Mouse ES-derived motor neurons (ES-MN) exposed to SOD1 mutant mouse astrocyte conditioned media
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE49023 | GEO | 2014/12/17
SECONDARY ACCESSION(S): PRJNA212701
REPOSITORIES: GEO
Similar Datasets
Project description:This datased was used to obtain a genome-wide expression signature for the early response of mouse motor neurons to mutant SOD1 astrocytes conditioned media. Neurons, far from living in isolation, are surrounded by a host of other neuronal and non-neuronal cells, such as astrocytes. The latter entertain complex functional interactions with neighboring neurons, which, under normal conditions, are important for the their well-being. In pathological situations, however, altered astrocyte behavior may contribute to the demise of neighboring neurons. Such non-cell autonomous pathogenic scenario is increasingly considered in a variety of disorders, including amyotrophic lateral sclerosis (ALS), the most frequent adult-onset paralytic disorder. Assembly and interrogation of gene regulatory models has helped elucidate causal mechanisms responsible for the presentation of several tumor-related phenotypes. To systematically elucidate the effectors of neurodegeneration in a model of ALS, we first developed techniques for the efficient purification of motor neurons (MNs), the primary target of ALS neurodegenerative process. We then generated gene expression profiles to fully characterize the critical timepoints associated with initiation and commitment of MN degenerative progression in an in vitro murine mutant SOD1 (mSOD1) model of ALS. ES cells were derived from transgenic Hlxb9-GFP1Tmj mice expressing eGFP and CD2 driven by the mouse HB9 promoter. These cells were then differentiated into motor neurons (ES-MN) as described previously [PMID 12176325] ES-MN were exposed to non-transgenic (NTg), G93A mutant SOD1 (mSOD1) or wtSOD1 over-expression astrocytes conditioned media for 0 days (time zero control), 1 day, and 3 days. Total RNA was extracted and profiled by RNAseq.
2014-12-17 | E-GEOD-49023 | biostudies-arrayexpress
Project description:Gene expression profiling has been performed previously on motor cortex and spinal cord homogenates and of sporadic ALS cases and controls, to identify genes and pathways differentially expressed in ALS. More recent studies have combined the use of laser capture microdissection (LCM) with gene expression profiling to isolate the motor neurons from the surrounding cells, such as microglia and astrocytes, in order to determine those genes differentially expressed in the vulnerable cell population – i.e. motor neuron. The aim of the present study is to combine LCM and microarray analysis to determine those genes and pathways differentially expressed in MNs from human SOD1-related MND and to establish potential pathways for therapeutic intervention. Keywords: Human motor neurons The aim of this study was to determine the gene expression profiles from a small subset of cases which all carry mutations in the SOD1 gene. Expression profiles from isolated motor neurons in SOD1-related ALS cases were compared to those from control motor neurons, in order to establish the pathways implicated in SOD1-related motor neuronal cell death. The 'control' samples were originally submitted to GEO as GSE19332.
2011-01-01 | E-GEOD-20589 | biostudies-arrayexpress
Project description:Ubiquitous expression of ALS-causing mutations in superoxide dismutase 1 (SOD1) provoke non-cell autonomous paralytic disease. By combining ribosome affinity purification and high-throughput sequencing, a cascade of mutant SOD1-dependent, cell type-specific changes are now identified. Initial mutant-dependent damage is restricted to motor neurons and includes synapse and metabolic abnormalities, endoplasmic reticulum (ER) stress, and selective activation of the PERK arm of the unfolded protein response. PERK activation correlates with what we identify to be a naturally low level of ER chaperones in motor neurons. Early changes in astrocytes are to genes involved in inflammation and metabolism and that are targets of the PPAR and LXR transcription factors. Dysregulation of myelination and lipid signaling pathways and activation of ETS transcription factors occur in oligodendrocytes only after disease initiation. Thus, pathogenesis involves a temporal cascade of cell type selective damage initiating in motor neurons, with subsequent damage within glia driving disease propagation. Cell type-specific mRNA was purified by ribosome affinity purification from the spinal cord of bacTRAP reporter mice that label selective cell types by EGFP-tagged ribosome RPL10A. Sequencing libraries were prepared from 3-6 biological replicates for each genotype to determine the mutant induced gene expression changes in specific cell types.
