Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1.

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Marcelo Vargas

PROVIDER: E-GEOD-7441 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1.

Vargas Marcelo R MR Pehar Mariana M Díaz-Amarilla Pablo J PJ Beckman Joseph S JS Barbeito Luis L

Journal of neuroscience research 20081201 16

Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in approximately 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although the molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in nonneuronal cells in shaping motor neuron degeneration. We have previousl ...[more]

PMID: 18683239

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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; PixCellÂ® 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).

Dataset Information

Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1.

Publications

Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1.

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