Comprehensive molecular characterisation of epilepsy-associated glioneuronal tumours.

Glioneuronal tumours are an important cause of treatment-resistant epilepsy. Subtypes of tumour are often poorly discriminated by histological features and may be difficult to diagnose due to a lack of robust diagnostic tools. This is illustrated by marked variability in the reported frequencies across different epilepsy surgical series. To address this, we used DNA methylation arrays and RNA sequencing to assay the methylation and expression profiles within a large cohort of glioneuronal tumours. By adopting a class discovery approach, we were able to identify two distinct groups of glioneuronal tumour, which only partially corresponded to the existing histological classification. Furthermore, by additional molecular analyses, we were able to identify pathogenic mutations in BRAF and FGFR1, specific to each group, in a high proportion of cases. Finally, by interrogating our expression data, we were able to show that each molecular group possessed expression phenotypes suggesting different cellular differentiation: astrocytic in one group and oligodendroglial in the second. Informed by this, we were able to identify CCND1, CSPG4, and PDGFRA as immunohistochemical targets which could distinguish between molecular groups. Our data suggest that the current histological classification of glioneuronal tumours does not adequately represent their underlying biology. Instead, we show that there are two molecular groups within glioneuronal tumours. The first of these displays astrocytic differentiation and is driven by BRAF mutations, while the second displays oligodendroglial differentiation and is driven by FGFR1 mutations.

Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons.

Developing sympathetic neurons depend on NGF for survival. When sympathetic neurons are deprived of NGF in vitro, a well documented series of events, including c-Jun N-terminal kinase (JNK) pathway activation, release of cytochrome c from the mitochondria, and caspase activation, culminates in the death of the neuron by apoptosis within 24-48 h. This process requires de novo gene expression, suggesting that increased expression of specific genes activates the cell death program. Using rat gene microarrays, we found that NGF withdrawal induces the expression of many genes, including mkp1, which encodes a MAPK phosphatase that can dephosphorylate JNKs. The increase in mkp1 mRNA level requires the MLK-JNK-c-Jun pathway, and we show that Mkp1 is an important regulator of JNK-dependent apoptosis in sympathetic neurons. In microinjection experiments, Mkp1 overexpression can inhibit JNK-mediated phosphorylation of c-Jun and protect sympathetic neurons from apoptosis, while Mkp1 knockdown accelerates NGF withdrawal-induced death. Accordingly, the number of superior cervical ganglion (SCG) neurons is reduced in mkp1-/- mice at P1 during the period of developmental sympathetic neuron death. We also show that c-Jun and ATF2 bind to two conserved ATF binding sites in the mkp1 promoter in vitro and in chromatin. Both of these ATF sites contribute to basal promoter activity and are required for mkp1 promoter induction after NGF withdrawal. These results demonstrate that Mkp1 is part of a negative feedback loop induced by the MLK-JNK-c-Jun signaling pathway that modulates JNK activity and the rate of neuronal death in rat sympathetic neurons following NGF withdrawal.

Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death.

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.

The MEK-ERK pathway negatively regulates bim expression through the 3' UTR in sympathetic neurons.

BACKGROUND: Apoptosis plays a critical role during neuronal development and disease. Developing sympathetic neurons depend on nerve growth factor (NGF) for survival during the late embryonic and early postnatal period and die by apoptosis in its absence. The proapoptotic BH3-only protein Bim increases in level after NGF withdrawal and is required for NGF withdrawal-induced death. The regulation of Bim expression in neurons is complex and this study describes a new mechanism by which an NGF-activated signalling pathway regulates bim gene expression in sympathetic neurons. RESULTS: We report that U0126, an inhibitor of the prosurvival MEK-ERK pathway, increases bim mRNA levels in sympathetic neurons in the presence of NGF. We find that this effect is independent of PI3-K-Akt and JNK-c-Jun signalling and is not mediated by the promoter, first exon or first intron of the bim gene. By performing 3' RACE and microinjection experiments with a new bim-LUC+3'UTR reporter construct, we show that U0126 increases bim expression via the bim 3' UTR. We demonstrate that this effect does not involve a change in bim mRNA stability and by using PD184352, a specific MEK1/2-ERK1/2 inhibitor, we show that this mechanism involves the MEK1/2-ERK1/2 pathway. Finally, we demonstrate that inhibition of MEK/ERK signalling independently reduces cell survival in NGF-treated sympathetic neurons. CONCLUSIONS: These results suggest that in sympathetic neurons, MEK-ERK signalling negatively regulates bim expression via the 3' UTR and that this regulation is likely to be at the level of transcription. This data provides further insight into the different mechanisms by which survival signalling pathways regulate bim expression in neurons.

The proapoptotic dp5 gene is a direct target of the MLK-JNK-c-Jun pathway in sympathetic neurons.

The death of sympathetic neurons after nerve growth factor (NGF) withdrawal requires de novo gene expression. Dp5 was one of the first NGF withdrawal-induced genes to be identified and it encodes a proapoptotic BH3-only member of the Bcl-2 family. To study how dp5 transcription is regulated by NGF withdrawal we cloned the regulatory regions of the rat dp5 gene and constructed a series of dp5-luciferase reporter plasmids. In microinjection experiments with sympathetic neurons we found that three regions of dp5 contribute to its induction after NGF withdrawal: the promoter, a conserved region in the single intron, and sequences in the 3' untranslated region of the dp5 mRNA. A construct containing all three regions is efficiently activated by NGF withdrawal and, like the endogenous dp5, its induction requires mixed-lineage kinase (MLK) and c-Jun N-terminal kinase (JNK) activity. JNKs phosphorylate the AP-1 transcription factor c-Jun, and thereby increase its activity. We identified a conserved ATF site in the dp5 promoter that binds c-Jun and ATF2, which is critical for dp5 promoter induction after NGF withdrawal. These results suggest that part of the mechanism by which the MLK-JNK-c-Jun pathway promotes neuronal apoptosis is by activating the transcription of the dp5 gene.

