RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

• Published in: PloS one 2016-08, Vol.11 (8), p.e0160520-e0160520
• Main Authors: Brohawn, David G, O'Brien, Laura C, Bennett, Jr, James P
• Format: Article
• Language: English
• Subjects: Abnormalities; Activation; Adults; Algorithms; Amyotrophic lateral sclerosis; Amyotrophic Lateral Sclerosis - complications; Amyotrophic Lateral Sclerosis - genetics; Amyotrophic Lateral Sclerosis - pathology; Analysis; Autopsy; Bioinformatics; Biology and Life Sciences; Biophysics; Case-Control Studies; Caspase; Caspase-3; Cell death; Chemokines; Computer and Information Sciences; Cytotoxicity; Development and progression; DNA binding proteins; Gene expression; Gene Regulatory Networks; Gene sequencing; Genes; Genome-Wide Association Study; Genomes; Genomics; High-Throughput Nucleotide Sequencing; Humans; Inflammation; Inflammation - genetics; Inflammation - pathology; Lymphocytes T; Macrophages; Medicine and Health Sciences; Molecular biology; Motor neurons; Motor Neurons - metabolism; Motor Neurons - pathology; Mutation; Necrosis; Network analysis; Neurons; NF-κB protein; Pathogenesis; Pathology; Physiology; Proteins; Research and Analysis Methods; Retinoid X receptor α; Ribonucleic acid; RNA; RNA sequencing; rRNA; Sequence Analysis, RNA; Signal Transduction - genetics; Signaling; Spasticity; Spinal cord; Spinal Cord - immunology; Spinal Cord - metabolism; Spinal Cord - pathology; Studies; Transcription (Genetics); Transcription factors; Tumor necrosis factor; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - physiology; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0160520

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 TNFα-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 TNFα signaling may be helpful in ALS patients.