression of bim is negatively regulated by the PI3K-Akt pathway in the presence of NGF. After NGF withdrawal, Akt-dependent phosphorylation of FOXO3a decreases and FOXO3a translocates from the cytoplasm into the nucleus, binds to two conserved FOXO binding sites in the promoter and first intron of bim and activates bim transcription (Gilley et al., 2003; Hughes et al., 2011).
In addition, we recently used Affymetrix Exon arrays to identify other JNK-c-Jun pathway target genes that are induced in sympathetic neurons undergoing apoptosis after NGF deprivation, and are studying their function and how they are regulated by the JNK pathway and other intracellular signalling pathways (Kristiansen et al., 2010; Kristiansen et al., 2011).
Schematic overview of Exon array analysis of gene expression in sympathetic neurons (Kristiansen et al., 2011).
Eilers, A., Whitfield, J., Babij, C., Rubin, L.L. and Ham, J. (1998) Role of the Jun kinase pathway in the regulation of c-Jun expression and apoptosis in sympathetic neurons. Journal of Neuroscience 18, 1713-1724.
Eilers, A., Whitfield, J., Shah, B., Spadoni, C., Desmond, H. and Ham, J. (2001) Direct inhibition of c-Jun N-terminal kinase in sympathetic neurones prevents c-jun promoter activation and NGF withdrawal-induced death. Journal of Neurochemistry 76, 1439-1454.
Gilley, J., Coffer, P.J. and Ham, J. (2003) FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. Journal of Cell Biology 162, 613-622.
Ham, J., Babij, C., Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv, M. and Rubin, L.L. (1995) A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 14, 927-939.
Hughes, R., Kristiansen, M., Lassot, I., Desagher, S., Mantovani, R. and Ham, J. (2011) NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons. Cell Death and Differentiation 18, 937-947.
Kristiansen, M., Hughes, R., Patel, P., Jacques, T.S., Clark, A.R. and Ham, J. (2010) Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons. Journal of Neuroscience 30, 10820-10832.
Kristiansen, M., Menghi, F., Hughes, R., Hubank, M. and Ham, J. (2011) Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death. BMC Genomics 12: 551.
Kristiansen, M. and Ham, J. (2014) Programmed cell death during neuronal development: the sympathetic neuron model. Cell Death and Differentiation, 21, 1025-1035.
Towers, E., Gilley, J., Randall, R., Hughes, R., Kristiansen, M., Ham, J. (2009) The pro-apoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons. Nucleic Acids Research 37, 3044-3060.
Whitfield, J., Neame, S.J., Paquet, L., Bernard, O. and Ham, J. (2001) Dominant negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release. Neuron 29, 629-643.