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

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity that arise 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). Transient expression in these neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and caspase 3/7 activation, signs suggestive of cell death. 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. (255 words) Amyotrophic lateral sclerosis (ALS) is a disease characterized by degeneration of upper and lower motor neurons (MNs) in the brainstem and spinal cord. Average life expectancy after diagnosis is between 2-5 years, and current treatment (riluzole) extends life by only a few months. ~5% of ALS occurs in families (fALS), with 68% of European fALS cases explained by mutations in 9 loci (as of 2014). However, only 11% of European sporadic ALS (sALS) patients are accounted for by mutations in these loci (1). Sporadically occurring ALS (sALS) represents ~95% of all ALS cases (2) and is widely thought to have both polygenic and environmental contributions (3-4). “Agnostic” exploratory association studies have been adopted to elucidate the genetic contributions to sALS (1,5-6). Gene expression studies identify differentially expressed genes (DEG’s), which are genes with statistically significantly changed expression values between groups. Over-representation analyses can be used to infer their likely biological relevance. These analyses detect statistically significant associations between an input list of genes compared to predefined groups of genes known to influence various cellular processes Shaw et al (6) published a comprehensive review of ALS gene expression studies dating back to 2001. Tissue types compared between ALS and control samples included: human bicep, human lymphocytes, rodent gastrocnemius, human or rodent spinal tissue containing disease-vulnerable MNs, and isolated spinal MNs. There was a broad range in the number (14 to 1,182) and identity of ALS group-specific DEG’s discovered in each study. Interestingly, a recurrent set of associated cellular processes emerged across over-representation analyses using these sets of DEGs. These included oxidative stress, mitochondrial dysfunction, apoptosis, cytoskeletal architecture, inflammation, RNA processing, and protein aggregation. Separate molecular biology assays revealed increased oxidative damage (7-11), abnormal mitochondrial morphology (12-18), and elevated inflammation (19-33) in various ALS tissues from human patients and fALS rodents. Increased inflammatory tumor necrosis factor (TNF) signaling (28-33) in ALS tissues may have therapeutic relevance, as it known to carry out cell fate decisions that may contribute to MN death (34). Taken together, it is likely aberrations in these processes contribute to ALS onset, progression, and symptoms. Exploratory gene expression studies comparing disease and control groups allow us to form novel hypotheses about what genes may functionally impact disease pathology. Candidate gene selection for downstream molecular biology experiments is rarely trivial, as these high throughput experiments often yield considerable options. Commonly, researchers select a candidate gene that 1) was identified as a DEG, 2) was in a group of DEG’s associated with cellular processes relevant to disease pathology, and 3) has known structural or functional properties plausibly connecting it to disease features. Systems-level gene co-expression network analyses provide separate criteria for candidate gene selection that can be used in conjunction with DEG analysis results. These analyses typically follow a step-wise process that involves 1) prediction of interconnected gene networks using all samples’ gene expression measurements, 2) identification of networks statistically associated with phenotypic traits (i.e. disease status), 3) over-representation analyses to identify cellular processes associated with these networks, and 4) identification of highly connected “hub” genes predicted to organize each prioritized network’s activity. This approach has identified networks associated with a polygenic trait and plausibly related cellular processes, and several of these networks’ hub genes were already associated with the trait studied using separate molecular biology techniques (35-36). Arguably more compelling, Horvath et al showed reducing expression of ASPM (a hub gene identified in a network associated with glioblastoma and mitosis) via siRNA significantly reduced proliferation rates in glioblastoma tumor cells in vitro (37). In this study, we gathered >50 million 2X150 RNA-Sequencing reads in 15 postmortem spinal section tissues (7 ALS patients and 8 neurologically healthy controls) to characterize transcriptome-wide gene expression differences specific to our ALS group. We aligned reads to the hg19 reference genome using Bowtie2/Tophat2 (38). We next performed three DEG analyses, a weighted gene co-expression network analysis, over-representation analyses of prioritized gene sets, and molecular assays testing a candidate hub gene’s potential to increase motor neuron death. 74 DEGs (56 up-regulated and 18 down-regulated) were identified in common across three analyses (Cuffdiff2, EdgeR, and DeSeq2, 39-41) at an FDR of 0.10. Our gene network analysis (WGCNA, 42) identified 37 different gene networks, and 2 were positively correlated with disease status at a p-value <0.01. QIAGEN’s Ingenuity Pathway Analysis (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity), an over-representation analysis tool, revealed the 56 DEG’s up-regulated in the ALS group and genes comprising the gene network most strongly correlated to disease status were both associated with numerous inflammatory processes. Additionally, tumor necrosis factor (TNF) was identified as an "activated" upstream regulator in both gene groups. We prioritized this gene network for candidate gene selection, as 1) it was associated with disease status and 2) inflammatory processes and TNF signaling were recurrently associated with ALS pathology in previous literature (19-33). To identify “hub” genes, we used three WGCNA metrics calculated for every gene within this network. We identified 12 “hub” genes (out of 495 total network genes) with scores in the top quartile for all three metrics. 9 of these genes were separately identified as up-regulated DEG’s in the ALS group across the three DEG analyses. TNFAIP2, a gene that encodes an intracellular protein of the tumor necrosis factor family, was one of them. Based on these findings and previous literature, we hypothesized TNFAIP2 functionally mediates motor neuron death via TNF signaling in motor neurons. To test this, we compared cell viability and activated caspase 3/7 levels in human iPSC-derived motor neurons that overexpressed TNFAIP2-GFP or GFP for 24 hours. We show motor neurons that overexpressed TNFAIP2-GFP had decreased cell viability and increased activated caspase 3/7 levels compared to those overexpressing GFP alone. Taken together, these results suggest TNF signaling may play an important role in ALS pathology and reducing its activity could be of therapeutic relevance.