Impact of NRSF variant over-expression in SK-N-AS human neuroblastoma cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE22467 | GEO | 2010/07/01
SECONDARY ACCESSION(S): PRJNA128615
REPOSITORIES: GEO
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Project description:Neuron-restrictive silencer factor (NRSF) and its isoforms are differentially regulated in rodent models of self-sustaining status epilepticus (SSSE). NRSF isoforms regulate genes associated with SSSE, including the proconvulsant tachykinins, brain-derived neurotrophic factor and multiple ion channels. NRSF isoforms may direct distinct gene expression patterns during SSSE and the ratio of each isoform may be a causative factor in traumatic damage to the CNS. Here we analysed global gene expression changes by microarray in human SK-N-AS neuroblastoma cells following the over expression of NRSF and a truncated isoform, HZ4. We used bioinformatics software to analyse the microarray dataset and correlated these data with epilepsy candidate gene pathways. Findings were validated by RT-PCR. We demonstrated that NRSF and HZ4 direct overlapping as well as distinct gene expression patterns and that they differentially modulated gene expression patterns associated with epilepsy. Finally we revealed that NRSF gene expression may be modulated by the anticonvulsant, phenytoin. This study provides fundamental information on networks of genes that may be altered during SSSE, following altered NRSF expression, which may be important in future therapeutic research and clinical analysis of genetic variation predisposing to epilepsy. Human SK-N-AS cells were treated with expression constructs over-expressing either the full length human transcription factor, NRSF, (via the RE-EX1 construct) or a truncated variant (via the HZ4 construct). Cells were treated for either 0hrs (base line controls), 24hrs or 48hrs, before being immediately processed for RNA extraction. An affymetrix microarray was employed to investigate gene expression patterns, comparing each time point (24hrs or 48hrs) againsts its baseline ()hrs) control. This experiment was performed in triplicate samples per batch, and over three batches.
2010-07-30 | E-GEOD-22467 | biostudies-arrayexpress
Project description:Epilepsy is a major neurological disorder that affects approximately 1% of the population. The processes that lead to the development of epilepsy (epileptogenesis) are largely unknown. Levetiracetam is a novel antiepileptic drug (AED) that in the kindling model inhibits epileptogenesis in addition to being effective in controlling established epilepsy. The mechanisms of action of levetiracetam as an AED and an antiepileptogenic drug are unknown. By identifying the effect of chronic levetiracetam therapy on gene expression in the brain we hope to be able to identify genes that are involved in epileptogenesis. By comparing the gene expression profiles of levetiracetam and phenytoin treatments, we hope to be able to distinguish between genes that are important for the antiepileptic (anti-seizure) effect and genes that are important for the antiepileptogenic effect of levetiracetam. Phenytoin is a well-established AED; its mechanism of action involves inhibition of sodium channels. In contrast to levetiracetam, available data suggest that phenytoin in certain situations may enhance rather than inhibit the development of epilepsy. We will identify changes in gene expression in the hippocampus, frontal cortex, and brain stem caused by chronic treatment (3 months) with levetiracetam or phenytoin. We will search for differences in the gene expression profiles after chronic treatment with the two drugs, to explain the different clinical effects of the two drugs. We hypothesize that chronic treatment with levetiracetam leads to changes in the expression of genes that are involved in both the antiepileptogenetic and antiepileptic effect of the drug, whereas chronic treatment with phenytoin affects genes that may be related to pro-epileptogenetic as well as antiepileptic effects of that drug. We further hypothesize that i) regional differences in the effects of levetiracetam and phenytoin may reflect important functional differences in the actions of these two drugs, and ii) there may be different mechanisms involved in their anti-seizure effect. Male Wistar rats receive levetiracetam (150 mg/kg x 2), phenytoin (75 mg/kg x 2) or control solution (N=7 in each group) through a gastric tube twice daily for 90 days. Experimental animals and controls are housed together in the same cages in groups of three, facilitating paired comparisons among the three groups in animals that have experienced the same environment throughout the 3 month experimental period. Four hrs after the last dose animals are anesthetized, decapitated, and total RNA is extracted from brain stem, frontal cortex, and hippocampus. RNA will be sent to the TGEN facility for