Spatially and temporally distributed data foraging decisions in disciplinary field science.

How do scientists generate and weight candidate queries for hypothesis testing, and how does learning from observations or experimental data impact query selection? Field sciences offer a compelling context to ask these questions because query selection and adaptation involves consideration of the spatiotemporal arrangement of data, and therefore closely parallels classic search and foraging behavior. Here we conduct a novel simulated data foraging study-and a complementary real-world case study-to determine how spatiotemporal data collection decisions are made in field sciences, and how search is adapted in response to in-situ data. Expert geoscientists evaluated a hypothesis by collecting environmental data using a mobile robot. At any point, participants were able to stop the robot and change their search strategy or make a conclusion about the hypothesis. We identified spatiotemporal reasoning heuristics, to which scientists strongly anchored, displaying limited adaptation to new data. We analyzed two key decision factors: variable-space coverage, and fitting error to the hypothesis. We found that, despite varied search strategies, the majority of scientists made a conclusion as the fitting error converged. Scientists who made premature conclusions, due to insufficient variable-space coverage or before the fitting error stabilized, were more prone to incorrect conclusions. We found that novice undergraduates used the same heuristics as expert geoscientists in a simplified version of the scenario. We believe the findings from this study could be used to improve field science training in data foraging, and aid in the development of technologies to support data collection decisions.

FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons.

Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal-induced death. Here, we have investigated the involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors in the regulation of BIM expression by NGF. We find that overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, we find that FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, we show that FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons.

Evidence for increased complexity in the regulation of Bim expression in sympathetic neurons: involvement of novel transcriptional and translational mechanisms.

The BH3-only protein Bim is induced following NGF deprivation in developing sympathetic neurons and contributes to their death by apoptosis. The regulation of Bim activity is complex, and involves both transcriptional and posttranslational mechanisms. We have previously shown that both the FOXO subfamily of Forkhead transcription factors and the JNK/c-Jun pathway contribute to the transcriptional induction of Bim expression and subsequent apoptosis of sympathetic neurons following NGF deprivation. Bim activity can also be modulated by JNK-mediated phosphorylation after NGF deprivation in these cells. Here, we provide evidence for additional complexity in the transcriptional and translational control of Bim expression. We show that the first intron of the bim gene contains elements with silencer and enhancer properties that can modulate the basal activity and NGF deprivation-induced activity of the previously characterized bim promoter. Surprisingly, we find that some of the elements responsible for these effects are linked to two novel, alternative promoters located towards the 3' end of the intron that have minimal, or no activity in sympathetic neurons. Finally, we provide evidence that Bim expression is reduced in sympathetic neurons by the presence of an upstream open reading frame in the 5' leader of bim transcripts.

Methods for culturing primary sympathetic neurons and for determining neuronal viability.

Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary sympathetic neurons and how to induce apoptosis by NGF deprivation. In addition, a simple method for measuring neuronal viability based on the live/dead assay is also described. This can be used for assessing the effect of small molecule inhibitors of protein kinases, caspases and other enzymes, on NGF withdrawal-induced death.

Immunocytochemical techniques for studying apoptosis in primary sympathetic neurons.

Developing sympathetic neurons, which depend on nerve growth factor for survival, are one of the best studied in vitro models of neuronal apoptosis and have been extensively used for cellular and molecular studies of the neuronal death pathway. Important apoptotic events after nerve growth factor withdrawal include the release of proapoptotic proteins, such as cytochrome c, from the mitochondria and the activation of caspases, followed by nuclear DNA fragmentation and chromatin condensation. In this chapter, we describe immunocytochemical techniques for studying apoptotic DNA fragmentation, changes in nuclear morphology, and mitochondrial cytochrome c release at the single cell level using sympathetic neurons cultured on glass coverslips.

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development.

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between pediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1,262 genes identified as potentially generating tumor-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by reverse transcriptase PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of sonic hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumor and normal fetal cerebellar samples shared significantly lower exon inclusion rates than normal adult cerebellum. We investigated whether tumor-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCP) and found that Shh caused a decrease in the cassette exon inclusion rate of five of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between postnatal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into postmitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signaling pathways and a failure of cerebellar precursor cells to differentiate.

Hrk/DP5 contributes to the apoptosis of select neuronal populations but is dispensable for haematopoietic cell apoptosis.

The pro-apoptotic BH3-only members of the Bcl2 family, crucial initiators of cell death, are activated by a diverse array of developmental cues or experimentally applied stress stimuli. We have investigated, through gene targeting in mice, the biological roles for the BH3-only family member HRK (also known as DP5) in apoptosis regulation. Hrk gene expression was found to be restricted to cells and tissues of the central and peripheral nervous systems. Sensory neurons from mice lacking Hrk were less sensitive to apoptosis induced by nerve growth factor (NGF) withdrawal, consistent with the induction of Hrk following NGF deprivation. By contrast, cerebellar granule neurons that upregulate Hrk upon transfer to low-K+ medium underwent apoptosis normally under these conditions in the absence of Hrk. Furthermore, loss of Hrk was not sufficient to rescue the neuronal degeneration in lurcher mutant mice. Despite previous reports, no evidence was found for Hrk expression or induction in growth-factor-dependent haematopoietic cell lines following withdrawal of their requisite cytokine, and haematopoietic progenitors lacking HRK died normally in response to cytokine deprivation. These results demonstrate that HRK contributes to apoptosis signalling elicited by trophic factor withdrawal in certain neuronal populations but is dispensable for apoptosis of haematopoietic cells.