肌萎缩侧索硬化转基因鼠模型腰髓差异表达基因及可变剪接研究
|
摘要
肌萎缩侧索硬化(Amyotrophic lateral sclerosis, ALS)是一种最常见的成人发病的神经退变性疾病,其特征是脊髓、脑干及运动皮质上、下运动神经元进行性及相对选择性的退变。病人表现为进行性的瘫痪与肌肉萎缩,约半数在发病3～5年内死亡,死因多为呼吸衰竭。每年全球发病率约为(1～2)/100 000,其终生发病率约为1/600至1/2 000。
90%的ALS病例为散发性,其余10%为家族性,二者的临床表现及病理特点难以区分。20%的家族性病例与细胞质中铜/锌超氧化物歧化酶(SOD1)发生的某种基因突变相关联。这种SOD1基因突变属于发挥主导作用的错义突变,致使一种氨基酸被另一种氨基酸所替代,例如甘氨酸被丙氨酸所替代(SOD1-G93A)。因为表达人SOD1突变的动物亦在成年期发病,且临床表现及病理特点与人类患者非常相似,故这种转基因动物已成为广泛采用的ALS动物模型。实际上,绝大多数的突变SOD1并未影响酶学活性,其引发的病理效应可能源于生物学功能方面的毒性获得。尽管在对ALS深入研究的基础上,学者们提出了解释SOD1突变效应的几种假说,包括氧化应激、谷氨酸毒性、大分子聚集体形成、线粒体功能失调及凋亡等假说,但是,目前人们对SOD1突变促发ALS的确切机制依然所知甚少。
高效的基因组学技术为阐释复杂的疾病发病机制及其调控提供了宝贵的机遇。mRNA转录本的可变剪接是转录后调控的重要形式,它将mRNA前体中的内含子选择性移除,同时将外显子组合连接成多种形式的成熟RNA。数量有限的基因可扩展表达为非常复杂的蛋白质组,因此可变剪接在产生蛋白质组学的多态性方面发挥着重要的作用。据估算,在所有的人类基因中发生可变剪接者约占3/4。目前资料显示,90%以上的人类基因发生了可变剪接。ASTD数据库(http://www.ebi.ac.uk)资料显示57%的小鼠基因发生可变剪接。研究者越来越认识到mRNA剪接在许多重要的生理过程如发育、生理和疾病中,发挥着主导作用。Affymetrix外显子芯片迅速获得业界认可并成为同时检测基因水平和外显子水平表达的操作标准。
新近的研究表明可变剪接在神经系统尤其普遍。已有报道ALS中TAR DNA-结合蛋白43(TDP-43)的异常剪接可增加聚集和细胞毒性作用。啮齿类动物中的谷氨酸转运体1(在人类称为EAAT2)以一种复杂的模式发生可变剪接,包括5′端与3′非翻译区及几个编码区异构体。尽管可变剪接在神经系统普遍存在,但在ALS中的研究甚少。
本研究应用Affymetrix小鼠外显子1.0 ST芯片来检测SOD1-G93A转基因小鼠及其同窝同性别非转基因对照动物腰髓基因的差异表达与外显子的可变剪接,并通过RT-PCR方法对上述检测结果进行验证。每张芯片大约包含5.4兆个探针(1.4兆个探针组),覆盖了约1百万个已知的和预测的外显子。大约90%的外显子被4个探针所覆盖,这些针对同一个外显子设计的探针组成的集合称为探针组(probesets)。对每一个基因而言,平均的探针数是30～40个,通常分布于整个转录本序列的全长。上述外显子芯片可整合每一个已知的和预测的外显子的表达,为研究可变剪接(外显子表达分析)提供了强有力的工具,同时可对基因水平的表达进行分析。
本研究首次对SOD1-G93A转基因小鼠的腰髓进行了外显子芯片分析,以确定在突变SOD1存在的情况下差异表达基因及可变剪接事件。本课题分为三部分。
第一部分肌萎缩侧索硬化转基因鼠模型腰髓差异表达基因研究
目的:研究在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的基因表达谱。
方法:应用Affymetrix小鼠外显子1.0 ST芯片检测发病期的雌性SOD1-G93A小鼠及非转基因同性别的同窝对照小鼠腰髓组织的基因转录本,并对差异表达基因进行分层聚类分析、GO分类及生物学通路分析。
结果:(1)在突变SOD1蛋白存在的情况下,有322个转录本的差异表达具有统计学意义,其中309个(占95.96%)上调,13个(占4.04%)下调。基于T检验的结果,经可视化处理生成一个火山图以对比发病组与对照组之间的差异表达基因。(2)通过分层聚类分析,我们可见各样品之间有很好的可重复性,而且发病组与对照组之间的基因可成功区分。(3)GO分类。对这些发生差异表达的基因根据其分子功能进行了分类,其中满足P值< 0.001的上述基因类别包括肝素结合、胰岛素样生长因子1结合、超氧化物引发的NADPH氧化活性、整合素结合、脂蛋白酯酶活性及化学因子活性等。从差异表达基因的生物学过程考虑,主要涉及细胞黏附、炎症反应及免疫反应。同时还涉及白细胞黏附、整合素复合体、整合素介导的信号途径以及MAPK活性等。(4)生物学通路分析。基于KEGG数据库分析,差异表达基因主要涉及如下生物学通路:细胞因子及其受体相互作用、actin细胞骨架调节、自然杀伤细胞介导的细胞毒作用、细胞黏附、补体及凝血瀑布、白细胞跨内皮迁移、细胞黏附分子及凋亡等。
结论:肌萎缩侧索硬化模型SOD1-G93A转基因小鼠腰髓组织的基因表达与非转基因同性别的同窝对照小鼠相比,具有显著的统计学差异,且发生差异表达的基因可明确区分并进行相应的归类与分析。
第二部分肌萎缩侧索硬化转基因鼠模型腰髓可变剪接研究
目的:研究在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的可变剪接。
方法:应用小鼠外显子1.0 ST芯片检测发病期的雌性SOD1-G93A小鼠及非转基因同性别的同窝对照小鼠的腰髓组织的可变剪接事件,并对可变剪接基因进行分层聚类分析、GO分类及生物学通路分析。
结果: (1)在全部44 087个探针组中,有333个探针组符合质量控制标准,可用于后继的分析处理。这些探针组分属于297个发生可变剪接的基因,提示单个转录本可能发生多个可变剪接事件。我们的研究结果显示,有63个发生可变剪接的外显子源于27个转录本。在多个编码蛋白的转录本中检测到3个或3个以上出现可变剪接的外显子,提示了小鼠体内剪接调控模式的复杂性。(2)根据其相似性用聚类分析3.0软件进行了HC分析。结果显示两组间具有良好的相关性。(3)剪接基因GO分类。根据其分子学功能分类,这些发生可变剪接的基因主要涉及铁离子结合、化学因子活性、基质金属蛋白酶活性及分子活性。而从生物学过程考虑,这些基因主要与炎症反应、免疫反应、趋化作用、MAPK活性的活化及抗凋亡等相关。(4)生物学路径分析。以KEGG数据库分析为基础,发现297个可变剪接基因中的95个与已知的生物学路径相关,并且鉴定出其中具有统计学差异的通路,包括细胞因子及其受体的相互作用、MAPK信号路径、细胞黏附分子、actin细胞骨架的调控、细胞凋亡、T细胞受体信号路径及自然杀伤细胞介导的细胞毒等。
结论:单个转录本可能发生多个可变剪接事件,提示了小鼠体内剪接调控模式的复杂性。发生可变剪接的基因与发生差异表达的基因相比,虽然重叠部分很少,但其GO归类与生物学通路重叠比例很高,提示相应的生物学效应受到转录表达与外显子剪接两个水平的共同调控。
第三部分肌萎缩侧索硬化转基因鼠模型腰髓差异表达基因及可变剪接的验证研究
目的:验证在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的差异表达基因及可变剪接事件。
方法:应用RT-PCR方法对发病G93A转基因小鼠及其同窝对照的腰髓组织中感兴趣差异表达基因及可变剪接进行验证。
结果:发病动物腰髓样本中CD68抗原(CD68 antigen,CD68)与脂蛋白脂酶(lipoprotein lipase,LPL)的mRNA表达水平明显增高(P < 0.001),提示在疾病的早期阶段存在炎症反应与脂类高代谢状态。我们也对ALS中程序性细胞死亡1(programmed cell death 1, PDCD1)mRNA转录本的可变剪接进行了RT-PCR验证。结果表明包含外显子2的异构体主要存在于发病动物,而不含外显子2的异构体主要存在于对照组。芯片预测发生可变剪接与不发生可变剪接的外显子均通过RT-PCR得以证实。
结论:上述结果证实了外显子芯片技术的可靠性与敏感性,其可用以检测基因水平及外显子水平的差异表达。
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 model
Objective: 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.
