Dataset Information

Profiling wt SOD versus ALS SOD1(G93A) mutant

ABSTRACT: Whole-genome profiling of SH-SY5Y cells was done on neuroblastoma SH-SY5Y stably transfected with cDNAs coding for SOD1WT or the mutant SOD1(G93A) protein. Five wt SOD versus five mutant SOD

ORGANISM(S): Homo sapiens

SUBMITTER: Mauro Cozzolino Tilmann Achsel Valentina Romeo Giuseppe Lamorte Donatella Biancolini Francesca Cordero Alessia Maracchioni Silvia C Lenzken MariaTeresa Carrì Silvia Barabino Remo Sanges Davide Bonanno Raffaele A Calogero R A Calogero Francesca Zolezzi

PROVIDER: E-GEOD-21298 | ArrayExpress | 2010-12-22

SECONDARY ACCESSION(S): GSE21298PRJNA129447

REPOSITORIES: GEO, ArrayExpress

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Publications

Mutant SOD1 and mitochondrial damage alter expression and splicing of genes controlling neuritogenesis in models of neurodegeneration.

Lenzken Silvia C SC Romeo Valentina V Zolezzi Francesca F Cordero Francesca F Lamorte Giuseppe G Bonanno Davide D Biancolini Donatella D Cozzolino Mauro M Pesaresi Maria Grazia MG Maracchioni Alessia A Sanges Remo R Achsel Tilmann T Carrì Maria Teresa MT Calogero Raffaele A RA Barabino Silvia M L SM

Human mutation 20110125 2

Mitochondrial dysfunction has been implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson, Alzheimer, and Amyotrophic Lateral Sclerosis (ALS). In addition, aberrant mRNA splicing has been documented in neurodegeneration. To characterize the cellular response to mitochondrial perturbations at the level of gene expression and alternative pre-mRNA splicing we used splicing-sensitive microarrays to profile human neuroblastoma SH-SY5Y cells treated with paraquat ...[more]

PMID: 21120952

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Project description:This SuperSeries is composed of the following subset Series: GSE21298: Profiling wt SOD versus ALS SOD1(G93A) mutant GSE21305: Profiling neuroblastoma SH-SY5Y with Paraquat treatment Mitochondrial dysfunction has been directly or indirectly implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis (ALS). We used exon-sentive microarrays to characterize the responses to different mitochondrial perturbations in cellular models. We examined human SH-SY5Y neuroblastoma cells treated with paraquat, a neurotoxic herbicide which both catalyzes the formation of reactive oxygen species (ROS) and induces mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant SOD1(G93A) protein, one of the genetic causes of ALS. We identified a common set of genes that have a deregulated transcription and alternative splicing in both models. Noticeably, pathway analysis revealed that the expression of a subset of genes involved in neuritogenesis and axon guidance is perturbed, suggesting that alterations of axonal function may descend directly from mitochondrial damage and be responsible for neurodegenerative conditions. Paraquat treatment on cell lines derived from human neuroblastoma SH-SY5Y and the same cell line stably transfected with cDNAs coding for wild type SOD1 or ALS mutant SOD1 G93A was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Human neuroblastoma SH-SY5Y untransfected or stably transfected with cDNAs coding for wild type SOD1 or the mutant SOD1(G93A) , were cultured in D-MEM/F-12 media with GlutaMAX™ (Gibco, Invitrogen, UK), 10% FBS, 100 Units/ml penicillin G, 100µg/ml streptomycin/ penicillin (Euroclone, Milano, Italy). Stably transfected cells were also maintained in the presence of 400µg/ml Geneticin (G418 sulphate, Euroclone). Cells were fed every 2–3 days and passed once a week. For the microarray experiment, after an initial amplification of each cell type [(SH-SY5Y; SOD1(WT), SOD1(G93A)], cells were aliquoted and cryopreservated in liquid nitrogen. For every experiment an aliquot of each cell line was thawed and seeded. After having reached confluence, cells were reseeded at 3x106cells in 100mm dish. Paraquat (N,N’-dimethyl-4,4’-bipyridinium dichloride, Sigma-Aldrich) treatment was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Total RNA was extracted using TRIzol® Reagent (Invitrogen), and subsequently purified using silica membrane spin columns from RNeasy Mini kit (Qiagen). RNA quantity and purity were assessed using a NanoDrop® instrument (Thermo Fisher Scientific Inc.). Total RNA integrity was assessed by using a 2100 Bioanalyzer (Agilent Technologies) and the RNA Integrity Number (RIN) was calculated. Each condition was replicated 5 times. After extraction and quality check 1.5 µg of total RNA was subjected to removal of ribosomal RNA following the procedure suggested by the manufacturer (Affymetrix). The resulting total RNA was then used to created the biotin-labeled library to be hybridized on GeneChip® Exon 1.0 ST human microarrays following the procedure described by the manufacturer (Affymetrix). The CEL files resulting from the hybridization were analyzed using oneChannelGUI 1.6.5 (Sanges et al. Bioinformatics 2007, 23, 3406-3408). Exon and gene-level probeset summarization was done by mean of RMA and sketch quantile normalization. Gene-level differential expression: to assess differential expression at gene-level, we used an empirical Bayes method together with a false discovery rate (FDR) correction of the p-value Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 together with an absolute log2(fold-change) threshold of 1. Exon-level analysis: an intensity filter was subsequently applied at gene-level to remove not expressed and low expressed genes, i.e. genes were retained for exon-level analysis if in all biological replication gene-level signal was greater than 5. Subsequently, only genes characterized to have at least two RNA isoforms annotated in Ensembl database were retained for further analysis. The Splicing Index value was calculated by taking the log2 ratio of the normalized exon intensity (NI) in Sample 1 and the NI in Sample 2. The normalized exon intensity (NI) is the ratio of the probe set intensity to the gene intensity. Alternative splicing events (ASEs) were detected as described in (Della Beffa et al. BMC Genomics 2008, 9, 571).

