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. 2008 Dec;86(16):3515-25.
doi: 10.1002/jnr.21797.

Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1

Marcelo R Vargas  1 , Mariana Pehar, Pablo J Díaz-Amarilla, Joseph S Beckman, Luis Barbeito
Affiliations

Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1

Marcelo R Vargas et al. J Neurosci Res. 2008 Dec.

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 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 previously found that in contrast to nontransgenic cells, SOD1(G93A)-expressing astrocytes induced apoptosis of cocultured 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 oligonucleotide microarrays, we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix synthesis, response to stress, and steroid and lipid metabolism. The most up-regulated gene was decorin (Dcn), a small multifunctional extracellular proteoglycan. Down-regulated genes included the insulin-like growth factor-1 receptor (Igf-1r) and the RNA binding protein ROD1. Rod1 was also found down-regulated in purified motor neurons expressing SOD1(G93A). Changes in the expression of Dcn, Igf-1r, and Rod1 were found in the spinal cord of asymptomatic animals, suggesting these changes occur before overt neuronal degeneration and potentially influence astrocyte-motor neuron interaction in the course of the disease. The astrocyte-specific gene expression profile might contribute to the identification of possible candidates for cell type-specific therapies in ALS.

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Figures

Figure 1
Figure 1
A) Microphotography showing morphological aspect of non-transgenic (NonTG) and SOD1G93A (G93A) spinal cord astrocyte monolayers. Cultures were stained for GFAP (green) and α-tubulin (red). Nuclei were stained with DAPI. Scale bar: 30 μm. B) GFAP protein expression analyzed by western blot in non-transgenic (NonTG) and SOD1G93A spinal cord astrocytes. β-Actin is shown as a loading control. C) Scatter plot of the fold-change (FC G93A vs. NonTG) versus the result p-value of significance analysis. Probe intensity data were normalized using GC-RMA. Following variance stabilization and Log transformation, the fold-change versus the p-value was calculated using non-transgenic samples as base values. Probe sets with significant change (p<0.05) of at least 1.2 fold are marked in gray.
Figure 2
Figure 2
Verification of selected differentially expressed genes in SOD1G93A astrocytes. Up-regulation of decorin (Dcn) mRNA (A) and protein (B) was confirmed by relative quantitative reverse transcriptase-PCR and Western blot as described under Materials and Methods. C) insulin-like growth factor-1 receptor (Igf-1r) and regulator of differentiation (Rod1) mRNA down-regulation in SOD1G93A astrocytes. D) Decreased IGF-1R expression in SOD1G93A astrocytes as determined by Western blot. In (A) and (C) mRNA levels are expressed as % of non-transgenic (NonTG) controls and corrected by 18S rRNA cDNA amplification. * Significantly different from control (p<0.05).
Figure 3
Figure 3
Expression of decorin (Dcn), insulin-like growth factor-1 receptor (Igf-1r) and regulator of differentiation (Rod1) in the spinal cord of SOD1G93A rats. Comparative mRNA levels from spinal cord of age-matched non-transgenic (NonTG) rats and A, asymptomatic or B, early symptomatic SOD1G93A rats (G93A). mRNA levels were corrected by 18S rRNA cDNA amplification. Data for each stage were expressed as % of age-matched non-transgenic littermates. * Significantly different from control (p<0.05). Decorin (C) and IGF-1R (D) expression levels analysed by Western blot in the spinal cord of asymptomatic (AS) and early symptomatic (ES) SOD1G93A rats compared to age-matched non-transgenic (NonTG) animals.
Figure 4
Figure 4
Expression of decorin (Dcn), insulin-like growth factor-1 receptor (Igf-1r) and regulator of differentiation (Rod1) in isolated spinal cord motor neurons from non-transgenic (NonTG) and SOD1G93A-expressing (G93A) E15 embryos. The mRNA levels were corrected by 18S rRNA cDNA amplification and expressed as % of non-transgenic (NonTG) controls. * Significantly different from control (p<0.05).
Figure 5
Figure 5
Schematic representation of some of the changes in gene expression found in SOD1G93A-astrocytes and its putative consequences.
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