PartⅡAlternative splicing of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse model
Objective: 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 model
Objective: 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.
90%的ALS病例为散发性,其余10%为家族性,二者的临床表现及病理特点难以区分。20%的家族性病例与细胞质中铜/锌超氧化物歧化酶(SOD1)发生的某种基因突变相关联。这种SOD1基因突变属于发挥主导作用的错义突变,致使一种氨基酸被另一种氨基酸所替代,例如甘氨酸被丙氨酸所替代(SOD1-G93A)。因为表达人SOD1突变的动物亦在成年期发病,且临床表现及病理特点与人类患者非常相似,故这种转基因动物已成为广泛采用的ALS动物模型。实际上,绝大多数的突变SOD1并未影响酶学活性,其引发的病理效应可能源于生物学功能方面的毒性获得。尽管在对ALS深入研究的基础上,学者们提出了解释SOD1突变效应的几种假说,包括氧化应激、谷氨酸毒性、大分子聚集体形成、线粒体功能失调及凋亡等假说,但是,目前人们对SOD1突变促发ALS的确切机制依然所知甚少。
高效的基因组学技术为阐释复杂的疾病发病机制及其调控提供了宝贵的机遇。mRNA转录本的可变剪接是转录后调控的重要形式,它将mRNA前体中的内含子选择性移除,同时将外显子组合连接成多种形式的成熟RNA。数量有限的基因可扩展表达为非常复杂的蛋白质组,因此可变剪接在产生蛋白质组学的多态性方面发挥着重要的作用。据估算,在所有的人类基因中发生可变剪接者约占3/4。目前资料显示,90%以上的人类基因发生了可变剪接。ASTD数据库(http://www.ebi.ac.uk)资料显示57%的小鼠基因发生可变剪接。研究者越来越认识到mRNA剪接在许多重要的生理过程如发育、生理和疾病中,发挥着主导作用。Affymetrix外显子芯片迅速获得业界认可并成为同时检测基因水平和外显子水平表达的操作标准。
新近的研究表明可变剪接在神经系统尤其普遍。已有报道ALS中TAR DNA-结合蛋白43(TDP-43)的异常剪接可增加聚集和细胞毒性作用。啮齿类动物中的谷氨酸转运体1(在人类称为EAAT2)以一种复杂的模式发生可变剪接,包括5′端与3′非翻译区及几个编码区异构体。尽管可变剪接在神经系统普遍存在,但在ALS中的研究甚少。
本研究应用Affymetrix小鼠外显子1.0 ST芯片来检测SOD1-G93A转基因小鼠及其同窝同性别非转基因对照动物腰髓基因的差异表达与外显子的可变剪接,并通过RT-PCR方法对上述检测结果进行验证。每张芯片大约包含5.4兆个探针(1.4兆个探针组),覆盖了约1百万个已知的和预测的外显子。大约90%的外显子被4个探针所覆盖,这些针对同一个外显子设计的探针组成的集合称为探针组(probesets)。对每一个基因而言,平均的探针数是30～40个,通常分布于整个转录本序列的全长。上述外显子芯片可整合每一个已知的和预测的外显子的表达,为研究可变剪接(外显子表达分析)提供了强有力的工具,同时可对基因水平的表达进行分析。
本研究首次对SOD1-G93A转基因小鼠的腰髓进行了外显子芯片分析,以确定在突变SOD1存在的情况下差异表达基因及可变剪接事件。本课题分为三部分。
第一部分肌萎缩侧索硬化转基因鼠模型腰髓差异表达基因研究
目的:研究在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的基因表达谱。
方法:应用Affymetrix小鼠外显子1.0 ST芯片检测发病期的雌性SOD1-G93A小鼠及非转基因同性别的同窝对照小鼠腰髓组织的基因转录本,并对差异表达基因进行分层聚类分析、GO分类及生物学通路分析。
结果:(1)在突变SOD1蛋白存在的情况下,有322个转录本的差异表达具有统计学意义,其中309个(占95.96%)上调,13个(占4.04%)下调。基于T检验的结果,经可视化处理生成一个火山图以对比发病组与对照组之间的差异表达基因。(2)通过分层聚类分析,我们可见各样品之间有很好的可重复性,而且发病组与对照组之间的基因可成功区分。(3)GO分类。对这些发生差异表达的基因根据其分子功能进行了分类,其中满足P值< 0.001的上述基因类别包括肝素结合、胰岛素样生长因子1结合、超氧化物引发的NADPH氧化活性、整合素结合、脂蛋白酯酶活性及化学因子活性等。从差异表达基因的生物学过程考虑,主要涉及细胞黏附、炎症反应及免疫反应。同时还涉及白细胞黏附、整合素复合体、整合素介导的信号途径以及MAPK活性等。(4)生物学通路分析。基于KEGG数据库分析,差异表达基因主要涉及如下生物学通路:细胞因子及其受体相互作用、actin细胞骨架调节、自然杀伤细胞介导的细胞毒作用、细胞黏附、补体及凝血瀑布、白细胞跨内皮迁移、细胞黏附分子及凋亡等。
结论:肌萎缩侧索硬化模型SOD1-G93A转基因小鼠腰髓组织的基因表达与非转基因同性别的同窝对照小鼠相比,具有显著的统计学差异,且发生差异表达的基因可明确区分并进行相应的归类与分析。
第二部分肌萎缩侧索硬化转基因鼠模型腰髓可变剪接研究
目的:研究在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的可变剪接。
方法:应用小鼠外显子1.0 ST芯片检测发病期的雌性SOD1-G93A小鼠及非转基因同性别的同窝对照小鼠的腰髓组织的可变剪接事件,并对可变剪接基因进行分层聚类分析、GO分类及生物学通路分析。
结果: (1)在全部44 087个探针组中,有333个探针组符合质量控制标准,可用于后继的分析处理。这些探针组分属于297个发生可变剪接的基因,提示单个转录本可能发生多个可变剪接事件。我们的研究结果显示,有63个发生可变剪接的外显子源于27个转录本。在多个编码蛋白的转录本中检测到3个或3个以上出现可变剪接的外显子,提示了小鼠体内剪接调控模式的复杂性。(2)根据其相似性用聚类分析3.0软件进行了HC分析。结果显示两组间具有良好的相关性。(3)剪接基因GO分类。根据其分子学功能分类,这些发生可变剪接的基因主要涉及铁离子结合、化学因子活性、基质金属蛋白酶活性及分子活性。而从生物学过程考虑,这些基因主要与炎症反应、免疫反应、趋化作用、MAPK活性的活化及抗凋亡等相关。(4)生物学路径分析。以KEGG数据库分析为基础,发现297个可变剪接基因中的95个与已知的生物学路径相关,并且鉴定出其中具有统计学差异的通路,包括细胞因子及其受体的相互作用、MAPK信号路径、细胞黏附分子、actin细胞骨架的调控、细胞凋亡、T细胞受体信号路径及自然杀伤细胞介导的细胞毒等。
结论:单个转录本可能发生多个可变剪接事件,提示了小鼠体内剪接调控模式的复杂性。发生可变剪接的基因与发生差异表达的基因相比,虽然重叠部分很少,但其GO归类与生物学通路重叠比例很高,提示相应的生物学效应受到转录表达与外显子剪接两个水平的共同调控。
第三部分肌萎缩侧索硬化转基因鼠模型腰髓差异表达基因及可变剪接的验证研究
目的:验证在突变SOD1存在的情况下,G93A转基因小鼠腰髓组织的差异表达基因及可变剪接事件。
方法:应用RT-PCR方法对发病G93A转基因小鼠及其同窝对照的腰髓组织中感兴趣差异表达基因及可变剪接进行验证。
结果:发病动物腰髓样本中CD68抗原(CD68 antigen,CD68)与脂蛋白脂酶(lipoprotein lipase,LPL)的mRNA表达水平明显增高(P < 0.001),提示在疾病的早期阶段存在炎症反应与脂类高代谢状态。我们也对ALS中程序性细胞死亡1(programmed cell death 1, PDCD1)mRNA转录本的可变剪接进行了RT-PCR验证。结果表明包含外显子2的异构体主要存在于发病动物,而不含外显子2的异构体主要存在于对照组。芯片预测发生可变剪接与不发生可变剪接的外显子均通过RT-PCR得以证实。
结论:上述结果证实了外显子芯片技术的可靠性与敏感性,其可用以检测基因水平及外显子水平的差异表达。
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 model
Objective: 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.
