RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord
RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord
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DOI：
10.1371/journal.pone.0160520
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发表时间：
2016-08-03
期刊：
影响因子：
3.7
通讯作者：
Bennett, James P., Jr.
中科院分区：
文献类型：
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作者：
Brohawn, David G.;O'Brien, Laura C.;Bennett, James P., Jr.
肌萎缩侧索硬化症是一种进展迅速、破坏性严重的成年人神经退行性疾病，由于上下运动神经元的死亡而导致残疾性虚弱和痉挛。目前还没有有意义的治疗方法来减缓ALS的进展，迫切需要对发病机制和进展的分子见解。在这种情况下，我们使用高深度的下一代RNA测序（RNAseq，Illumina）来定义从7个ALS和8个CTL样品的颈脊髓切片中分离的缺失rRNA的RNA样品中的基因网络异常。我们将> 5000万个2X 150 bp配对末端序列/样品与hg 19人类基因组进行比对，并应用三种不同的算法（Cuffdiff 2、DEseq 2、EdgeR）来鉴定差异表达基因（DEG）。免疫途径分析（IPA）和加权基因共表达网络分析（WGCNA）鉴定出在我们的ALS样品中炎症过程显著升高，发现肿瘤坏死因子（TNF）是主要途径调节因子（IPA），并且TNF α诱导蛋白2（TNFAIP 2）是主要网络“枢纽”基因（WGCNA）。使用oPOSSUM算法，我们分析了ALS样品中控制9个DEG/hub基因表达的转录因子（TF），并鉴定了参与炎症（NFkB、REL、NFkB 1）和巨噬细胞功能（NR 1H 2：：RXRA异二聚体）的TF。TNFAIP 2（也是通过所有三种算法鉴定的DEG）在人iPSC衍生的运动神经元中的瞬时表达降低了细胞活力并诱导半胱天冬酶3/7活化。使用高密度RNAseq、DEG鉴定的多种算法和无监督基因共表达网络方法，我们鉴定了ALS脊髓中炎症过程的显著升高，其中TNF作为主要调节分子。DEG TNFAIP 2在人运动神经元中的过表达，最容易在ALS中死亡的群体，增加了细胞死亡和半胱天冬酶3/7激活。我们认为，针对减少炎症性TNF α信号传导的治疗可能对ALS患者有帮助。
ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned > 50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNF alpha-induced protein 2 (TNFAIP2) as a major network "hub" gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF's involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNF alpha signaling may be helpful in ALS patients.