2015-12-09 | E-GEOD-74724 | biostudies-arrayexpress
Project description:Ubiquitous expression of ALS-causing mutations in superoxide dismutase 1 (SOD1) provoke non-cell autonomous paralytic disease. By combining ribosome affinity purification and high-throughput sequencing, a cascade of mutant SOD1-dependent, cell type-specific changes are now identified. Initial mutant-dependent damage is restricted to motor neurons and includes synapse and metabolic abnormalities, endoplasmic reticulum (ER) stress, and selective activation of the PERK arm of the unfolded protein response. PERK activation correlates with what we identify to be a naturally low level of ER chaperones in motor neurons. Early changes in astrocytes are to genes involved in inflammation and metabolism and that are targets of the PPAR and LXR transcription factors. Dysregulation of myelination and lipid signaling pathways and activation of ETS transcription factors occur in oligodendrocytes only after disease initiation. Thus, pathogenesis involves a temporal cascade of cell type selective damage initiating in motor neurons, with subsequent damage within glia driving disease propagation.
2015-12-09 | GSE74724 | GEO
Project description:Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in about 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although, molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in non-neuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to non-transgenic, SOD1G93A-expressing astrocytes induced apoptosis of co-cultured motor neurons. This prompted us to investigate whether the effect on motor neuron survival was related to a change in the gene expression profile. Through high-density oligonucletide microarrays we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix construction, response to stress and steroid and lipid metabolism. Decorin, a small multifunctional proteoglycan, was the most up-regulated gene. Down-regulated genes included the insulin-like growth factor-1 receptor and the RNA binding protein ROD1. We also analyzed the expression of selected genes in purified motor neurons expressing SOD1G93A and in spinal cord of asymptomatic and early symptomatic ALS-rodent model. The expression of mutated SOD1 in astrocytes cause gene expression changes with potential consequences for its interaction with motor neurons. The astrocyte-specific gene expression profile contributes to the identification of possible candidates for cell type-specific therapies in ALS Keywords: Cell type comparison
2008-12-18 | GSE7441 | GEO
Project description:Microarray analysis has been applied to the study of ALS in order to investigate gene expression in whole spinal cord homogenates of SOD1 G93A mice and human ALS cases, although the massive presence of glial cells and inflammatory factors has made it difficult to define which gene expression changes were motor neuron specific. Recently, laser capture microdissection (LCM), combined with microarray analysis, has allowed the identification of motor neuron specific changes in gene expression in human ALS cases. The aim of the present study is to combine LCM and microarray analysis to study how motor neurons in the spinal cord of transgenic SOD1 G93A mice and transgenic SOD1 WT respond to stimuli determined by the presence of the human mutant protein throughout the evolution of the stages in motor neuron injury Experiment Overall Design: Motor neurons have been isolated from the spinal cord of G93A mice and non transgenic littermates at different time points and the transcription expression profile of the isolated motor neurons has been analysed
2008-06-11 | E-GEOD-10953 | biostudies-arrayexpress
Project description:Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in about 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although, molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in non-neuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to non-transgenic, SOD1G93A-expressing astrocytes induced apoptosis of co-cultured motor neurons. This prompted us to investigate whether the effect on motor neuron survival was related to a change in the gene expression profile. Through high-density oligonucletide microarrays we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix construction, response to stress and steroid and lipid