PartⅡAlternative splicing of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse model
Objective: 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 model
Objective: 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.
引文
1 Shaw PJ. Molecular and cellular pathways of neurodegeneration in motor neurone disease. J Neurol Neurosurg Psychiatry, 2005, 76(8): 1 046~1 057
2 Ince PG, Lowe J, Shaw PJ. Amyotrophic lateral sclerosis: current issues in classification, pathogenesis and molecular pathology. Neuropathol Appl Neurobiol, 1998, 24: 104~117
3 Boillée S, Vande Velde C, Cleveland DW. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron, 2006, 52(1): 39~59
4 Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci, 2006, 7(9): 710~723
5 Cozzolino M, Ferri A, CarrìMT. Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications. Antioxid Redox Signal, 2008, 10(3): 405~443
6 Simpson EP, Yen AA, Appel SH. Oxidative Stress: a common denominator in the pathogenesis of amyotrophic lateral sclerosis. Curr Opin Rheumatol, 2003, 15(6): 730~736
7 Andersen PM. Amyotrophic lateral sclerosis genetics with Mendelian inheritance. In: Brown Jr., R.H., Swash, M., Pasinelli, P. (Eds.), Amyotrophic Lateral Sclerosis. Informa Healthcare, London, 2006, pp. 187~207
8 Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 1993, 362(6415): 59~62
9 Fodor SP, Read JL, Pirrung MC, et al. Light-directed, spatially addressable parallel chemical synthesis. Science, 1991, 251(4 995): 767~773
10 Weydt P, Hong SY, Kliot M, et al. Assessing disease onset and progression in the SOD1 mouse model of ALS. Neuroreport, 2003. 14(7): 1 051~1 054
11 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
12 Jiang YM, Yamamoto M, Kobayashi Y, et al. Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol, 2005, 57(2): 236~251
13 Perrin FE, Boisset G, Docquier M, et al. No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model. Hum Mol Genet, 2005, 14(21): 3 309~3 320
14 Ferraiuolo L, Heath PR, Holden H, et al. Microarray analysis of the cellular pathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS. J Neurosci, 2007, 27(34): 9 201~9 219
15 Lobsiger CS, Boillée S, Cleveland DW. Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proc Natl Acad Sci USA 2007, 104(18): 7 319~7 326
16 Kirby J, Halligan E, Baptista MJ, et al. Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS. Brain, 2005, 128(Pt 7): 1 686~1 706
17 Vargas MR, Pehar M, Díaz-Amarilla PJ, et al. Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1. J Neurosci Res, 2008, 86(16): 3 515~3 525
18 Olsen MK, Roberds SL, Ellbrock BR, et al. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol, 2001, 50: 730~740
19 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
20 Hensley K, Floyd RA, Gordon B, et al. Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis. J Neurochem, 2002, 82(2): 365~374
21 Malaspina A, Kaushik N, de Belleroche J. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem, 2001, 77: 132~145
22 Ishigaki S, Niwa J, Ando Y, et al. Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords—screening by molecular indexing and subsequent cDNA microarray analysis. FEBS Lett, 2002, 531(2): 354~358
23 Dangond F, Hwang D, Camelo S, et al. Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics, 2004, 16(2): 229~239
24 Offen D, Barhum Y, Melamed E, et al. Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant. J Mol Neurosci, 2009, 38(2): 85~93
25 Wang XS, Simmons Z, Liu W, et al. Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex. Amyotroph Lateral Scler, 2006, 7(4): 201~210
26 Lederer CW, Torrisi A, Pantelidou M, et al. Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis. BMC Genomics, 2007, 8: 26
27 Guo Y, Duan W, Li Z, et al. Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice. FEBS Lett, 2010, 584(8): 1 615~1 622
28 Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease—a double-edged sword. Neuron, 2002, 35: 419~432
29 Di Giorgio FP, Carrasco MA, Siao MC, et al. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neuroscience, 2007, 10: 608~614
30 Marchetto MC, Muotri AR, Mu Y, et al. Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell, 2008, 3: 649~657
31 King AE, Dickson TC, Blizzard CA, et al. Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology. Neurobiol Aging, 2009 May 6
32 González-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci, 1999, 22: 219~240
33 Tikka TM, Vartiainen NE, Goldsteins G, et al. Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease. Brain, 2002, 125(Pt 4): 722~731
34 Cleveland DW, Rothstein JD. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci, 2001, 2: 806~819
35 Yasojima K, Tourtellotte WW, McGeer EG, et al. Marked increase in cyclooxygenase-2 in ALS spinal cord: implications for therapy. Neurology, 2001, 57(6): 952~956
36 Goldknopf IL, Sheta EA, Bryson J, et al. Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson’s disease. Biochem Biophys Res Commun, 2006, 342(4): 1 034~1 039
37 McGeer PL, McGeer EG. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26(4): 459~470
38 Nguyen MD, Julien JP, Rivest S. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci, 2002, 3: 216~227
39 Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol, 2001, 2(4): 285~293
40 Gonzalez NC, Wood JG. Leukocyte-endothelial interactions in environmental hypoxia. Adv Exp Med Biol, 2001, 502: 39~60
41 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
42 Gibson RM, Craig SE, Heenan L, et al. Activation of integrin alpha5beta1 delays apoptosis of Ntera2 neuronal cells. Mol Cell Neurosci, 2005, 28: 588~598
43 Suwan K, Choocheep K, Hatano S, et al. Versican/PG-M assembles hyaluronan into extracellular matrix and inhibits CD44-mediated signaling toward premature senescence in embryonic fibroblasts. J Biol Chem, 2009, 284: 8 596~8 604
44 Wang MJ, Kuo JS, Lee WW, et al. Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages. J Neurochem, 2006, 97: 857~871
45 Wolburg H, Noell S, Wolburg-Buchholz K, et al. Agrin, aquaporin-4, and astrocyte polarity as an important feature of the blood-brain barrier. Neuroscientist, 2009, 15: 180~193
46 Stritt C, Stern S, Harting K, et al. Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression. Nat Neurosci, 2009, 12: 418~427
47 Cayre M, Canoll P, Goldman JE. Cell migration in the normal and pathological postnatal mammalian brain. Prog Neurobiol, 2009, 88: 41~63
1 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
2 Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics, 2006, 7: 325
3 Namshin Kim, Seokmin Shin, Sanghyuk Lee1 ECgene: Genome-based EST clustering and gene modeling for alternative splicing. Genome Research, 2005, 15(4): 566~576
4 Johnson JM, Castle J, Garrett-Engele P, et al. Genomewide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003, 302(5653): 2 141~2 144
5 Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature, 2008, 456(7221): 470~476
6 Pan Q, Shai O, Lee LJ, et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet, 2008, 40(12): 1 413~1 415
7 Novoyatleva T, Tang Y, Rafalska I, et al. Pre-mRNA missplicing as a cause of human disease. Prog Mol Subcell Biol, 2006, 44: 27~46
8 Licatalosi DD, Darnell RB. Splicing regulation in neurologic disease, Neuron, 2006, 52(1): 93~101
9 Zhang YJ, Xu YF, Cook C, et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA, 2009, 106(18): 7 607~7 612
10 Lauriat TL, Richler E, McInnes LA. A quantitative regional expression profile of EAAT2 known and novel splice variants reopens the question of aberrant EAAT2 splicing in disease. Neurochem Int, 2007, 50(1): 271~280
11 Yeo GW, Xu X, Liang TY, et al. Alternative splicing events identified in human embryonic stem cells and neural progenitors. PLoS Comput Biol, 2007, 3(10): 1 951~1 967
12 Thorsen K, S?rensen KD, Brems-Eskildsen AS, et al. Alternative splicing in colon, bladder, and prostate cancer identified by exon-array analysis. Mol Cell Proteomics, 2008, 7(7): 1 214~1 224
13 Hang X, Li P, Li Z, et al. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array. BMC Genomics, 2009, 10: 126