Project description:Mitochondrial dysfunction has been directly or indirectly implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis (ALS). We used exon-sentive microarrays to characterize the responses to different mitochondrial perturbations in cellular models. We examined human SH-SY5Y neuroblastoma cells treated with paraquat, a neurotoxic herbicide which both catalyzes the formation of reactive oxygen species (ROS) and induces mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant SOD1(G93A) protein, one of the genetic causes of ALS. We identified a common set of genes that have a deregulated transcription and alternative splicing in both models. Noticeably, pathway analysis revealed that the expression of a subset of genes involved in neuritogenesis and axon guidance is perturbed, suggesting that alterations of axonal function may descend directly from mitochondrial damage and be responsible for neurodegenerative conditions. This SuperSeries is composed of the SubSeries listed below. Overall design: Paraquat treatment on cell lines derived from human neuroblastoma SH-SY5Y and the same cell line stably transfected with cDNAs coding for wild type SOD1 or ALS mutant SOD1 G93A was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Human neuroblastoma SH-SY5Y untransfected or stably transfected with cDNAs coding for wild type SOD1 or the mutant SOD1(G93A) , were cultured in D-MEM/F-12 media with GlutaMAX™ (Gibco, Invitrogen, UK), 10% FBS, 100 Units/ml penicillin G, 100µg/ml streptomycin/ penicillin (Euroclone, Milano, Italy). Stably transfected cells were also maintained in the presence of 400µg/ml Geneticin (G418 sulphate, Euroclone). Cells were fed every 2–3 days and passed once a week. For the microarray experiment, after an initial amplification of each cell type [(SH-SY5Y; SOD1(WT), SOD1(G93A)], cells were aliquoted and cryopreservated in liquid nitrogen. For every experiment an aliquot of each cell line was thawed and seeded. After having reached confluence, cells were reseeded at 3x106cells in 100mm dish. Paraquat (N,N’-dimethyl-4,4’-bipyridinium dichloride, Sigma-Aldrich) treatment was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Total RNA was extracted using TRIzol® Reagent (Invitrogen), and subsequently purified using silica membrane spin columns from RNeasy Mini kit (Qiagen). RNA quantity and purity were assessed using a NanoDrop® instrument (Thermo Fisher Scientific Inc.). Total RNA integrity was assessed by using a 2100 Bioanalyzer (Agilent Technologies) and the RNA Integrity Number (RIN) was calculated. Each condition was replicated 5 times. After extraction and quality check 1.5 µg of total RNA was subjected to removal of ribosomal RNA following the procedure suggested by the manufacturer (Affymetrix). The resulting total RNA was then used to created the biotin-labeled library to be hybridized on GeneChip® Exon 1.0 ST human microarrays following the procedure described by the manufacturer (Affymetrix). The CEL files resulting from the hybridization were analyzed using oneChannelGUI 1.6.5 (Sanges et al. Bioinformatics 2007, 23, 3406-3408). Exon and gene-level probeset summarization was done by mean of RMA and sketch quantile normalization. Gene-level differential expression: to assess differential expression at gene-level, we used an empirical Bayes method together with a false discovery rate (FDR) correction of the p-value Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 together with an absolute log2(fold-change) threshold of 1. Exon-level analysis: an intensity filter was subsequently applied at gene-level to remove not expressed and low expressed genes, i.e. genes were retained for exon-level analysis if in all biological replication gene-level signal was greater than 5. Subsequently, only genes characterized to have at least two RNA isoforms annotated in Ensembl database were retained for further analysis. The Splicing Index value was calculated by taking the log2 ratio of the normalized exon intensity (NI) in Sample 1 and the NI in Sample 2. The normalized exon intensity (NI) is the ratio of the probe set intensity to the gene intensity. Alternative splicing events (ASEs) were detected as described in (Della Beffa et al. BMC Genomics 2008, 9, 571).

Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Keywords: Cell type comparison, time course Overall design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.