Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death
© Kristiansen et al; licensee BioMed Central Ltd. 2011
Received: 4 August 2011
Accepted: 8 November 2011
Published: 8 November 2011
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.
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.
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.
During normal nervous system development, neurons depend on growth factors secreted by their target tissues for survival . These neurotrophic factors bind to cell surface receptors on developing neurons and activate intracellular signalling pathways that inhibit programmed cell death and promote neuronal growth. The regulation of programmed cell death by survival factors plays an integral part in ensuring that neuronal populations of the correct size are established [1–3]. In 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 disorders, such as Parkinson's disease and Alzheimer's disease . 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 cycloheximide suggesting that new gene expression is required for cell death to occur . The key prediction of this hypothesis is that the transcription of specific genes increases after NGF withdrawal and that the proteins encoded by these induced genes trigger cell death.
To date only a limited number of induced genes that promote apoptosis have been identified, either by studying the expression of candidate genes (c-jun, bim, tp63, puma) or in mRNA differential display experiments (egln3). In the case of each of these genes the mRNA and protein increases in level after NGF withdrawal and experiments with knockout mice have demonstrated that the gene is required for NGF withdrawal-induced death [13–23]. However, the intracellular signalling pathways that are altered by NGF withdrawal - the MLK-JNK-c-Jun pathway is activated and the PI3K-Akt and Raf-MEK-ERK pathways are inactivated - are likely to regulate the expression of a much larger number of genes. Some of these genes, like bim and puma, will directly regulate the intrinsic pathway of caspase activation. However, other genes induced after NGF withdrawal may be involved in other aspects of NGF withdrawal-induced death, e.g. alterations in signalling pathways, changes in cell shape, the decrease in the rate of protein synthesis or neurite fragmentation. No previous study has comprehensively addressed these issues in sympathetic neurons. Recent advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons for the first time. The Affymetrix Rat Exon 1.0ST microarray allows more accurate measurement of gene expression at the whole gene level because there are several oligonucleotide probes for each exon of a gene. In addition, exon arrays can be used to measure the expression of individual exons, which provides information about alternative splicing. Microarray analysis represents an unbiased approach to the investigation of NGF-withdrawal induced apoptosis and will identify the majority of the genes that are up- or down-regulated after NGF withdrawal.
Using developing sympathetic neurons as a model system, we have carried out a genome-wide analysis of gene expression at 16 hours following NGF withdrawal. Furthermore we have analysed gene expression in NGF-deprived sympathetic neurons in the presence or absence of the MLK inhibitor, CEP-11004 . By including CEP-11004 in our experimental design we were able to identify which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun signalling pathway, which is activated after NGF withdrawal and required for NGF deprivation-induced death [13, 14, 25–30]. To provide further insight into the molecular mechanisms that underlie NGF withdrawal-induced apoptosis in sympathetic neurons we also performed functional analysis that identified highly enriched genetic pathways. Our data provides a comprehensive overview of how NGF withdrawal alters signalling pathways and global gene expression. This will increase our understanding of the basic mechanisms of neuronal apoptosis and may also identify new targets for the development of neuroprotective drugs.
Temporal analysis of NGF withdrawal induced apoptosis in sympathetic neurons
To comprehensively study the expression of all known genes in rat sympathetic neurons we used Affymetrix Exon arrays to profile gene expression at 16 hours after NGF withdrawal compared to +NGF as a control. We selected 16 hours because this was previously defined as the transcriptional commitment point  and induced genes known to be required for NGF withdrawal-induced death, e.g. c-jun, bim, egln3, are expressed at a high level at this time.