14 Xi L, Feber A, Gupta V, et al. Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer. Nucleic Acids Res, 2008, 36(20): 6 535~6 547
15 McKee AE, Neretti N, Carvalho LE, et al. Exon expression profiling reveals stimulus-mediated exon use in neural cells. Genome Biol, 2007, 8(8): R159
16 Zhang Z, Lotti F, Dittmar K, et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 2008, 133(4): 585~600
17 Zeng C, Berget SM. Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol Cell Biol, 2000, 20: 8 290~8 301
18 Kadener S, Fededa JP, Rosbash M, et al. Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation. Proc Natl Acad Sci USA, 2002, 99: 8 185~8 190
19 Kornblihtt AR, de la Mata M, Fededa JP, et al. Multiple links between transcription and splicing. RNA, 2004, 10(10): 1 489~1 498
20 Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics, 2006, 7(1): 325
21 Ip JY, Tong A, Pan Q, et al. Globar analysis of alternative splicing during T-cell activation. RNA, 2007, 13(4): 563~572
22 Wu MH. Endothelial focal adhesions and barrier function. J Physiol, 2005, 569(Pt 2): 359~366
23 Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease—a double-edged sword. Neuron, 2002, 35: 419~432
24 Di Giorgio FP, Carrasco MA, Siao MC, et al. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neuroscience, 2007, 10: 608~614
25 Marchetto MC, Muotri AR, Mu Y, et al. Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell, 2008, 3: 649~657
26 King AE, Dickson TC, Blizzard CA, et al. Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology. Neurobiol Aging, 2009 May 6
27 González-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci, 1999, 22: 219~240
28 McGeer PL, McGeer EG. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26(4): 459~470
29 Suwan K, Choocheep K, Hatano S, et al. Versican/PG-M assembles hyaluronan into extracellular matrix and inhibits CD44-mediated signaling toward premature senescence in embryonic fibroblasts. J Biol Chem, 2009, 284: 8 596~8 604
30 Wang MJ, Kuo JS, Lee WW, et al. Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages. J Neurochem, 2006, 97: 857~871
31 Wolburg H, Noell S, Wolburg-Buchholz K, et al. Agrin, aquaporin-4, and astrocyte polarity as an important feature of the blood-brain barrier. Neuroscientist, 2009, 15: 180~193
32 Stritt C, Stern S, Harting K, et al. Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression. Nat Neurosci, 2009, 12: 418~427
33 Cayre M, Canoll P, Goldman JE. Cell migration in the normal and pathological postnatal mammalian brain. Prog Neurobiol, 2009, 88: 41~63
34 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
1 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
2 Chiu IM, Phatnani H, Kuligowski M, et al. Activation of innate and humoral immunity in the peripheral nervous system of ALS transgenic mice. Proc Natl Acad Sci U S A, 2009, 106(49): 20 960~20 965
3 Wainwright DA, Xin J, Mesnard NA, et al. Toll-like receptor 2 and facial motoneuron survival after facial nerve axotomy. Neurosci Lett, 2010, 471(1): 10~14
4 Graber DJ, Hickey WF, Harris BT. Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis. J Neuroinflammation, 2010, 7: 8
5 Yiangou Y, Facer P, Durrenberger P, et al. COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. BMC Neurol, 2006, 6: 12
6 Henkel JS, Beers DR, Siklós L, et al. The chemokine MCP-1 and the dendritic and myeloid cells it attracts are increased in the mSOD1 mouse model of ALS. Mol Cell Neurosci, 2006 , 31(3): 427~437
7 Ahtoniemi T, Goldsteins G, Keksa-Goldsteine V, et al. Pyrrolidine dithiocarbamate inhibits induction of immunoproteasome and decreases survival in a rat model of amyotrophic lateral sclerosis. Mol Pharmacol, 2007, 71: 30~37
8 Beers DR, Henkel JS, Xiao Q, et al. Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA, 2006, 103: 16 021~16 026
9 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
10 Kriz J, Nguyen MD, Julien JP. Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis, 2002, 10: 268~278
11 Kiaei M, Petri S, Kipiani K, et al. Thalidomide and lenalidomide extend survival in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci, 2006, 26: 2 467~2 473
12 Klivenyi P, Kiaei M, Gardian G, et al. Additive neuroprotective effects ofcreatine and cyclooxygenase 2 inhibitors in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurochem, 2004, 88: 576~582
13 Ignacio S, Moore DH, Smith AP, et al. Effect of neuroprotective drugs on gene expression in G93A/SOD1 mice. Ann N Y Acad Sci, 2005, 1 053: 121~136
14 Beers DR, Henkel JS, Zhao W, et al. CD4 T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS. Proc Natl Acad Sci USA, 2008, 105: 15558~15 563
15 Chiu IM, Chen A, Zheng Y, et al. T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS. Proc Natl Acad Sci USA, 2008, 105: 17 913~17 918
16 Zhang L, Lookene A, Wu G, et al. Calcium triggers folding of lipoprotein lipase into active dimers. J Biol Chem, 2005, 280(52): 42 580~42 591
17 Xie C, Wang ZC, Liu XF, et al. The common biological basis for common complex diseases: evidence from lipoprotein lipase gene. Eur J Hum Genet. 2010V18N1:3~7
18 Baum L, Wiebusch H, Pang CP. Roles for lipoprotein lipase in Alzheimer's disease: an association study. Microsc Res Tech, 2000, 50(4): 291~296
19 Scacchi R, Gambina G, Broggio E, et al. The H+ allele of the lipoprotein lipase (LPL) HindIII intronic polymorphism and the risk for sporadic late-onset Alzheimer’s disease. Neurosci Lett, 2004, 367: 177~180
20 Blain JF, Aumont N, Théroux L, et al. A polymorphism in lipoprotein lipase affects the severity of Alzheimer's disease pathophysiology. Eur J Neurosci, 2006, 24(5): 1 245~1 251
21 Paradis E, Clavel S, Julien P, et al. Lipoprotein lipase and endothelial lipase expression in mouse brain: regional distribution and selective induction following kainic acid-induced lesion and focal cerebral ischemia. Neurobiol Dis, 2004, 15: 312~325
22 Blain JF, Paradis E, Gaudreault SB, et al. A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain. Neurobiol Dis, 2004,15: 510~519
23 Fergani A, Oudart H, Gonzalez De Aguilar JL, et al. Increased peripheral lipid clearance in an animal model of amyotrophic lateral sclerosis. J Lipid Res, 2007, 48(7): 1 571~1 580
24 Dupuis L, Corcia P, Fergani A, et al. Dyslipidemia is a protective factor in amyotrophic lateral sclerosis. Neurology, 2008, 70(13): 1 004~1 009
25 Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol, 2002, 2: 116~126
26 Folkl A, Bienzle D. Structure and function of programmed death (PD) molecules. Vet Immunol Immunopathol, 2010, 134(1-2): 33~38
27 Zhang X, Schwartz JC, Guo X, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20(3): 337~347
28 Riley JL. PD-1 signaling in primary T cells. Immunol Rev, 2009, 229(1): 114~125
29 Lázár-Molnár E, Yan Q, Cao E, et al. Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2. Proc Natl Acad Sci U S A, 2008, 105(30): 10 483~10 488
30 Parry RV, Chemnitz JM, Frauwirth KA, et al. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol, 2005, 25: 9543~9 553
31 Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci USA, 2001, 98: 13 866~13 871
32 Chemnitz JM, Parry RV, Nichols KE, et al. SHP-1 and SHP-2 associate with immunoreceptor tyrosine based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol, 2004, 173: 945~954
33 Ishida Y, Agata Y, Shibahara K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed celldeath. EMBO J, 1992, 11(11): 3 887~3 895
34 Simone R, Piatti G, Saverino D. The inhibitory co-receptors: a way to save from anergy the HIV-specific T cells. Curr HIV Res, 2009, 7(3): 266~272
35 Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med, 2000, 192(7): 1 027~1034
36 Sharpe AH, Wherry EJ, Ahmed R, et al. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol, 2007, 8(3): 239~245
37 Boussiotis VA, Freeman GJ, Gribben JG, et al. The role of B7-1/B7-2:CD28/CTLA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response. Immunol Rev, 1996, 153: 5~26
38 Keir ME, Butte MJ, Freeman GJ, et al. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 2008, 26: 677~704
