| Amyotrophic lateral sclerosis (ALS) is one of the most common adult-onset neurodegenerative diseases, characterized by progressive and relatively selective degeneration of the upper and lower motor neurons in the spinal cord, brainstem, and motor cortex. Patients have progressive paralysis and muscle atrophy, and half of the patients die on average within 3 to 5 years of symptom onset, mostly resulting from respiratory failure. The global incidence of ALS is about 1-2 per 100 000 each year and the lifetime risk of developing ALS is estimated to be approximately 1/600 to 1/2 000.Ninety perccent of ALS are sporadic and the remaining 10% is familial, but the two forms of ALS are clinically and pathologically indistinguishable. Of the familial cases, 20% are associated with a mutation in the cytosolic copper/zinc superoxide dismutase gene (SOD1). The SOD1 mutations are predominantly missense mutations leading to substitution of one amino acid by another, for example glycine by alanine (SOD1-G93A). Because expression of a hSOD1 mutant in animals causes an adult-onset phenotype recapitulating closely the human disease both clinically and histopathologically, these transgenic animals have become a widely used ALS model. In fact, the effects of the mutant are due to the toxic gain of function, while most of SOD1 mutants retain enzymatic activity. Yet, at the present time, the actual mechanism by which mutant SOD1 initiates ALS is poorly understood, although several hypotheses have been proposed to explain the toxic effect of mutated SOD1, on the basis of extensive research on ALS: oxidative stress, glutamate excitotoxicity, formation of high-molecular-weight aggregates, mitochondrial dysfunction and apoptosis.The availability of powerful genomics technologies provides the opportunity to unravel complex regulatory and disease mechanisms. Alternative splicing of mRNA transcripts is a major form of post-transcriptional gene regulation, during which introns in a pre-mRNA are differentially removed and exons are joined to form multiple forms of mature RNA. Alternative splicing plays an important role in generating proteomic diversity, as a relatively limited number of genes may expand into very complex proteomes. It has been estimated that about three-quarters of all human genes exhibit alternative splicing. Current data suggest that greater than 90% of all human genes undergo alternative splicing. Statistics report of ASTD database Mus musculus shows that 57% genes undergo alternative splicing. Researchers are becoming aware of the dominant role of mRNA splicing in many important physiological processes such as development, physiology and disease. Affymetrix Exon Arrays are rapidly gaining popularity and becoming a standard for both gene- and exon-level expression analysis.Recent studies have shown that alternative splicing is especially prevalent in the nervous system. Zhang et al. reported that aberrant cleavage of TAR DNA-binding protein-43 (TDP-43) enhances aggregation and cellular toxicity in ALS. The glutamate transporter 1 in rodents, or EAAT2 in humans, is alternatively spliced in a complex manner, including the use of multiple 5' and 3' untranslated regions and several coding variants. Though alternative splicing is prevalent in the nervous system, little is known in ALS.In the present study, we used Affymetrix GeneChip? Mouse Exon 1.0 ST Array to identify genes and exons that are differentially expressed in SOD1-G93A transgenic mice. The array contains approximately 5.4 million probes grouped into 1.4 million probe sets and interrogates more than 1 million known and predicted exons. For each gene, the median number of probes is 30 to 40, usually distributed along the entire transcript sequence. The commercial availability of specific software provides a powerful tool for the study of alternative splicing (exon-level expression analysis) and interrogation of the expression of every known and predicted exon. At the same time, exon arrays provide robust gene-level expression analysis.For the first time, we have identified not only genes that are altered in expression in response to mutant SOD1, but also genes that undergo alternative splicing. The study was focused on the lumbar spinal cord which is the most affected tissue in the SOD1-G93A transgenic mice. Our study includes three parts.Part I Differential expression of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse modelObjective: The study was aimed to identify the transcription profiles of the lumbar spinal cord removed from SOD1-G93A mice at the onset of the disease in the presence of the mutant SOD1 protein.Methods: Using the mouse exon 1.0 ST, we analyzed the transcription profiles of the lumbar spinal cord removed from three female SOD1-G93A mice at the onset of the disease and three female non-transgenic littermates as the same age. Hierarchical cluster (HC) analysis, the gene ontology (GO) hierarchy analysis and a pathway-based analysis were then carried out on the differentially expressed genes.Results: (1) The analyses identified a total of 322 transcripts that were statistically differential in the presence of the mutant SOD1 protein: 309 (95.96%) transcripts were up-regulated and 13 (4.04%) transcripts were down-regulated. Based on T-test of the results, we generated a valcano plot to compare visually the differentially expressed genes between the onset group and the control group. (2) Cluster analysis 3.0 software was used for hierarchical cluster (HC) analysis. There is a good reproducibility between samples, and the genes can be successfully distinguish between the onset group and the control