hybridization to the rat U230A chips. The number of chips needed is (2 drugs + 1 control) * (3 brain regions) * (7 animals per group) = 63 chips. We will initially select those genes for analysis that are called “present” by the Affymetrix software in 4 or more animals for at least one of the 6 experimental groups. Then t-tests with Benjamini & Hochberg correction for protection against multiple comparisons will be used to identify genes that are differentially expressed at a false discovery rate of .05 among any of the six groups. We will be primarily interested in the following expression profiles, analyzed as paired comparisons by the Affymetrix software: 1. phenytoin vs control in hippocampus 2. phenytoin vs control in brain stem 3. levetiracetam vs control in hippocampus 4. levetiracetam vs control in brain stem We will then compare the profiles of both drugs in each brain region (e.g., phenytoin vs levetiracetam in hippocampus) to select genes that are drug-specific. We will also take a Venn diagram approach to identify genes that are drug-specific across both brain regions tested. Keywords: other
2005-07-06 | GSE2880 | GEO
Project description:Epilepsy is a major neurological disorder that affects approximately 1% of the population. The processes that lead to the development of epilepsy (epileptogenesis) are largely unknown. Levetiracetam is a novel antiepileptic drug (AED) that in the kindling model inhibits epileptogenesis in addition to being effective in controlling established epilepsy. The mechanisms of action of levetiracetam as an AED and an antiepileptogenic drug are unknown. By identifying the effect of chronic levetiracetam therapy on gene expression in the brain we hope to be able to identify genes that are involved in epileptogenesis. By comparing the gene expression profiles of levetiracetam and phenytoin treatments, we hope to be able to distinguish between genes that are important for the antiepileptic (anti-seizure) effect and genes that are important for the antiepileptogenic effect of levetiracetam. Phenytoin is a well-established AED; its mechanism of action involves inhibition of sodium channels. In contrast to levetiracetam, available data suggest that phenytoin in certain situations may enhance rather than inhibit the development of epilepsy. ,We will identify changes in gene expression in the hippocampus, frontal cortex, and brain stem caused by chronic treatment (3 months) with levetiracetam or phenytoin. We will search for differences in the gene expression profiles after chronic treatment with the two drugs, to explain the different clinical effects of the two drugs.,We hypothesize that chronic treatment with levetiracetam leads to changes in the expression of genes that are involved in both the antiepileptogenetic and antiepileptic effect of the drug, whereas chronic treatment with phenytoin affects genes that may be related to pro-epileptogenetic as well as antiepileptic effects of that drug. We further hypothesize that i) regional differences in the effects of levetiracetam and phenytoin may reflect important functional differences in the actions of these two drugs, and ii) there may be different mechanisms involved in their anti-seizure effect. Male Wistar rats receive levetiracetam (150 mg/kg x 2), phenytoin (75 mg/kg x 2) or control solution (N=7 in each group) through a gastric tube twice daily for 90 days. Experimental animals and controls are housed together in the same cages in groups of three, facilitating paired comparisons among the three groups in animals that have experienced the same environment throughout the 3 month experimental period. Four hrs after the last dose animals are anesthetized, decapitated, and total RNA is extracted from brain stem, frontal cortex, and hippocampus. RNA will be sent to the TGEN facility for hybridization to the rat U230A chips. The number of chips needed is (2 drugs + 1 control) * (3 brain regions) * (7 animals per group) = 63 chips. We will initially select those genes for analysis that are called present by the Affymetrix software in 4 or more animals for at least one of the 6 experimental groups. Then t-tests with Benjamini & Hochberg correction for protection against multiple comparisons will be used to identify genes that are differentially expressed at a false discovery rate of .05 among any of the six groups. We will be primarily interested in the following expression profiles, analyzed as paired comparisons by the Affymetrix software: 1. phenytoin vs control in hippocampus, 2. phenytoin vs control in brain stem, 3. levetiracetam vs control in hippocampus, 4. levetiracetam vs control in brain stem. We will then compare the profiles of both drugs in each brain region (e.g. phenytoin vs levetiracetam in hippocampus) to select genes that are drug-specific. We will also take a Venn diagram approach to identify genes that are drug-specific across both brain regions tested.