39 Lee YH, Woo JH, Choi SJ, et al. Association of programmed cell death 1 polymorphisms and systemic lupus erythematosus: a meta-analysis. Lupus, 2009, 18(1): 9~15
40 Wang L, Han R, Hancock WW. Programmed cell death 1 (PD-1) and its ligand PD-L1 are required for allograft tolerance. Eur J Immunol, 2007, 37(10): 2 983~2 990
41 Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity, 1999, 11: 141~151
42 Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001, 291: 319~322
43 Wang J, Yoshida T, Nakaki F, et al. Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes. Proc. Natl Acad Sci USA, 2005, 102: 11 823
44 Nielsen C, Ohm-Laursen L, Barington T, et al. Alternative splice variantsof the human PD-1 gene. Cell Immunol, 2005, 235(2): 109~116
1 Kirby J, Heath PR, Shaw PJ, et al. Gene expression assays. Adv Clin Chem, 2007, 44: 247~292
2 Kelz MB, Dent GW, Therianos S, et al. Single-cell antisense RNAamplification and microarray analysis as a tool for studying neurological degeneration and restoration. Sci Aging Knowledge Environ, 2002, 1: 1~10
3 Ginsberg SD, Che S. RNA amplification in brain tissues. Neurochem Res, 2002, 27(10): 981~992
4 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
5 Shaw PJ. Molecular and cellular pathways of neurodegeneration in motor neurone disease. J. Neurol. Neurosurg. Psychiatry, 2005, 76(8): 1046~1057
6 Ince PG, Lowe J, Shaw PJ. Amyotrophic lateral sclerosis: current issues in classification, pathogenesis and molecular pathology. Neuropathol Appl Neurobiol, 1998, 24: 104~117
7 Bilsland LG, Dick JR, Pryce G, et al. Increasing cannabinoid levels by pharmacological and genetic manipulation delay disease progression in SOD1 mice. FASEB J , 2006, 20: 1 003~1 005
8 Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci, 2006, 7(9): 710~723
9樊东升,张俊,邓敏,等.肌萎缩侧索硬化/运动神经元病的基础与临床研究.北京大学学报(医学版), 2009, 41(3): 279~281
10 Andersen PM. Amyotrophic lateral sclerosis genetics with Mendelian inheritance. In: Brown Jr., R.H., Swash, M., Pasinelli, P. (Eds.), Amyotrophic Lateral Sclerosis. Informa Healthcare, London, 2006, 187~207
11 Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 1993, 362(6 415): 59~62
12 Valdmanis PN, Rouleau GA. Genetics of familial amyotrophic lateral sclerosis. Neurology, 2008, 70: 144~152
13 Simpson CL, Al-Chalabi A. Amyotrophic lateral sclerosis as a complex genetic disease. Biochim Biophys Acta, 2006, 1 762: 973~985
14 Hirschhorn JN, Daly MJ. Genome-wide association studies for commondiseases and complex traits. Nat Rev Genet, 2005, 6(2): 95~108
15 Schymick JC, Scholz SW, Fung HC, et al. Genome-wide genotyping in amyotrophic lateral sclerosis and neurologically normal controls: first stage analysis and public release of data. Lancet Neurol, 2007, 6(4): 322~328
16 Dunckley T, Huentelman MJ, Craig DW, et al. Whole-genome analysis of sporadic amyotrophic lateral sclerosis. N Engl J Med, 2007, 357(8): 775~788
17 van Es MA, Van Vught PW, Blauw HM, et al. ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study. Lancet Neurol, 2007, 6(10): 869~877
18 van Es MA, van Vught PW, Blauw HM, et al. Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nat Genet, 2008, 40(1): 29~31
19 Cronin S, Berger S, Ding J, et al. A genome-wide association study of sporadic ALS in a homogenous Irish population. Hum Mol Genet, 2008, 17(5): 768~774
20 Garber K. Genetics. The elusive ALS genes. Science, 2008, 319(5859): 20
21 Olsen MK, Roberds SL, Ellbrock BR, et al. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol, 2001, 50: 730~740
22 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
23 Hensley K, Floyd RA, Gordon B, et al. Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis. J Neurochem, 2002, 82(2): 365~374
24 Malaspina A, Kaushik N, de Belleroche J. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem, 2001, 77: 132~145
25 Ishigaki S, Niwa J, Ando Y, et al. Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords—screening by molecular indexing and subsequent cDNA microarray analysis. FEBS Lett, 2002, 531(2): 354~358
26 Dangond F, Hwang D, Camelo S, et al. Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics, 2004, 16(2): 229~239
27 Offen D, Barhum Y, Melamed E, et al. Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant. J Mol Neurosci, 2009, 38(2): 85~93
28 Wang XS, Simmons Z, Liu W, et al. Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex. Amyotroph Lateral Scler, 2006, 7(4): 201~210
29 Lederer CW, Torrisi A, Pantelidou M, et al. Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis. BMC Genomics, 2007, 8: 26
30 Gonzalez de Aguilar JL, Niederhauser-Wiederkehr C, Halter B, et al. Gene profiling of skeletal muscle in an amyotrophic lateral sclerosis mouse model. Physiol Genomics, 2008, 32(2): 207~218
31 Jiang YM, Yamamoto M, Kobayashi Y, et al. Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol, 2005, 57(2): 236~251
32 Jiang YM, Yamamoto M, Tanaka F, et al. Gene expressions specifically detected in motor neurons (dynactin 1, early growth response 3, acetyl-CoA transporter, death receptor 5, and cyclin C) differentially correlate to pathologic markers in sporadic amyotrophic lateral sclerosis. J Neuropathol Exp Neurol, 2007, 66(7): 617~627
33 Perrin FE, Boisset G, Docquier M, et al. No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model. Hum Mol Genet, 2005, 14(21): 3 309~3 320
34 Ferraiuolo L, Heath PR, Holden H, et al. Microarray analysis of the cellularpathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS. J Neurosci, 2007, 27(34): 9 201~9 219
35 Perrin FE, Boisset G, Lathuilière A, et al. Cell death pathways differ in several mouse models with motoneurone disease: analysis of pure motoneurone populations at a presymptomatic age. J Neurochem, 2006, 98(6): 1 959~1 972
36 Lobsiger CS, Boillée S, Cleveland DW. Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proc Natl Acad Sci USA, 2007, 104(18): 7 319~7 326
37 Kirby J, Halligan E, Baptista MJ, et al. Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS. Brain, 2005, 128(Pt 7): 1 686~1 706
38 Vargas MR, Pehar M, Díaz-Amarilla PJ, et al. Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1. J Neurosci Res, 2008, 86(16): 3 515~3 525
39 Namshin Kim, Seokmin Shin, Sanghyuk Lee1 ECgene: Genome-based EST clustering and gene modeling for alternative splicing. Genome Research, 2005, 15(4): 566~576
40 Johnson JM, Castle J, Garrett-Engele P, et al. Genomewide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003, 302(5 653): 2141~2144
41 Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature, 2008, 456(7 221): 470~476
42 Pan Q, Shai O, Lee LJ, et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet, 2008, 40(12): 1 413~1 415
43 Novoyatleva T, Tang Y, Rafalska I, et al. Pre-mRNA missplicing as a cause of human disease. Prog Mol Subcell Biol, 2006, 44: 27~46
44 Zhang YJ, Xu YF, Cook C, et al. Aberrant cleavage of TDP-43 enhancesaggregation and cellular toxicity. Proc Natl Acad Sci USA, 2009, 106(18): 7 607~7 612
45 Lauriat TL, Richler E, McInnes LA. A quantitative regional expression profile of EAAT2 known and novel splice variants reopens the question of aberrant EAAT2 splicing in disease. Neurochem Int, 2007, 50(1): 271~280
46 Zhang Z, Lotti F, Dittmar K, et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 2008, 133(4): 585~600
48 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
49 Chen H, Guo Y, Hu M, et al. Differential expression and alternative splicing of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse model. Brain Res, 2010 Mar 31. [Epub ahead of print]
50 Mehler MF, Mattick JS. Non-coding RNAs in the nervous system. J Physiol, 2006, 575(Pt 2): 333~341
51 Williams AH, Valdez G, Moresi V, et al. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science, 2009, 326(5 959): 1 549~1 554
52 Shioya M, Obayashi S, Tabunoki H, et al. Aberrant microRNA expression in the brains of neurodegenerative diseases: miR-29a decreased in Alzheimer disease brains targets neuron navigator-3. Neuropathol Appl Neurobiol, 2010, Feb 25
2 Ince PG, Lowe J, Shaw PJ. Amyotrophic lateral sclerosis: current issues in classification, pathogenesis and molecular pathology. Neuropathol Appl Neurobiol, 1998, 24: 104~117
