Exon Array Analysis Of Lumbar Spinal Cord From HSOD1-G93A Transgenic Mice And Exploration Of Pathogenesis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons in the cortex, brainstem, and spinal cord. The clinical manifestations of ALS include chronic, progressive weakness and muscle atrophy, together with signs of corticospinal tracts. Most of ALS patients died of respiratory failure within 3⁵ years after the onset of symptoms. Research finded that neurodegenerative disease, such as ALS, Parkinson's disease,Huntington's disease and Alzheimer's disease and so on, have the same characteristics of the pathophysiology and the same mode of cell death (apoptosis) except for the difference of apoptotic cell distribution in the central nervous system. Therefore, the in-depth study of one disease may reveal a number of neurodegenerative diseases in the pathogenesis and lay a theoretical foundation for the treatment and prevention of these diseases. Familial ALS (FALS) accounts for 5%¹0% of diagnosed cases, with SALS comprising the remainder of diagnoses. Approximately 20% of FALS cases are associated with mutations in the gene for copper/zinc superoxide dismutase, SOD1. ALS occurs in both sporadic (SALS) and familial (FALS) forms, which are clinically and pathologically similar, mutant SOD1 transgenic mice appear to the pathogenesis of ALS provide a good disease model.Although the pathogenesis of ALS remains unknown, many theories have been proposed to account for the selective degeneration of motoneurons in ALS, including 1) the involvement of autoimmune mechanisms, 2) oxidative stress, 3) excitotoxicity caused by elevated extracelluar glutamate concentration, 4) excessive phosphorylation of neurofilaments 5) mitochondria dysfunction, and so on. But any kind of mechanism can not satisfactorily explain the characteristics of selective damage of ALS motor neuron. Therefore, the pathogenic mechanism of ALS can not be a single factor, which is the result of many factors. Protein disorders have been confirmed that is the final reasons leading to the disease, and the abnormal change of the gene level is pathogenic basis reasons. It is estimated that over 70% genes in the human genome involves alternative splicing of mRNA, which makes the human proteome complexity is much higher than other organisms. Some recent studies have reported that the exon splicing in RNA level is a pathogenic key, especially in ALS. But because of technical limitations research in this area can not be in-depth till today, so research on pathogenesis of ALS and protection of motor neurons from RNA level is necessary.It is limited of application of PCR technique to study on nucleic acid level. The completion of human genome project has greatly boosted the study on the human disease gene mapping, cloning, structure and function. The focus of life science research has risen from the gene sequence to the gene function. Gene chips are developed in this context. Compared with traditional methods, it has a fast, high throughput, high accuracy, low cost, and can meet the requirements of clinical genetic testing. Today the birth of a more advanced and more reliable chip technology——Affymetrix GeneChip? Mouse Exon 1.0 ST Array, is applied to high-throughput and high-density scanning on genetic level, prediction of transcript splicing method. The chip showed a more broad application at the transcriptional level. The issue is the application of this chip to in-depth study of the pathogenic mechanism of ALS, in order to find effective treatment.There are four parts in this study. The first part: using the exon array, we studied the differential expression of genes and the amount of alternative exon splicing events in spinal cord samples of mouse model of ALS, which were in asymptomatic stage. The second part: using the exon array, we studied the differential expression of genes and the amount of alternative exon splicing events in spinal cord samples. The compares were asymptomatic with symptomatic hSOD1-G93A transgenic mice. The third part: based on the exon array, we studied the expression of Nox2, PI3K, and the alternative splicing event of Fyb using RT-PCR technology, which was also a validation chip reliability of the results.Partâ… Changes in spinal cord samples of asymptomatic hSOD1-G93A mouse and exploration of the emergence processes of ALSObjective: using the exon array, we studied the differential expression of genes and the amount of alternative exon splicing events in spinal cord samples of mouse model of ALS, which were in asymptomatic stage. Signal pathway analysis and gene function analysis were performed on these abnormal genes. The changes were discussed in