metabolism. Decorin, a small multifunctional proteoglycan, was the most up-regulated gene. Down-regulated genes included the insulin-like growth factor-1 receptor and the RNA binding protein ROD1. We also analyzed the expression of selected genes in purified motor neurons expressing SOD1G93A and in spinal cord of asymptomatic and early symptomatic ALS-rodent model. The expression of mutated SOD1 in astrocytes cause gene expression changes with potential consequences for its interaction with motor neurons. The astrocyte-specific gene expression profile contributes to the identification of possible candidates for cell type-specific therapies in ALS Keywords: Cell type comparison Astrocytes were plated at a density of 2x104 cells/cm2 and maintained as described in Cassina P, et al.J Neurosci Res. 2002;67(1):21-9. Confluent astrocytes monolayers were changed to supplemented L15 medium (Vargas et al., 2006) for 24h before RNA isolation. Total RNA was isolated with RNeasy kit/RNase-Free DNase Set (Qiagen, CA, USA). RNA quality was assessed with the A260/280 ratio and the 2100 Bioanalyzer (Agilent Technologies, CA, USA) to ensure integrity of the samples used for microarray analysis. Double-stranded cDNA was synthesized from 5 μg of total RNA and used for microarray analysis with the Rat Genome 230 2.0 array (Affymetrix, CA, USA). A total of six arrays divided in three control and three transgenic samples were used. Labeling was performed with one-cycle target labeling assay according to Affymetrix, including the eukaryotic poly-A RNA control and the eukaryotic hybridization control kit. Hybridization, washing and scanning were carried out as described in the Affymetrix GeneChip expression analysis technical manual at the Oregon State University’s Center for Genome Research and Biocomputing. Image processing was done using Affymetrix GCOS 1.4 software. The quality of hybridization and overall chip performance was determined by visual inspection of the raw scanned data and the GCOS-generated report file. Microarray data (.CEL files) was normalized using GC-RMA probe-level analysis in ArrayAssist 4.0 (Stratagene, CA, USA). Following variance stabilization and Log transformation, fold chance versus p-value was calculated using nontransgenic (NonTG) samples as base values.
2008-12-18 | E-GEOD-7441 | biostudies-arrayexpress
Project description:Downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is thought to be implicated in motor neuron excitotoxicity in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 is cleaved by caspase-3 at the cytosolic C-terminus domain, impairing the transport activity and generating a proteolytic fragment found to be SUMO1 conjugated (CTE-SUMO1). We show here that this fragment accumulates in the nucleus of spinal cord astrocytes in vivo throughout the disease stages of the SOD1-G93A mouse model of ALS. In vitro expression in spinal cord astrocytes of the C-terminus peptide of EAAT2 (CTE), which was artificially fused to SUMO1 (CTE-SUMO1fus) to mimic the endogenous SUMOylation reaction, recapitulates the nuclear accumulation of the fragment seen in vivo and causes caspase-3 activation and axonal growth impairment in motor neuron-derived NSC-34 cells and primary motor neurons co-cultured with CTE-SUMO1fus-expressing spinal cord astrocytes. This indicates that CTE-SUMO1fus could trigger non-cell autonomous mechanisms of neurodegeneration. Prolonged nuclear accumulation of CTE-SUMO1fus in astrocytes leads to their degeneration, although the time frame of the cell-autonomous toxicity is longer than the one for the indirect toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a SUMOylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could take part to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration in ALS. Comparison is made between the toxic fragment (SUMO) and the same fragment without lysines that can be sumo-ylated (CTE). Four replicates have been performed for each sample group.
2011-07-19 | E-GEOD-26070 | biostudies-arrayexpress
Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from fibroblasts grown from neurologically healthy controls (n=6) and 3 groups of patients with ALS: 1) sporadic cases (n=6); 2) cases due to mutations of SOD1 (n=4); 3) cases due to mutations of TARDBP (n=3). The three ALS groups were compared to the controls.