3 Boillée S, Vande Velde C, Cleveland DW. ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron, 2006, 52(1): 39~59
4 Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci, 2006, 7(9): 710~723
5 Cozzolino M, Ferri A, CarrìMT. Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications. Antioxid Redox Signal, 2008, 10(3): 405~443
6 Simpson EP, Yen AA, Appel SH. Oxidative Stress: a common denominator in the pathogenesis of amyotrophic lateral sclerosis. Curr Opin Rheumatol, 2003, 15(6): 730~736
7 Andersen PM. Amyotrophic lateral sclerosis genetics with Mendelian inheritance. In: Brown Jr., R.H., Swash, M., Pasinelli, P. (Eds.), Amyotrophic Lateral Sclerosis. Informa Healthcare, London, 2006, pp. 187~207
8 Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 1993, 362(6415): 59~62
9 Fodor SP, Read JL, Pirrung MC, et al. Light-directed, spatially addressable parallel chemical synthesis. Science, 1991, 251(4 995): 767~773
10 Weydt P, Hong SY, Kliot M, et al. Assessing disease onset and progression in the SOD1 mouse model of ALS. Neuroreport, 2003. 14(7): 1 051~1 054
11 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
12 Jiang YM, Yamamoto M, Kobayashi Y, et al. Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol, 2005, 57(2): 236~251
13 Perrin FE, Boisset G, Docquier M, et al. No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model. Hum Mol Genet, 2005, 14(21): 3 309~3 320
14 Ferraiuolo L, Heath PR, Holden H, et al. Microarray analysis of the cellular pathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS. J Neurosci, 2007, 27(34): 9 201~9 219
15 Lobsiger CS, Boillée S, Cleveland DW. Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proc Natl Acad Sci USA 2007, 104(18): 7 319~7 326
16 Kirby J, Halligan E, Baptista MJ, et al. Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS. Brain, 2005, 128(Pt 7): 1 686~1 706
17 Vargas MR, Pehar M, Díaz-Amarilla PJ, et al. Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1. J Neurosci Res, 2008, 86(16): 3 515~3 525
18 Olsen MK, Roberds SL, Ellbrock BR, et al. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol, 2001, 50: 730~740
19 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
20 Hensley K, Floyd RA, Gordon B, et al. Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis. J Neurochem, 2002, 82(2): 365~374
21 Malaspina A, Kaushik N, de Belleroche J. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem, 2001, 77: 132~145
22 Ishigaki S, Niwa J, Ando Y, et al. Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords—screening by molecular indexing and subsequent cDNA microarray analysis. FEBS Lett, 2002, 531(2): 354~358
23 Dangond F, Hwang D, Camelo S, et al. Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics, 2004, 16(2): 229~239
24 Offen D, Barhum Y, Melamed E, et al. Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant. J Mol Neurosci, 2009, 38(2): 85~93
25 Wang XS, Simmons Z, Liu W, et al. Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex. Amyotroph Lateral Scler, 2006, 7(4): 201~210
26 Lederer CW, Torrisi A, Pantelidou M, et al. Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis. BMC Genomics, 2007, 8: 26
27 Guo Y, Duan W, Li Z, et al. Decreased GLT-1 and increased SOD1 and HO-1 expression in astrocytes contribute to lumbar spinal cord vulnerability of SOD1-G93A transgenic mice. FEBS Lett, 2010, 584(8): 1 615~1 622
28 Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease—a double-edged sword. Neuron, 2002, 35: 419~432
29 Di Giorgio FP, Carrasco MA, Siao MC, et al. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neuroscience, 2007, 10: 608~614
30 Marchetto MC, Muotri AR, Mu Y, et al. Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell, 2008, 3: 649~657
31 King AE, Dickson TC, Blizzard CA, et al. Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology. Neurobiol Aging, 2009 May 6
32 González-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci, 1999, 22: 219~240
33 Tikka TM, Vartiainen NE, Goldsteins G, et al. Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease. Brain, 2002, 125(Pt 4): 722~731
34 Cleveland DW, Rothstein JD. From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci, 2001, 2: 806~819
35 Yasojima K, Tourtellotte WW, McGeer EG, et al. Marked increase in cyclooxygenase-2 in ALS spinal cord: implications for therapy. Neurology, 2001, 57(6): 952~956
36 Goldknopf IL, Sheta EA, Bryson J, et al. Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson’s disease. Biochem Biophys Res Commun, 2006, 342(4): 1 034~1 039
37 McGeer PL, McGeer EG. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26(4): 459~470
38 Nguyen MD, Julien JP, Rivest S. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci, 2002, 3: 216~227
39 Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol, 2001, 2(4): 285~293
40 Gonzalez NC, Wood JG. Leukocyte-endothelial interactions in environmental hypoxia. Adv Exp Med Biol, 2001, 502: 39~60
41 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
42 Gibson RM, Craig SE, Heenan L, et al. Activation of integrin alpha5beta1 delays apoptosis of Ntera2 neuronal cells. Mol Cell Neurosci, 2005, 28: 588~598
43 Suwan K, Choocheep K, Hatano S, et al. Versican/PG-M assembles hyaluronan into extracellular matrix and inhibits CD44-mediated signaling toward premature senescence in embryonic fibroblasts. J Biol Chem, 2009, 284: 8 596~8 604
44 Wang MJ, Kuo JS, Lee WW, et al. Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages. J Neurochem, 2006, 97: 857~871
45 Wolburg H, Noell S, Wolburg-Buchholz K, et al. Agrin, aquaporin-4, and astrocyte polarity as an important feature of the blood-brain barrier. Neuroscientist, 2009, 15: 180~193
46 Stritt C, Stern S, Harting K, et al. Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression. Nat Neurosci, 2009, 12: 418~427
47 Cayre M, Canoll P, Goldman JE. Cell migration in the normal and pathological postnatal mammalian brain. Prog Neurobiol, 2009, 88: 41~63
1 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
2 Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics, 2006, 7: 325
3 Namshin Kim, Seokmin Shin, Sanghyuk Lee1 ECgene: Genome-based EST clustering and gene modeling for alternative splicing. Genome Research, 2005, 15(4): 566~576
4 Johnson JM, Castle J, Garrett-Engele P, et al. Genomewide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003, 302(5653): 2 141~2 144
5 Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature, 2008, 456(7221): 470~476
6 Pan Q, Shai O, Lee LJ, et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet, 2008, 40(12): 1 413~1 415
7 Novoyatleva T, Tang Y, Rafalska I, et al. Pre-mRNA missplicing as a cause of human disease. Prog Mol Subcell Biol, 2006, 44: 27~46
8 Licatalosi DD, Darnell RB. Splicing regulation in neurologic disease, Neuron, 2006, 52(1): 93~101
9 Zhang YJ, Xu YF, Cook C, et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA, 2009, 106(18): 7 607~7 612
10 Lauriat TL, Richler E, McInnes LA. A quantitative regional expression profile of EAAT2 known and novel splice variants reopens the question of aberrant EAAT2 splicing in disease. Neurochem Int, 2007, 50(1): 271~280
11 Yeo GW, Xu X, Liang TY, et al. Alternative splicing events identified in human embryonic stem cells and neural progenitors. PLoS Comput Biol, 2007, 3(10): 1 951~1 967
12 Thorsen K, S?rensen KD, Brems-Eskildsen AS, et al. Alternative splicing in colon, bladder, and prostate cancer identified by exon-array analysis. Mol Cell Proteomics, 2008, 7(7): 1 214~1 224
13 Hang X, Li P, Li Z, et al. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array. BMC Genomics, 2009, 10: 126
14 Xi L, Feber A, Gupta V, et al. Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer. Nucleic Acids Res, 2008, 36(20): 6 535~6 547
15 McKee AE, Neretti N, Carvalho LE, et al. Exon expression profiling reveals stimulus-mediated exon use in neural cells. Genome Biol, 2007, 8(8): R159
16 Zhang Z, Lotti F, Dittmar K, et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 2008, 133(4): 585~600
17 Zeng C, Berget SM. Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol Cell Biol, 2000, 20: 8 290~8 301