Gene expression profiling in sympathetic neurons after NGF withdrawal
Genes known to be regulated by NGF withdrawal
BH3-interacting domain-containing protein 3; Dp5; harakiri
tribbles homologue 3 (Drosophila)
dual specificity phosphatase 1;MAP kinase phosphatase 1
BCL2-like 11 (apoptosis facilitator)
EGL nine homologue 3 (C. elegans)
growth arrest and DNA-damage- inducible protein 45 gamma
DNA damage inducible
Jun oncogene; c-Jun
myelocytomatosis viral oncogene homologue (avian); c-Myc
nerve growth factor receptor (TNFR superfamily, member 16); p75NTR
Gene Ontology analysis and unsupervised hierarchical clustering
Selection of genes regulated by NGF withdrawal identified in this study
cAMP responsive element modulator
Jun D proto-onocogene
Bcl2 modifying factor
caspase 3, apoptosis related cysteine protease
caspase 4, apoptosis related cysteine protease
cytochrome c, somatic
Growth arrest and DNA damage-inducible 45 alpha
activating transcription factor 3
DNA damage-inducible transcript 3/CHOP10
eukaryotic translation initiation factor 2 alpha kinase 3/PERK
homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
myeloid differentiation primary response gene 116
Fatty acid/cholesterol metabolism
3-hydroxy-3-methylglutaryl-coenzyme A reductase
insulin induced gene 1
thioredoxin interacting protein/Vdup1
dual specificity phosphatase 6/Mkp3
Ras homologue gene family, member Q
basic helix-loop-helix domain containing, class B2
B-cell translocation gene 1, anti-proliferative
B-cell translocation gene 2, anti-proliferative
cholinergic receptor, nicotinic, alpha polypeptide 7
Cnksr family member 3 (Magi1)
protein kinase (cAMP-dependent, catalytic) inhibitor beta
developmentally regulated protein TPO1
Inhibitor of DNA binding 2
Max interacting protein 1
v-myc myelocytomatosis viral related oncogene, neuroblastoma derived
N-myc downstream regulated gene 1
similar to HGFL protein
phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1/p85
Validation of gene induction after NGF withdrawal by real time PCR
The protein levels of selected regulated genes increase after NGF withdrawal
The txnip and trib3 promoters contain potential c-Jun binding sites
In 1988, it was first proposed by Martin et al. that new RNA and protein synthesis is required for NGF withdrawal-induced apoptosis in sympathetic neurons. However, since then only a small number of genes have been shown to be regulated in this system and these were identified either by candidate gene approaches or the differential display technique. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. However, advances in technology have now allowed us to identify the majority of the genes regulated by NGF withdrawal in sympathetic neurons. Using Affymetrix exon arrays and RNA isolated from rat sympathetic neurons, we investigated the global pattern of gene expression at 16 hours after NGF withdrawal. This time-point represents the transcriptional commitment point for sympathetic neurons undergoing NGF withdrawal-induced apoptosis and induced genes known to be required for NGF withdrawal-induced death, e.g. c-jun, bim, and egln3, are expressed at a high level at this time. We were able to detect almost all of the genes known to be regulated after NGF withdrawal (Table 1) indicating the reliability of the microarray data. However, one exception was the previously described up-regulated gene puma which is required for NGF withdrawal-induced death . On further investigation, we found that no probe sets matching the puma gene were represented on the rat Affymetrix exon 1.0ST microarray. Nevertheless, microarray technology remains a reliable tool and represents the best method for obtaining a complete overview of patterns of gene expression in this system. In addition, microarray studies can identify candidate genes for functional studies. For example, in the microarray experiments described in this paper we identified mkp1 as a gene induced after NGF withdrawal that could be a target of the MLK-JNK-c-Jun pathway. We subsequently showed that mkp1 is a direct transcriptional target of the MLK-JNK-c-Jun pathway in sympathetic neurons and an important regulator of JNK activity and the rate of NGF withdrawal-induced death . Microarrays have previously been used to study gene expression in potassium-deprived cerebellar granule neurons undergoing apoptosis . The most highly up-regulated gene in this study, trim17, was subsequently shown to encode a novel E3-ubiquitin ligase that can initiate neuronal apoptosis in several in vitro models of transcription-dependent apoptosis, including cerebellar granule neurons and NGF-deprived sympathetic neurons .
Approximately 95% of the genes identified in our microarray study have never been shown before to be transcriptionally regulated during NGF withdrawal-induced apoptosis. We have been able to identify potential targets of the MLK-JNK-c-Jun pathway by including CEP-11004 in our experimental design. We selected 16 hours as the optimal time-point for our microarray study and therefore identifying those genes and pathways 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 significantly up-regulated (FDR, p < 0.01) after NGF withdrawal 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 analysis 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 cascades, 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  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 according to their pattern of expression, especially in the presence 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 targets 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 transcription of these genes may be regulated by other transcription 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 withdrawal. Many of these genes encode proteins involved in fatty acid metabolism and cholesterol metabolism, 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 cytochrome c and prothymosin alpha (ptma; an inhibitor of the apoptosome; ) decreases after NGF withdrawal (Table 2). Thus in sympathetic neurons, as previously described for cerebellar granule neurons , 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.
NGF-deprived sympathetic neurons undergo several biochemical and morphological changes before committing to the neuronal death programme and some of these are likely to play an important role in triggering apoptosis. Interestingly, levels of mitochondrial-produced reactive oxygen species (ROS) are known to increase early after NGF withdrawal  and this causes a cellular pro-oxidant state which appears to be required for the release of cytochrome c . 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 . 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 oxidation 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-dependent differentiated PC6-3 cells (data not shown). Importantly, 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.