2014-06-01 | E-GEOD-33855 | biostudies-arrayexpress
Project description:Familial amyotrophic lateral sclerosis (ALS) represents about 10% of ALS cases. In about 20% of familial ALS patients, a mutation in superoxide dismutase-1 (SOD1) can be found. The ubiquitous SOD1 protein converts superoxide radical anions to oxygen and hydrogen peroxide. Patients with familial ALS caused by mutations in SOD1 can show comorbidity with frontotemporal dementia and develop cognitive impairment, including apathy, inattention, verbal deficits, and hypersexuality. At the cellular level, pathological signs of ALS may include tau immunoreactive astrocytic and neuronal inclusions, suggesting that cognitive dysfunction in ALS may also reflect abnormal protein metabolism of the microtubule associated protein (MAP), tau. To identify cell-specific expression changes, we performed laser capture microdissection (LCM) to isolate anterior horn motor neurons and surrounding cells. To determine common pathways for the development of ALS due to dysfunction of SOD1 or TAU, we performed whole transcriptome analysis in SOD1 and TAU mouse models. Because ALS is a neurodegenerative disease that specifically affects motor neurons, these cells were the main investigative target. Global transcriptomes of glial cells surrounding the motor neurons were also assessed, because these cells have been implicated as triggers of neurodegeneration. Mouse experiments were performed at the presymptomatic stage, prior to the onset of cell loss, in order to reduce false positive signals due to tissue reactive changes. Lumbar anterior horn anterior horn motor neurons and surrounding cells (glia) were isolated from presymptomatic TAU-P301L and SOD1-G93A transgenic mice using laser capture microdissection (LCM; PixCell® IIe LCM System, Arcturus, Molecular Devices, CA). On average, 500 motor neuron bodies or surrounding glial cells from 20 tissue sections per animal were collected. RNA isolation from LCM collected cells was performed using the Qiagen RNeasy Micro Kit. Only RNA with RNA Integrity Numbers (RIN) above 7.5 were taken for further analysis (Agilent Bioanalyzer 2100). A two-round T7-based amplification and labeling protocol (Agilent Low RNA Input Linear Amplification Kit PLUS) was used to generate high quality labeled cRNA. Agilent Whole Mouse Genome Microarray comparisons of motor neurons and surrounding glia were performed between transgenic (SOD1G93A and TAUP301L) and corresponding nontransgenic (control) littermate animals, producing four independent comparison groups: SOD1G93A motor neurons versus control motor neurons (SOD1mn), SOD1G93A motor neuron surrounding glia versus control glia (SOD1gl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Raw microarray data were acquired using the Agilent DNA Microarray scanner and processed with the accompanying Agilent Feature Extraction 10.5 Image Analysis software using default settings. Normalized signal intensities were used to identify gene expression changes in SOD1G93A and TAUP301L motor neurons and surrounding glial cells, generating four partially overlapping sets of data. For the identification of differential expression, the genes were required to pass two conservative criteria: a ratio beyond the 99.5% confidence interval observed in homotypic comparisons, which corresponded to an approximately 1.5-fold expression change, and a paired t-test (P<0.01) computed using 100 permutations of the data for each gene. Correction for multiple comparisons was performed using the adjusted Bonferroni test. The analysis was performed in the TM4: Microarray Software Suite. These techniques identified 251 transcripts representing 186 known genes for which expression was altered in at least one of the four comparisons. Four-condition experiment, SOD1G93A motor neurons versus control motor neurons (SODmn), SOD1G93A motor neuron surrounding glia versus control glia (SODgl), TAUP301L motor neurons versus control motor neurons (TAUmn) and TAUP301L glia versus control glia (TAUgl). Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Biological replicates: 4 SOD1G93A motor neurons (SODmn), 4 non SOD littermate motor neurons (nonSODmn), 4 SOD1G93A motor neuron surrounding glia (SODgl), 4 non SOD littermate motor neuron surrounding glia (nonSODgl), 4 TAUP301L motor neurons (TAUmn), 4 non TAU littermate motor neurons (nonTAUmn), 4 TAUP301L motor neuron surrounding glia (TAUgl), 4 non TAU littermate motor neuron surrounding glia (nonTAUgl). Four independent comparison groups were generated: SOD1G93A motor neurons versus control nonSOD motor neurons, SOD1G93A motor neuron surrounding glia versus control nonSOD glia, TAUP301L motor neurons versus control nonTAU motor neurons and TAUP301L glia versus control nonTAU glia. Each comparison used four pairs of transgenic (SOD1G93A or TAUP301L) vs. corresponding control littermate animals, producing four biological replicates. Each replicate was repeated twice with dye flip to correct for unequal dye incorporation rates. Therefore, eight microarray hybridizations were performed for each biological comparison, for a total of 32 microarray slides and generating four groups of differentially expressed genes in SOD1G93A motor neurons (SODmn), SOD1G93A motor neuron surrounding glial cells (SODgl), TAUP301L motor neurons (TAUmn), and TAUP301L surrounding glial cells (TAUgl).
2010-07-30 | E-GEOD-22482 | biostudies-arrayexpress