18 Kadener S, Fededa JP, Rosbash M, et al. Regulation of alternative splicing by a transcriptional enhancer through RNA pol II elongation. Proc Natl Acad Sci USA, 2002, 99: 8 185~8 190
19 Kornblihtt AR, de la Mata M, Fededa JP, et al. Multiple links between transcription and splicing. RNA, 2004, 10(10): 1 489~1 498
20 Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics, 2006, 7(1): 325
21 Ip JY, Tong A, Pan Q, et al. Globar analysis of alternative splicing during T-cell activation. RNA, 2007, 13(4): 563~572
22 Wu MH. Endothelial focal adhesions and barrier function. J Physiol, 2005, 569(Pt 2): 359~366
23 Wyss-Coray T, Mucke L. Inflammation in neurodegenerative disease—a double-edged sword. Neuron, 2002, 35: 419~432
24 Di Giorgio FP, Carrasco MA, Siao MC, et al. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neuroscience, 2007, 10: 608~614
25 Marchetto MC, Muotri AR, Mu Y, et al. Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell, 2008, 3: 649~657
26 King AE, Dickson TC, Blizzard CA, et al. Neuron-glia interactions underlie ALS-like axonal cytoskeletal pathology. Neurobiol Aging, 2009 May 6
27 González-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci, 1999, 22: 219~240
28 McGeer PL, McGeer EG. Inflammatory processes in amyotrophic lateral sclerosis. Muscle Nerve, 2002, 26(4): 459~470
29 Suwan K, Choocheep K, Hatano S, et al. Versican/PG-M assembles hyaluronan into extracellular matrix and inhibits CD44-mediated signaling toward premature senescence in embryonic fibroblasts. J Biol Chem, 2009, 284: 8 596~8 604
30 Wang MJ, Kuo JS, Lee WW, et al. Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages. J Neurochem, 2006, 97: 857~871
31 Wolburg H, Noell S, Wolburg-Buchholz K, et al. Agrin, aquaporin-4, and astrocyte polarity as an important feature of the blood-brain barrier. Neuroscientist, 2009, 15: 180~193
32 Stritt C, Stern S, Harting K, et al. Paracrine control of oligodendrocyte differentiation by SRF-directed neuronal gene expression. Nat Neurosci, 2009, 12: 418~427
33 Cayre M, Canoll P, Goldman JE. Cell migration in the normal and pathological postnatal mammalian brain. Prog Neurobiol, 2009, 88: 41~63
34 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
1 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
2 Chiu IM, Phatnani H, Kuligowski M, et al. Activation of innate and humoral immunity in the peripheral nervous system of ALS transgenic mice. Proc Natl Acad Sci U S A, 2009, 106(49): 20 960~20 965
3 Wainwright DA, Xin J, Mesnard NA, et al. Toll-like receptor 2 and facial motoneuron survival after facial nerve axotomy. Neurosci Lett, 2010, 471(1): 10~14
4 Graber DJ, Hickey WF, Harris BT. Progressive changes in microglia and macrophages in spinal cord and peripheral nerve in the transgenic rat model of amyotrophic lateral sclerosis. J Neuroinflammation, 2010, 7: 8
5 Yiangou Y, Facer P, Durrenberger P, et al. COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. BMC Neurol, 2006, 6: 12
6 Henkel JS, Beers DR, Siklós L, et al. The chemokine MCP-1 and the dendritic and myeloid cells it attracts are increased in the mSOD1 mouse model of ALS. Mol Cell Neurosci, 2006 , 31(3): 427~437
7 Ahtoniemi T, Goldsteins G, Keksa-Goldsteine V, et al. Pyrrolidine dithiocarbamate inhibits induction of immunoproteasome and decreases survival in a rat model of amyotrophic lateral sclerosis. Mol Pharmacol, 2007, 71: 30~37
8 Beers DR, Henkel JS, Xiao Q, et al. Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA, 2006, 103: 16 021~16 026
9 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
10 Kriz J, Nguyen MD, Julien JP. Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis, 2002, 10: 268~278
11 Kiaei M, Petri S, Kipiani K, et al. Thalidomide and lenalidomide extend survival in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci, 2006, 26: 2 467~2 473
12 Klivenyi P, Kiaei M, Gardian G, et al. Additive neuroprotective effects ofcreatine and cyclooxygenase 2 inhibitors in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurochem, 2004, 88: 576~582
13 Ignacio S, Moore DH, Smith AP, et al. Effect of neuroprotective drugs on gene expression in G93A/SOD1 mice. Ann N Y Acad Sci, 2005, 1 053: 121~136
14 Beers DR, Henkel JS, Zhao W, et al. CD4 T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS. Proc Natl Acad Sci USA, 2008, 105: 15558~15 563
15 Chiu IM, Chen A, Zheng Y, et al. T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS. Proc Natl Acad Sci USA, 2008, 105: 17 913~17 918
16 Zhang L, Lookene A, Wu G, et al. Calcium triggers folding of lipoprotein lipase into active dimers. J Biol Chem, 2005, 280(52): 42 580~42 591
17 Xie C, Wang ZC, Liu XF, et al. The common biological basis for common complex diseases: evidence from lipoprotein lipase gene. Eur J Hum Genet. 2010V18N1:3~7
18 Baum L, Wiebusch H, Pang CP. Roles for lipoprotein lipase in Alzheimer's disease: an association study. Microsc Res Tech, 2000, 50(4): 291~296
19 Scacchi R, Gambina G, Broggio E, et al. The H+ allele of the lipoprotein lipase (LPL) HindIII intronic polymorphism and the risk for sporadic late-onset Alzheimer’s disease. Neurosci Lett, 2004, 367: 177~180
20 Blain JF, Aumont N, Théroux L, et al. A polymorphism in lipoprotein lipase affects the severity of Alzheimer's disease pathophysiology. Eur J Neurosci, 2006, 24(5): 1 245~1 251
21 Paradis E, Clavel S, Julien P, et al. Lipoprotein lipase and endothelial lipase expression in mouse brain: regional distribution and selective induction following kainic acid-induced lesion and focal cerebral ischemia. Neurobiol Dis, 2004, 15: 312~325
22 Blain JF, Paradis E, Gaudreault SB, et al. A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain. Neurobiol Dis, 2004,15: 510~519
23 Fergani A, Oudart H, Gonzalez De Aguilar JL, et al. Increased peripheral lipid clearance in an animal model of amyotrophic lateral sclerosis. J Lipid Res, 2007, 48(7): 1 571~1 580
24 Dupuis L, Corcia P, Fergani A, et al. Dyslipidemia is a protective factor in amyotrophic lateral sclerosis. Neurology, 2008, 70(13): 1 004~1 009
25 Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol, 2002, 2: 116~126
26 Folkl A, Bienzle D. Structure and function of programmed death (PD) molecules. Vet Immunol Immunopathol, 2010, 134(1-2): 33~38
27 Zhang X, Schwartz JC, Guo X, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20(3): 337~347
28 Riley JL. PD-1 signaling in primary T cells. Immunol Rev, 2009, 229(1): 114~125
29 Lázár-Molnár E, Yan Q, Cao E, et al. Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2. Proc Natl Acad Sci U S A, 2008, 105(30): 10 483~10 488
30 Parry RV, Chemnitz JM, Frauwirth KA, et al. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol, 2005, 25: 9543~9 553
31 Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci USA, 2001, 98: 13 866~13 871
32 Chemnitz JM, Parry RV, Nichols KE, et al. SHP-1 and SHP-2 associate with immunoreceptor tyrosine based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol, 2004, 173: 945~954
33 Ishida Y, Agata Y, Shibahara K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed celldeath. EMBO J, 1992, 11(11): 3 887~3 895
34 Simone R, Piatti G, Saverino D. The inhibitory co-receptors: a way to save from anergy the HIV-specific T cells. Curr HIV Res, 2009, 7(3): 266~272
35 Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med, 2000, 192(7): 1 027~1034
36 Sharpe AH, Wherry EJ, Ahmed R, et al. The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol, 2007, 8(3): 239~245
37 Boussiotis VA, Freeman GJ, Gribben JG, et al. The role of B7-1/B7-2:CD28/CTLA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune response. Immunol Rev, 1996, 153: 5~26
38 Keir ME, Butte MJ, Freeman GJ, et al. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol, 2008, 26: 677~704
39 Lee YH, Woo JH, Choi SJ, et al. Association of programmed cell death 1 polymorphisms and systemic lupus erythematosus: a meta-analysis. Lupus, 2009, 18(1): 9~15
40 Wang L, Han R, Hancock WW. Programmed cell death 1 (PD-1) and its ligand PD-L1 are required for allograft tolerance. Eur J Immunol, 2007, 37(10): 2 983~2 990
