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

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 Overall design: 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.

肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）由运动神经元进行性变性所引发。约20%的家族性ALS患者携带有铜/锌超氧化物歧化酶（Cu/Zn superoxide dismutase, SOD1）基因突变。突变型SOD1通过获得性毒性特性介导运动神经元死亡。尽管该毒性功能获得的分子机制仍未明确，但已有证据显示，非神经元细胞中突变型SOD1的表达可调控运动神经元变性进程。本团队既往研究表明，与非转基因星形胶质细胞相比，表达SOD1G93A的星形胶质细胞可诱导共培养运动神经元发生凋亡。这一发现促使我们探究其对运动神经元存活的影响是否与基因表达谱改变相关。

通过高密度寡核苷酸微阵列（high-density oligonucleotide microarrays）分析，我们发现与转录调控、信号通路、细胞增殖、细胞外基质构建、应激反应以及类固醇与脂质代谢相关的基因表达出现显著变化。其中，多功能小分子蛋白聚糖核心蛋白聚糖（Decorin）为上调幅度最高的基因；下调基因则包括胰岛素样生长因子-1受体（insulin-like growth factor-1 receptor）与RNA结合蛋白ROD1（RNA binding protein ROD1）。我们还对表达SOD1G93A的纯化运动神经元，以及无症状与早期症状期ALS啮齿动物模型的脊髓组织中部分基因的表达水平进行了分析。星形胶质细胞中突变型SOD1的表达可引发基因表达谱改变，进而可能影响其与运动神经元的相互作用。星形胶质细胞特异性基因表达谱有助于筛选ALS细胞类型特异性治疗的潜在候选靶点。

关键词：细胞类型比较
整体实验设计：
星形胶质细胞以2×10⁴细胞/cm²的密度接种并培养，培养方法参照Cassina P等发表于《Journal of Neurosci Res》2002;67(1):21-9的研究。汇合后的星形胶质细胞单层更换为添加了补充剂的L15培养基（Vargas等，2006），培养24小时后进行RNA提取。总RNA采用RNeasy试剂盒/无RNase DNase套装（Qiagen，美国加利福尼亚州）提取，通过A260/A280比值及2100生物分析仪（Agilent Technologies，美国加利福尼亚州）评估RNA质量，以确保用于微阵列分析的样本完整性。取5 μg总RNA合成双链cDNA，随后使用大鼠基因组230 2.0芯片（Rat Genome 230 2.0 array，Affymetrix，美国加利福尼亚州）进行微阵列分析。实验共设置6张芯片，分为3组对照组与3组转基因组样本。标记流程采用Affymetrix单循环靶标标记检测试剂盒，包含真核poly-A RNA对照品与真核杂交对照试剂盒。杂交、洗涤与扫描操作参照Affymetrix GeneChip表达分析技术手册，由俄勒冈州立大学基因组研究与生物计算中心完成。图像采用Affymetrix GCOS 1.4软件处理。通过对原始扫描数据及GCOS生成的报告文件进行目视检查，评估杂交质量与芯片整体性能。微阵列数据（.CEL文件）采用ArrayAssist 4.0（Stratagene，美国加利福尼亚州）中的GC-RMA探针水平分析方法进行标准化。经过方差稳定化与对数转换后，以非转基因（NonTG）样本为基准，计算倍数变化与p值。