Fig. 7

WGCNA of 3xTg-AD somatomotor cortex transcriptomes identifies modules of co-expressed genes associated with injury, GO biological processes, inflammatory protein expression, brain cell types, and AD genetic risk factors. A) WGCNA identified 14 modules of co-expressed genes. B) Expression scores for the 14 module eigengenes (MEs, the first principal component of each module) for each sample. MEs are clustered by Euclidean distance (columns) and expression scores are displayed by increasing injury number and time point from bottom to top of the heatmap (rows). C) Line plots show changing ME expression scores over increasing numbers of injuries. Time points for 30 min, 4 h, and 24 h after 1x, 3x, and 5xCHI are combined due to low sample sizes (n = 2–4). ME1 (turquoise) and ME2 (blue) show opposite behavior after 1xCHI, with ME1 significantly increasing (p = 0.047, permutation test) alongside trending decreases (p = 0.054) in ME2 after a single injury. ME 9 (magenta) shows significant increases after successive injuries that track closely with ME7 and 8 (black and pink) (p = 0.0015 after 3xCHI, p = 0.023 after 5xCHI; permutation tests compared to sham-injured; see Methods) (mean ± SEM). D) Gene ontology enrichment of biological processes was conducted for each module (Fisher’s exact test with FDR-corrected p-value < 0.05). ME3 and ME6 had fewer than two significant GO sets. Sets marked with asterisks had FDR-corrected p > 0.05 but uncorrected p < 0.0001. E) Protein expression data from the frontal cortex of the same 3xTg-AD animals were correlated against each ME. Selected proteins showed a correlation p-value of at least 0.01 against at least one ME. Red indicates a positive Pearson’s correlation coefficient (R) while blue indicates negative correlation. F) Cell type enrichment analysis showed significant enrichment of neuronal genes in ME2 (blue), oligodendrocyte genes in ME4 (yellow), endothelia genes in ME7 (black), astrocyte genes in ME8 (pink), and microglial genes in ME9 (magenta). G) MAGMA enrichment for GWAS AD genes showed significant over-representation within ME1 (turquoise), ME9 (magenta), and ME14 (cyan). The dotted red line indicates an enrichment z-score of 1.96, above which a module is considered significantly enriched (normal distribution, p < 0.05)