transcription levels of early stage in ALS, help us to explore the possible initiator hypothesis of ALS.Methods: We collected 6 female mice, of which 3 were derived from 30-day-old transgenic mice and 3 were derived from littermates (30-days of age) without hSOD1-G93A, namely 30A and 30C groups. Transgenic progeny and their normal littermates were identified by polymerase chain reaction (PCR) amplification of tail DNA. Animals were killed at 30 days to obtain fresh lumbar spinal cord tissues (L3 to L5 segment) for analysis. The specimens were immediatelty put into RNAlater (RNAlater RNA Stabilization Reagent, Qiagen), and additional specimens were instantly frozen in liquid nitrogen and then stored at -80°C until use. Total RNA was isolated from the spinal tissue stored in RNAlater using RNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. Purity and integrity of the RNA were assessed on the Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip? reagent set (Agilent Technologies, Santa Clara, CA, USA). The RNA specimens were quantified spectrophotometrically. Abnormal changes were determined using Affymetrix GeneChip? Mouse Exon 1.0ST arrays according to the manufacturer's instructions (Affymetrix, Inc.). General process as follows: double-Cycle, singal-stranded cDNA synthesis and biotin labeling --- hybridization --- washing --- scanning. The Affymetrix Expression Console Software Version 1.0 was used to create gene- and exon-level expression signal estimates. Using MAS (Molecule Annotation System) systems, the biological significance of high-throughput experimental data were further discussed, such as GO analysis ang pathway analysis.Results: Comparison of the 30A and 30C groups only identified 1 gene upregulated more than 2-fold (definitely 2.8-fold) which has no annotation and 4 genes upregulated more than 1.5-fold, only one of which has annotation, named receptor-like S scavenger receptor (NM₀30707). There are no genes down-regulated more than 1.5-fold. On exon level, 85 PSRs (probe select regions) were identified as alternatively spliced exons that belonged to 85 ASTs (alternatively spliced transcripts), and 36 transcripts have annotation. Using MAS system, the genes which have annotation were analyzed by GO analysis and pathway analysis. There are no definite known pathogenic factors related to ALS. Doing research on the new findings is of great worth.Conclusions: Compared with the control group, pathogenic hSOD1-G93A gene almost has no significant effect on gene expression profiles in the early stage of ALS. On transcriptional level, no foregone alternative splicing event was found. It was supposed that the new gene and new alternative splicing event might be correlated with the pathogenic effect of hSOD1-G93A genes in the early stage of ALS. These new findings will be further studied in the future.Partâ…¡Changes in spinal cord samples of symptomatic/asymptomatic hSOD1-G93A mouse and exploration of the emergence processes of ALSObjective: using the exon array, we studied the differential expression of genes and the alternative exon splicing events in spinal cord samples of mouse model of ALS, which were in symptomatic and asymptomatic stage. Signal pathway analysis and gene function analysis were performed on the abnormal genes. The changes were discussed in transcription levels of onset stage of ALS, help us to explore the possible pathogenic mechanism of ALS.Methods: We collected 6 female mice, of which 3 were derived from symptomatic transgenic mice and 3 were derived from 30-day-old transgenic mice, namely SYMPTOM and 30A groups. Transgenic progeny and their normal littermates were identified by polymerase chain reaction (PCR) amplification of tail DNA. Animals were killed at 30 days to obtain fresh lumbar spinal cord tissues (L3 to L5 segment) for analysis. The specimens were immediatelty put into RNAlater (RNAlater RNA Stabilization Reagent, Qiagen), and additional specimens were instantly frozen in liquid nitrogen and then stored at -80°C until use. Total RNA was isolated from the spinal tissue stored in RNAlater using RNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. Purity and integrity of the RNA were assessed on the Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip? reagent set (Agilent Technologies, Santa Clara, CA, USA). The RNA specimens were quantified spectrophotometrically. Abnormal changes were determined using Affymetrix GeneChip? Mouse Exon 1.0ST arrays according to the manufacturer's instructions (Affymetrix, Inc.). General process as follows: double-cycle, singal-stranded cDNA synthesis and biotin labeling --- hybridization --- washing --- scanning. The Affymetrix Expression Console Software