41 Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity, 1999, 11: 141~151
42 Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001, 291: 319~322
43 Wang J, Yoshida T, Nakaki F, et al. Establishment of NOD-Pdcd1-/- mice as an efficient animal model of type I diabetes. Proc. Natl Acad Sci USA, 2005, 102: 11 823
44 Nielsen C, Ohm-Laursen L, Barington T, et al. Alternative splice variantsof the human PD-1 gene. Cell Immunol, 2005, 235(2): 109~116
1 Kirby J, Heath PR, Shaw PJ, et al. Gene expression assays. Adv Clin Chem, 2007, 44: 247~292
2 Kelz MB, Dent GW, Therianos S, et al. Single-cell antisense RNAamplification and microarray analysis as a tool for studying neurological degeneration and restoration. Sci Aging Knowledge Environ, 2002, 1: 1~10
3 Ginsberg SD, Che S. RNA amplification in brain tissues. Neurochem Res, 2002, 27(10): 981~992
4 Affymetrix Inc. Identifying and validating alternative splicing events. Affymetrix Technical Note, 2006
5 Shaw PJ. Molecular and cellular pathways of neurodegeneration in motor neurone disease. J. Neurol. Neurosurg. Psychiatry, 2005, 76(8): 1046~1057
6 Ince PG, Lowe J, Shaw PJ. Amyotrophic lateral sclerosis: current issues in classification, pathogenesis and molecular pathology. Neuropathol Appl Neurobiol, 1998, 24: 104~117
7 Bilsland LG, Dick JR, Pryce G, et al. Increasing cannabinoid levels by pharmacological and genetic manipulation delay disease progression in SOD1 mice. FASEB J , 2006, 20: 1 003~1 005
8 Pasinelli P, Brown RH. Molecular biology of amyotrophic lateral sclerosis: insights from genetics. Nat Rev Neurosci, 2006, 7(9): 710~723
9樊东升,张俊,邓敏,等.肌萎缩侧索硬化/运动神经元病的基础与临床研究.北京大学学报(医学版), 2009, 41(3): 279~281
10 Andersen PM. Amyotrophic lateral sclerosis genetics with Mendelian inheritance. In: Brown Jr., R.H., Swash, M., Pasinelli, P. (Eds.), Amyotrophic Lateral Sclerosis. Informa Healthcare, London, 2006, 187~207
11 Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 1993, 362(6 415): 59~62
12 Valdmanis PN, Rouleau GA. Genetics of familial amyotrophic lateral sclerosis. Neurology, 2008, 70: 144~152
13 Simpson CL, Al-Chalabi A. Amyotrophic lateral sclerosis as a complex genetic disease. Biochim Biophys Acta, 2006, 1 762: 973~985
14 Hirschhorn JN, Daly MJ. Genome-wide association studies for commondiseases and complex traits. Nat Rev Genet, 2005, 6(2): 95~108
15 Schymick JC, Scholz SW, Fung HC, et al. Genome-wide genotyping in amyotrophic lateral sclerosis and neurologically normal controls: first stage analysis and public release of data. Lancet Neurol, 2007, 6(4): 322~328
16 Dunckley T, Huentelman MJ, Craig DW, et al. Whole-genome analysis of sporadic amyotrophic lateral sclerosis. N Engl J Med, 2007, 357(8): 775~788
17 van Es MA, Van Vught PW, Blauw HM, et al. ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study. Lancet Neurol, 2007, 6(10): 869~877
18 van Es MA, van Vught PW, Blauw HM, et al. Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nat Genet, 2008, 40(1): 29~31
19 Cronin S, Berger S, Ding J, et al. A genome-wide association study of sporadic ALS in a homogenous Irish population. Hum Mol Genet, 2008, 17(5): 768~774
20 Garber K. Genetics. The elusive ALS genes. Science, 2008, 319(5859): 20
21 Olsen MK, Roberds SL, Ellbrock BR, et al. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol, 2001, 50: 730~740
22 Yoshihara T, Ishigaki S, Yamamoto M, et al. Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis. J Neurochem, 2002, 80: 158~167
23 Hensley K, Floyd RA, Gordon B, et al. Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis. J Neurochem, 2002, 82(2): 365~374
24 Malaspina A, Kaushik N, de Belleroche J. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem, 2001, 77: 132~145
25 Ishigaki S, Niwa J, Ando Y, et al. Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords—screening by molecular indexing and subsequent cDNA microarray analysis. FEBS Lett, 2002, 531(2): 354~358
26 Dangond F, Hwang D, Camelo S, et al. Molecular signature of late-stage human ALS revealed by expression profiling of postmortem spinal cord gray matter. Physiol Genomics, 2004, 16(2): 229~239
27 Offen D, Barhum Y, Melamed E, et al. Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant. J Mol Neurosci, 2009, 38(2): 85~93
28 Wang XS, Simmons Z, Liu W, et al. Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex. Amyotroph Lateral Scler, 2006, 7(4): 201~210
29 Lederer CW, Torrisi A, Pantelidou M, et al. Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis. BMC Genomics, 2007, 8: 26
30 Gonzalez de Aguilar JL, Niederhauser-Wiederkehr C, Halter B, et al. Gene profiling of skeletal muscle in an amyotrophic lateral sclerosis mouse model. Physiol Genomics, 2008, 32(2): 207~218
31 Jiang YM, Yamamoto M, Kobayashi Y, et al. Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis. Ann Neurol, 2005, 57(2): 236~251
32 Jiang YM, Yamamoto M, Tanaka F, et al. Gene expressions specifically detected in motor neurons (dynactin 1, early growth response 3, acetyl-CoA transporter, death receptor 5, and cyclin C) differentially correlate to pathologic markers in sporadic amyotrophic lateral sclerosis. J Neuropathol Exp Neurol, 2007, 66(7): 617~627
33 Perrin FE, Boisset G, Docquier M, et al. No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model. Hum Mol Genet, 2005, 14(21): 3 309~3 320
34 Ferraiuolo L, Heath PR, Holden H, et al. Microarray analysis of the cellularpathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS. J Neurosci, 2007, 27(34): 9 201~9 219
35 Perrin FE, Boisset G, Lathuilière A, et al. Cell death pathways differ in several mouse models with motoneurone disease: analysis of pure motoneurone populations at a presymptomatic age. J Neurochem, 2006, 98(6): 1 959~1 972
36 Lobsiger CS, Boillée S, Cleveland DW. Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proc Natl Acad Sci USA, 2007, 104(18): 7 319~7 326
37 Kirby J, Halligan E, Baptista MJ, et al. Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS. Brain, 2005, 128(Pt 7): 1 686~1 706
38 Vargas MR, Pehar M, Díaz-Amarilla PJ, et al. Transcriptional profile of primary astrocytes expressing ALS-linked mutant SOD1. J Neurosci Res, 2008, 86(16): 3 515~3 525
39 Namshin Kim, Seokmin Shin, Sanghyuk Lee1 ECgene: Genome-based EST clustering and gene modeling for alternative splicing. Genome Research, 2005, 15(4): 566~576
40 Johnson JM, Castle J, Garrett-Engele P, et al. Genomewide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003, 302(5 653): 2141~2144
41 Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature, 2008, 456(7 221): 470~476
42 Pan Q, Shai O, Lee LJ, et al. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet, 2008, 40(12): 1 413~1 415
43 Novoyatleva T, Tang Y, Rafalska I, et al. Pre-mRNA missplicing as a cause of human disease. Prog Mol Subcell Biol, 2006, 44: 27~46
44 Zhang YJ, Xu YF, Cook C, et al. Aberrant cleavage of TDP-43 enhancesaggregation and cellular toxicity. Proc Natl Acad Sci USA, 2009, 106(18): 7 607~7 612
45 Lauriat TL, Richler E, McInnes LA. A quantitative regional expression profile of EAAT2 known and novel splice variants reopens the question of aberrant EAAT2 splicing in disease. Neurochem Int, 2007, 50(1): 271~280
46 Zhang Z, Lotti F, Dittmar K, et al. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 2008, 133(4): 585~600
48 Rabin SJ, Kim JM, Baughn M, et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum Mol Genet, 2010, 19(2): 313~328
49 Chen H, Guo Y, Hu M, et al. Differential expression and alternative splicing of genes in lumbar spinal cord of an amyotrophic lateral sclerosis mouse model. Brain Res, 2010 Mar 31. [Epub ahead of print]
50 Mehler MF, Mattick JS. Non-coding RNAs in the nervous system. J Physiol, 2006, 575(Pt 2): 333~341
51 Williams AH, Valdez G, Moresi V, et al. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science, 2009, 326(5 959): 1 549~1 554
52 Shioya M, Obayashi S, Tabunoki H, et al. Aberrant microRNA expression in the brains of neurodegenerative diseases: miR-29a decreased in Alzheimer disease brains targets neuron navigator-3. Neuropathol Appl Neurobiol, 2010, Feb 25