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

Abstract

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.

Fig 1. Genes pathogenic when mutated for fALS.

Fig 2

Shown are lists of common DEGs identified as “upregulated” (left) and “downregulated” (right) in the ALS group relative to controls across all three DEG analyses. Fold change values from Cuffdiff2 are listed.

Fig 3. Shown are all 13,301 genes (individual black lines at top) clustered by their topological overlap dissimilarity scores.

The multi-colored panel next to “Dynamic Tree Cut” shows 122 identified modules using the Dynamic Tree Cut algorithm. The second multi-colored panel shows 37 larger modules identified after highly correlated smaller modules were merged together.

Fig 4. Shown are each module eigengene’s correlation values with disease status, gender, and age.

The correlation score is listed above an associated p value.

Fig 5

Select IPA Core Analysis Results are displayed for DEGs (56) identified as upregulated in the ALS group by all three analyses (left) and genes (495) comprising the black module (right).

Fig 6. 6A shows all black module hub genes and which was an upregulated DEG in the ALS group.

6B shows each black module gene’s module membership vs. gene significance scores.

Fig 7

(left, “MTT”) shows absorbance readings reflecting MTT metabolism (a function viable cells perform) On day 21 of differentiation cells were transfected with TNFAIP2-GFP (TNFAIP2) vector or GFP alone vector (EV) and MTT cell viability assay was performed 24 hrs later. Activated caspase 3/7 levels (right, “Casp 3/7 assay”) were measured using fluorescently labeled antibodies. Day 21 motor neurons that overexpressed TNFAIP2-GFP (TNFAIP2) compared to GFP alone (EV) were significantly less viable and have significantly more caspase 3/7 staining 24 hours after introduction of the TNFAIP2 vector. Statistical analyses were carried out using unpaired t-tests and Prism® software.