Version 1.0 was used to create gene- and exon-level expression signal estimates. Using MAS (Molecule Annotation System) systems, the biological significance of high-throughput experimental data were further discussed, such as GO analysis ang pathway analysis.Results: Comparison of the SYMPTOM and 30A groups identified 2869 differentially expressed genes (DEGs), consisting of 263 (9.17%) filtered genes upregulated more than 2-fold and 71 (2.47%) filtered genes down regulated more than 2-fold (P <0.05). On exon level, 630 PSRs were identified as alternatively spliced exons that belonged to 563 ASTs. In addition, 16.2% (54/334) of the transcripts which were upregulated or down regulated significantly containing alternatively spliced exons. We selected top 90 upregulated genes and bottom 10 down regulated genes in SYMPTOM group vs 30A group were listed, and filtered 46 genes which exon'NI near 1.0 in the group predicted to have a higher inclusion rate and near 0 in the group predicted to have a lower inclusion rate to show. GO analysis and pathway analysis were performed on 100 DEGs and 46 ASTs showed in tables. 4 genes which were up-regulated 1.5-fold in the comparison of the 30A and 30C groups were all detected in the comparison of the SYMPTOM and 30A groups, but they have no statistical significance because of P >0.05. It can be seen from that 330 significantly changed genes (fold change≥2 or≤0.5, P<0.05) were emerging with the progression of the disease process. Only 7 of 85 ASTs in the comparison of the 30A and 30C groups were detected in the comparison of the SYMPTOM and 30A groups, that is, 556 ASTs were emerging with the progression of the disease process.Conclusions: Compared with the 30A, mice in SYMPTOM group appeared many cross changes on gene and exon level, indicating the possibility of combinatorial regulation of transcription and splicing. These two aspects of regulations may result in high degree of correlation between splicing patterns and transcriptional expression. The abnormal changes in symptomatic mice of two levels are much more than asymptomatic mice, indicating that the mutant SOD1 may be involved in a number of key steps in the pathological cascade of events leading to neuronal injury and this kind of ALS may be a cumulative result of mutant SOD1. It may triger the cascade amplification reaction. In addition, a few of these abnormal changes had been confirmed by previous research reports, but most of them were predicted by this study. It may provide a new direction for further study of possible pathogenic mechanism and treatment of ALS at the transcriptional level.Partâ…¢expression and investigation of PI3K/Rac1/Nox2/ROS signal pathway in hSOD1-G93A miceObjective: to understand the changes of PI3K/Rac1/Nox2/ROS pathway in the process of spinal cord motoneruon injury by examining the expression of PI3K and Nox2 and the splicing mode of Fyb in lumbar spinal cord, and discuss the possible mechanism of ALS from transcriptional level. At the same time validate the reliability of the results of exon chip.Methods: We collected 9 female mice, of which 6 were derived from transgenic mice (three 30-day-old, and three at symptomatic stage at approximately 120 days of age) and 3 were derived from littermates (30-days of age) without hSOD1-G93A, namely 30A, SYMPTOM and 30C groups, to used in this study. Transgenic progeny and their normal littermates were identified by polymerase chain reaction (PCR) amplification of tail DNA. Animals were killed at the two time points to obtain fresh lumbar spinal cord tissues (L3 to L5 segment) and were instantly frozen in liquid nitrogen, then stored at -80°C. RT-PCR was used to examine the expression of PI3K and Nox2 and the splicing mode of Fyb.Results: in the comparison of the SYMPTOM and 30A groups, PI3K and Nox2 genes were significantly up-regulated ,which was consistent with the results acquired by the exon array, but no significant change in 30A group vs 30C group. From the exon array, the exon 19 of Fyb gene (SI = 5.25, P = 0.042) was highly included in the symptom mice (NI = 0.9704) and lower expressed in 30A group (NI = 0.185), which is consistent with the results of the RT-PCR.Conclusions: In the progression process of ALS, PI3K and Nox2 were significantly up-regulated, which suggested the activation of PI3K/Rac1 /Nox2/ROS pathway, but had no obvious change in 30A vs 30C group. The alternative splicing of Fyb which was contained in this pathway supposed a combinatorial regulation between the two levels. All these results suggest that the exon array system maybe reliable and effective enough to detect differential expression at both the transcriptional and splicing levels.