2007-08-01 | E-GEOD-2880 | biostudies-arrayexpress
Project description:Epilepsy affects between 1-2% of people worldwide and is defined symptomatically by the appearance of spontaneous, recurrent seizures. The ketogenic diet (KD) is an anticonvulsant treatment that has been used to manage medically-intractable epilepsies. Both clinical and experimental reports show that the KD can be used to treat a variety of seizure types. During KD treatment, ketone bodies, rather than glucose, become the major energy substrate for the brain; somehow, metabolic conversion confers a potent anticonvulsant effect. The KD requires 10-12 days to become maximally effective, suggesting that changes in gene expression are involved in its anticonvulsant action. Despite its use for over 80 years, the mechanism of the diet remains unknown. It is expected that this work will help elucidate the underlying anticonvulsant action of the KD and contribute to future, rationale-based drug development. We propose to identify sets of genes activated/repressed within hippocampus after treatment with a KD. We hypothesize that gene expression patterns induced by alterations in metabolic pathways are critically involved with the anticonvulsant effects of the KD in mice. Mice (n=44, 129svJ) will be used for these experiments. Animals will be fed either a KD (n=22) or control diet (n=22) for 14 days. After this time, ten animals from each group will be subjected to flurothyl seizure testing to confirm seizure threshold elevation. Of the remaining KD- (n=12) or control-fed (n=12) animals, two left hippocampi will be pooled together such that six arrays from each treatment group can be tested. This is expected to reduce biological variability. Right hippocampi (n=12/group) will be maintained in our lab for corroborative analysis via RT-PCR and immunohistochemistry. All tissue samples will be rapidly dissected, frozen in liquid nitrogen, and stored at -80C until shipping/corroborative testing. Data analysis will be completed with Significance of Array Measurement (SAM) program using ANOVA from protection for multiple comparisons. Please use the Affy chip with known genes in lieu of the chip containing mostly EST's.
2008-06-11 | E-GEOD-1829 | biostudies-arrayexpress
Project description:Neuropsychiatric disorders are highly complex conditions and the risk of developing a disorder has been tied to hundreds of genomic variants that alter the expression and/or products (isoforms) made by risk genes. However, how these genes contribute to disease risk and onset through altered expression and RNA splicing is not well understood. Here we show our current understanding of gene isoforms is far from complete and reveal the precise splicing profiles of neuropsychiatric disorder risk genes. Combining our new bioinformatic pipeline IsoLamp with nanopore long-read amplicon sequencing, we deeply profiled the RNA isoform repertoire of 31 high-confidence neuropsychiatric disorder risk genes in human brain. We show most risk genes are more complex than previously reported, identifying 443 novel isoforms and 28 novel exons, including isoforms which alter protein domains, and genes such as ATG13 and GATAD2A where most expression was from previously undiscovered isoforms. The greatest isoform diversity was present in the schizophrenia risk gene ITIH4. Mass spectrometry of brain protein isolates confirmed translation of a novel exon skipping event in ITIH4, suggesting a new regulatory mechanism for this gene in brain. Our results emphasize the widespread presence of previously undetected RNA and protein isoforms in brain and provide an effective approach to address this knowledge gap. Uncovering the isoform repertoire of neuropsychiatric risk genes will underpin future analyses of the functional impact these isoforms have on neuropsychiatric disorders, enabling the translation of genomic findings into a pathophysiological understanding of disease.
| EGAS00001007744 | EGA
Project description:ChIP-seq was performed using Drosophila Kc167 cells using antibodies against the two isoforms of Fs(1)h, the Brd4 homologue. Differences in binding patterns between the two isoforms are described. We examined the differences in Fs(1)h isoform binding across the genome and describe the short isoform to be correlated with transcription at enhancers and promoters. The long isoform is found predominately at insulator binding sites where multiple insulators are bound.