group. (3) To reveal global functional differences, the gene ontology (GO) hierarchy analysis was then carried out on the differentially expressed genes. These differentially expressed genes were classified into molecular function with a P value cutoff of less than 0.001, including heparin binding, insulin-like growth factor 1 binding, superoxide-generating NADPH oxidase activity, integrin binding, lipoprotein lipase activity and chemokine activity. Taking into account the biological process of the differentially expressed genes, they were critically involved in cell adhesion, inflammatory response and immune response. Leukocyte adhesion, integrin complex, integrin-mediated signaling pathway and activation of MAPK activity were also identified. (4) We performed a pathway-based analysis of the differentially expressed genes. The differentially expressed genes based on the KEGG database analysis were involved in some pathways, such as cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, natural killer cell mediated cytotoxicity, focal adhesion, complement and coagulation cascades, leukocyte transendothelial migration, cell adhesion molecules and apoptosis.Conclusion: There is significant difference between the gene expression of SOD1-G93A transgenic mice and that of their littermate control. In addition, the differentially expressed genes can be defined clearly, clarified and analyzed.Partâ…¡Alternative splicing of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse modelObjective: The study was aimed to identify alternatively spliced genes of the lumbar spinal cord removed from SOD1-G93A mice at the onset of the disease in the presence of the mutant SOD1 protein.Methods: Using the mouse exon 1.0 ST, we analyzed alternatively spliced genes of the lumbar spinal cord removed from three female SOD1-G93A mice at the onset of the disease and three female non-transgenic littermates as the same age. Hierarchical cluster (HC) analysis, the gene ontology (GO) hierarchy analysis and a pathway-based analysis were then carried out on these alternatively spliced genes.Results: (1) Three hundred and thirty three probesets (44 087 probesets in all) passed our quality control criteria and were used for subsequent analyses. The three hundred and thirty three probesets belonged to 297 alternatively spliced genes, reflecting that multiple alternative splicing events might occur in a single transcript. Our result showed that 63 alternatively spliced exons were derived from 27 transcripts. Three or more alternatively spliced exons were detected individually in some transcripts, indicating a complicated pattern of splicing regulation of these transcripts in the mice. (2) HC analysis was carried out according to similarity by using cluster analysis 3.0 software. The result showed that a good correlation between two groups. (3) The GO hierarchy analysis was then carried out on the alternatively spliced genes. These alternatively spliced genes are involved in iron ion binding, chemokine activity, metalloendopeptidase activity or motor activity, according to their molecular function categories. Taking into account biological process, the genes are predominately associated with inflammatory response, immune response, chemotaxis, activation of MAPK activity and anti-apoptosis. (4) Applying the same bioinformatics analyses that were used for the differential gene expression, we found that 95 of 297 alternatively spliced genes were involved in known pathways, based on the KEGG database analysis. Significant pathways were identified, including cytokine-cytokine receptor interaction, MAPK signaling pathway, cell adhesion molecules, regulation of actin cytoskeleton, apoptosis, T cell receptor signaling pathway and natural killer cell mediated cytotoxicity.Conclusions: Two or more alternative splicing events may take place in a single transcript, which indicates the complexity of alternative splicing model. Though the overlapped part of alternative spliced genes and differently expressed ones is quite small, the GO attribution and biological pathways of the two are both much similar, which suggests the corresponding biological effects are co-regulated at the two different levels.Part III Validation of differentially expressed and alternatively spliced genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse modelObjective: The study was aimed to validate differentially expressed and alternatively spliced genes identified by exon array in the presence of the mutant SOD1 protein.Methods: Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to validate differentially expressed and alternatively spliced genes of the lumbar spinal cord removed from SOD1-G93A mice at the onset of the disease..Results: The mRNA levels of CD68 and lipoprotein lipase (LPL) were significantly increased in the lumbar spinal cords of SOD1-G93A transgenic mice compared to the controls (P < 0.001), suggesting that there existed inflammatory responses and a hypermetabolic state of lipid at the onset stage. We also carried out RT-PCR validation of differential splicing in programmed cell death 1 (PDCD1) mRNA transcription in ALS. The results showed that the exon2-skipped isoform predominated in control samples, while the exon2-included isoform predominated in disease samples. Both exon predicted to have alternative splicing and exon predicted not to have alternative splicing were confirmed by RT-PCR.Conclusions: The results we obtained indicate that the exon array technology is reliable and sensitive enough to detect differential expression at both the gene level and exon level. |
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