The endoplasmic reticulum (ER) plays a significant role in how cellular proteins are processed, folded, modified and transported. In neurodegenerative diseases, these cellular processes may go wrong leading to various levels of ER stress that may contribute to neuronal death . When sympathetic neurons are treated with the ER-stressor, tunicamycin, c-Jun becomes phosphorylated but this can be prevented using CEP-11004 . Bcl-2 and Bcl-xL have been found to associate with both the mitochondrial outer membrane and the ER membrane and it has been reported that crosstalk can occur between the ER and the mitochondria in cells undergoing 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 annotation 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 . A propapoptotic role for CHOP10 has been reported since its overexpression can lead to apoptosis , whilst MEFs derived from CHOP10-/- mice are resistant to ER stress-induced cell death . 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 knockdown of Bax . 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 promoter . Also, ER stress can activate bim through CHOP-C/EBPα dependent transcriptional activation  and in other studies CHOP has been found to bind to the promoter of the proapoptotic Bcl-2 family member puma. The relationship between ER stress, CHOP, Trib3 and BH3-only proteins may suggest an important role in the apoptotic pathway after NGF withdrawal. 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 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 translocates 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-SRα promoter . Mxi1 dimerises with Max and binds to E-boxes and represses c-Myc and MycN target genes by recruiting co-repressors to their promoters . 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 , 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  so it will be interesting to determine whether Ptma protein levels also decrease after NGF withdrawal. Conversely, the expression of genes repressed by c-Myc and MycN increases after NGF deprivation, for example, ndrg1 (Figures 5 and 6 and Table 2).
The sympathetic neuron model is one of the best studied 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 contribute 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.
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 Technologies, USA) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine (Invitrogen Ltd, UK) and penicillin-streptomycin as described previously (SCG medium; ). To limit the proliferation of non-neuronal cells, the antimitotic agents fluorodeoxyuridine and uridine (Sigma Aldrich) were added to the SCG medium at a final concentration of 20 μM. For some experiments, 2.5S NGF (Cedarlane, Canada) was also added to SCG medium at a final concentration of 50 ng/ml. Neurons were plated on 13 mm diameter glass coverslips coated with poly-L-lysine and laminin placed in 3.5 cm diameter dishes containing 2 ml of SCG medium and NGF for 5-7 days. In NGF withdrawal experiments, neurons were washed twice in SCG medium lacking NGF and then refed with SCG medium supplemented with a neutralising anti-NGF antibody at 100 ng/ml (Chemicon Europe Ltd, UK). The MLK inhibitor, CEP-11004 (Cephalon, Inc., USA) was dissolved in DMSO and used at a final concentration of 400 nM.
Total RNA was isolated from sympathetic neurons cultured for 7 days using an RNeasy mini kit (Qiagen). An on-column DNase digestion was performed to eliminate genomic DNA contamination using DNase I according to the manufacturer's instructions (Qiagen). RNA concentrations were determined using a NanoDrop spectrophotometer (ND-1000). RNA was further analysed for integrity and quality on an Agilent Bioanalyser.
Up to 2 μg of total RNA was processed and labelled using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay as outlined in the manufacturer's instructions. Hybridisation to Affymetrix Rat Exon 1.0 ST arrays was performed for 16 hours at 45°C with constant rotation. Exon array data are available from the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-696.
Analysis of array data
Signal estimates and normalisation for gene-level analysis were generated using the Probe Logarithmic Intensity Error Estimation (PLIER) algorithm implemented in the Expression Console software (Affymetrix). Only core, non cross-hybridising probe sets that map to well-annotated exons were included. To reduce noise, probe sets and transcript clusters which fell into the lowest quartile of the expression signal distribution across all samples were excluded from the dataset. Signal values were analysed using Bioconductor (R). Gene expression values were compared between the three sample groups (+NGF, -NGF and -NGF+CEP-11004) using the moderated t-statistic of the Bioconductor package, Limma . To correct for multiple testing at the gene level, the Benjamini-Hochberg (FDR) test was applied to identify statistically significant differentially expressed genes (FDR adjusted p-values < 0.05). Lists of significantly up- and down-regulated genes obtained from statistical comparisons were subjected to functional enrichment analysis using DAVID annotation tools .
Real-time quantitative PCR
Up to 1 μg of total RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) as described previously . Ten nanograms of cDNA template was used for real time quantitative PCR using the ABI-Prism 7900HT fast Sequence Detection System (Applied Biosystems, Warrington, UK). Txnip, ddit3, trib3, ndrg1 and mxi1 mRNA levels were normalised to the level of the control genes, gapdh or hprt1. Pre-optimised Taqman®gapdh (Rn99999916_s1), hprt1 (Mn00446968_m1), txnip (Rn01533885_g1), trib3 (Rn00595314_m1), ddit3 (Rn00492098_g1), ndrg1 (Rn01506130_m1), and mxi1 (Rn00565846_m1) primer/probe sets were supplied by Applied Biosystems. QPCR reactions were set up with 1 × primer/probe set and 1 × Taqman® PCR Master Mix (Applied Biosystems). PCR conditions were 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. QPCR data was analysed using the 2-ΔΔCT relative quantitation method .