2012-11-15 | E-GEOD-42086 | biostudies-arrayexpress
Project description:HNF4α is a nuclear receptor regulating the transcription of genes involved mainly in development, cell differentiation and metabolism. Opposite functions for the two classes of P1 and P2 isoforms of HNF4α have recently been highlighted. These classes include 12 variants of HNF4α that can be expressed by the use of two promoters and by alternative splicing. Until now, the characterization of this transcription factor has ignored this diversity and has remained confined to the study of a fraction of the isoforms. We therefore wanted to clarify the situation by specifically characterizing the transcriptional functions of the 12 isoforms of HNF4α. We have generated for this purpose stable lines expressing each isoform of HNF4α in HCT 116 cells. We analyzed the whole transcriptome associated with each isoform by sequencing RNA, as well as their proteome by a BioID approach coupled to quantitative mass spectrometry. We noted major differences in the transcriptional function of the 12 isoforms. The α4, α5 and α6 isoforms have been characterized for the first time, and show a greatly reduced transcriptional potential. We have shown that these isoforms are unable to recognize the consensus response element of HNF4α. The α1 and α2 isoforms are the most potent regulators of gene expression, while the α3 isoform exhibits significantly reduced activity. Several transcription factors and coregulators have been identified as potential specific partners for certain HFH4α isoforms. The IRF-2BP2 co-repressor interacts specifically with isoforms which include the long form of the F domain of HNF4α. This specific interaction could explain the large number of genes modulated negatively by α1 and α2 compared to α3. The analysis integrating the vast amount of transcriptomic and proteomic data allows the identification of transcriptional regulatory mechanisms specific to certain isoforms, demonstrating the importance of considering all isoforms which can have diverse functions.
2020-04-17 | PXD012146 | Pride
Project description:A novel model of drug-resistant epilepsy based on the selection of mice treated with phenytoin (PHT) in the 6 Hz seizure model was developed. It leads to the identification of subpopulations of drug-responder and non-responder mice. Transcriptional characterization of the hippocampi from these 2 sub-populations was performed and compared. No significant difference has been observed between the 2 sample groups.
2020-09-11 | GSE157797 | GEO
Project description:To fully understand cell type identity and function in the nervous system there is a need to understand neuronal gene expression at the level of isoform diversity. Here we applied Next Generation Sequencing of the transcriptome (RNA-Seq) to purified sensory neurons and cerebellar granular neurons (CGNs) grown on an axonal growth permissive substrate. The goal of the analysis was to uncover neuronal type specific isoforms as a prelude to understanding patterns of gene expression underlying their intrinsic growth abilities. Global gene expression patterns were comparable to those found for other cell types, in that a vast majority of genes were expressed at low abundance. Nearly 18% of gene loci produced more than one transcript. More than 8000 isoforms were differentially expressed, either to different degrees in different neuronal types or uniquely expressed in one or the other. Sensory neurons expressed a larger number of genes and gene isoforms than did CGNs. To begin to understand the mechanisms responsible for the differential gene/isoform expression we identified transcription factor binding sites present specifically in the upstream genomic sequences of differentially expressed isoforms, and analyzed the 3’ untranslated regions (3’ UTRs) for microRNA (miRNA) target sites. Our analysis defines isoform diversity for two neuronal types with diverse axon growth capabilities and begins to illuminate the complex transcriptional landscape in two neuronal populations.
2012-01-17 | GSE33343 | GEO
Project description:Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform change upon cell-state transitions during cell differentiation, and the determinants and functional consequences have largely remained unclear. Here, we used an improved model for human neurogenesis in vitro that we show is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct stages during neurogenesis. RNA isoforms mainly arise from the alternative usage of transcription start sites and poly-adenylation sites as well as the skipping of individual exons during human neurogenesis. The transcript isoform changes can remodel the abundance and functions of protein isoforms. Finally, our study identifies a set of RNA-binding proteins as a likely determinant of differentiation stage-specific global isoform changes.
2024-04-11 | PXD046084 | Pride