Immunoblotting was performed as described previously . Briefly, sympathetic neurons were harvested in 1 ml of ice-cold PBS, spun down and lysed in sample buffer (2% SDS, 2 mM β-mercaptoethanol, 60 mM Tris, pH 6.8, 0.01% bromophenol blue) for 10 minutes at 100°C. Proteins were separated on 12% SDS polyacrylamide gels and transferred to Immobilon-P (Millipore). After blocking for 45 min with 5% non-fat milk in TBS supplemented with 0.5% Tween-20, the membrane was incubated with different primary antibodies overnight at 4°C.
The following primary antibodies were used: rabbit polyclonal Trib3 antibody (M-165; Santa Cruz, USA), rabbit polyclonal Ndrg1 antibody (H-60; Santa Cruz), mouse monoclonal Txnip antibody (MBL International, Woburn, USA), mouse monoclonal CHOP10/Ddit3 antibody (B3; Santa Cruz), rabbit polyclonal Mxi1 antibody (G16; Santa Cruz), mouse monoclonal c-Jun antibody (BD Transduction Laboratories). Equivalent protein loading was confirmed by using a rabbit polyclonal ERK 1/2 antibody (Cell Signalling Technology).
Sympathetic neurons cultured on poly-L-lysine/laminin-coated glass coverslips were fixed using 4% paraformaldehyde at room temperature for 20 min, washed three times with PBS, and then permeabilised with 0.5% Triton X-100 in PBS at room temperature for 5 min. Neurons were then incubated in 50% normal goat serum in 1% BSA in PBS for 30 min at room temperature. After washing, neurons were incubated with primary antibody for 1 hour at room temperature, followed by a 45 min incubation with secondary antibody at room temperature. The following antibodies were used: mouse monoclonal phospho-c-Jun (ser63) antibody (Santa Cruz), rabbit polyclonal activated caspase-3 antibody (Abcam), mouse monoclonal cytochrome c antibody (BD Biosciences, Oxford, UK), rabbit polyclonal MAP2 antibody (Millipore) . Fluoroscein- or rhodamine-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Stratech, UK) were typically used at a dilution of 1:250. Neurons were rinsed in PBS and nuclei stained with DAPI dye in Antifade (DAKO) or Hoechst dye and mounted on glass slides. TUNEL staining was performed using an in situ cell death detection kit (Roche) according to the manufacturer's protocol.
Microscopy and image collection
Slides were viewed on a Zeiss Axioplan 2 microscope using a Plan-Apochromat 63x/1.40 oil objective. Images were captured at room temperature (20°C) using a Quantix digital camera (Photometrics, USA) and SmartCapture VP software. For the different treatments for each gene, the optimal exposure time was determined using the +NGF (0 hour) coverslip and was kept constant for all subsequent images for the remaining timepoints. For immunofluorescence time course experiments, all coverslips in each series for a particular gene were analysed in parallel and then saved as TIFF files and viewed using Adobe Photoshop CS4. Brightfield images were collected using a Zeiss Axiovert 200 M microscope with a Plan-Apochromat 63x/1.40 oil objective. The microscope stage was maintained at 37°C with 5% CO2. Images were captured using a Zeiss axiocam and Axiovision 4.0 software.
The statistical significance of differences between means was analysed by performing an unpaired Student's T-test (for two-tailed distributions). To compare normalised data to a control sample, that has no error associated to it (for example, where +NGF is set to 1), the log10 values of the data were taken and a one sample T-test was used (for two-tailed distributions) as previously described . All data are presented as the mean ± S.E. of multiple experiments and significance is expressed as follows *P< 0.01.
Acknowledgements and Funding
We would like to thank Nipurna Jina for sample processing and quality control, Dr. Sonia Shah for help with the microarray analysis and Cephalon, Inc. for CEP-11004. We would also like to thank Dr. Solange Desagher for critical reading of this manuscript. This work was supported by a Wellcome Trust Senior Research Fellowship awarded to JH (Grant number 057700) and a Wellcome Trust VIP award to MK. FM was funded by the Annabel McEnery Children's Cancer Fund. We declare that there are no conflicts of interest.
List of abbreviations
false discovery rate
mixed lineage kinase
nerve growth factor
probe logarithmic intensity error estimation
reactive oxygen species
superior cervical ganglion
standard error of the mean.
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