Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS

Abstract

Increasing evidence suggests that defective RNA processing contributes to the development of amyotrophic lateral sclerosis (ALS). This may be especially true for ALS caused by a repeat expansion in C9orf72 (c9ALS), in which the accumulation of RNA foci and dipeptide-repeat proteins are expected to modify RNA metabolism. We report extensive alternative splicing (AS) and alternative polyadenylation (APA) defects in the cerebellum of c9ALS subjects (8,224 AS and 1,437 APA), including changes in ALS-associated genes (for example, ATXN2 and FUS), and in subjects with sporadic ALS (sALS; 2,229 AS and 716 APA). Furthermore, heterogeneous nuclear ribonucleoprotein H (hnRNPH) and other RNA-binding proteins are predicted to be potential regulators of cassette exon AS events in both c9ALS and sALS. Co-expression and gene-association network analyses of gene expression and AS data revealed divergent pathways associated with c9ALS and sALS.

Figure 1

Differential regulation of gene expression in c9ALS and sALS. (a–d) MA plots showing up- and down-regulated gene expression (P < 0.05, |Log₂FC| ≥ 1) in c9ALS (a,b) and sALS (c,d) vs. controls in cerebellum (a,c) and frontal cortex (b, d). (e,f) Venn diagrams and graphs depicting the number up- and down-regulated transcripts (P < 0.05, |Log₂FC| ≥ 2,) that are unique or common to c9ALS and sALS cerebellum (e) or frontal cortex (f). The black section in graph (e) shows a DE gene common to both c9ALS and sALS, but with the expression change going in opposite directions between both forms of ALS. (g,h) Hierarchical clustering representation of c9ALS DE genes (P < 0.05, |Log₂FC| ≥ 2) in cerebellum (g) and frontal cortex (h). Each row of the heat maps corresponds to either a control, sALS or c9ALS case, as designated by the color-coded bar on the left. A legend is provided below the heat maps.

Figure 2

Weighted gene co-expression correlation network analyses (WGCNA) of top identified modules in cerebellum and frontal cortex of c9ALS. (a,b) Shown on the left side of panels are heat maps of genes and bar plots for eigengene values of top c9ALS cerebellum (MEpink, shown in blue) and frontal cortex (MEsalmon, shown in red) WGCNA co-expression modules. Network diagrams for these two modules are shown (right), with node color darkness proportional to the number of connections (degree). Note that eight genes within the top co-expression module in c9ALS frontal cortex were differentially expressed (those in red font as well as BAG3). The increase in expression for seven of the genes (those in red font) was validated by qRT-PCR in cerebellum and frontal cortex tissues (see Supplementary Fig. 4).

Figure 3

Widespread alternative splicing defects are found in c9ALS and sALS. (a) Venn diagrams depicting the number of unique and common total AS events in c9ALS and sALS cerebellum (left) and frontal cortex (right) (FDR < 0.05). (b) Pie charts showing the percentage (%) of different types of AS events in cerebellum and frontal cortex of c9ALS (top) and sALS (bottom) (FDR < 0.05). A color-coded legend of the different AS events is provided below the pie charts.

Figure 4

Extensive misregulation of cassette exon splicing occurs in the c9ALS cerebellum. (a, b) Venn diagrams showing the number of unique and common AS cassette exon events in c9ALS and sALS cerebellum (a) and frontal cortex (b) (FDR < 0.05). (c–f) Scatter plots showing exclusion (blue) and inclusion (red) of AS cassette exons in c9ALS (c,d) and sALS (e,f) in comparison to controls (FDR < 0.05, |dI| ≥ 0.1) in cerebellum (c,e) or frontal cortex (d,f). dl: differential index value. Exon inclusion indices (I) are plotted for control group (x-axis) vs. c9ALS (c,d, y-axis) or vs. sALS (e,f, y-axis).

Figure 5

Misregulation of cassette exon splicing in c9ALS affects transcripts with roles in diverse molecular pathways. (a) Wiggle plots from RNAseq data and qRT-PCR bar graphs (mean ± s.e.m.) of top differentially misregulated cassette exons (FDR < 0.05, |dI| ≥ 0.1) in c9ALS cerebellum (see additional examples in Supplementary Fig. 8). Shown is an example of the 3 technical qRT-PCR replicates performed for each AS event in c9ALS (N=9) and sALS (N=10) and compared to controls (N=9). (b) qRT-PCR bar graphs of the same differentially misregulated cassette exons shown in a in the cerebellum of c9ALS (N=9) and sALS (N=10) compared to a disease control group (PSP: progressive supranuclear palsy, N=13). Relative mRNA levels shown in all bar graphs were normalized to the endogenous control, RPLP0, and respective controls (mean value set to 1). The full list of primers used in this study can be found in Supplementary Table 11.Statistical differences were calculated by one-way ANOVA with Bonferroni post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.005, ^#P < 0.001). Exact P values can be found in Supplementary Fig. 8. (c) Gene-association network of the top most significant misregulated cassette exon events in c9ALS cerebellum (FDR < 0.05, |dI| ≥ 0.1). dl: differential index value. Genes are represented by nodes of different colors, which vary according to degree. The size of the node denotes neighborhood connectivity: nodes are bigger if they are connected to other nodes with higher connectivity. Edges are colored according to edge betweenness to indicate the proximity to other nodes, with low betweeness (closer proximity) meaning larger influence to other nodes. GO annotations for different interconnected cellular pathways are indicated. A more complete figure containing all gene names for each of the nodes can be found in Supplementary Figure 11.

Figure 6

Alternative polyadenylation changes are prominent in cerebellum of c9ALS and sALS cases. (a,b) Scatter plots representing differential PAS usage index (PDUI) in cerebellum of c9ALS (a) or sALS (b) compared to controls. PAS shifts are color coded to indicate significant distal (green) and proximal (purple) PAS usage (FDR < 0.05, |ΔPDUI| ≥ 0.2, |dPDUI| ≥ 0.2). (c,d) Wiggle plots of RNAseq cerebellum data for calcium/calmodulin-dependent protein kinase 1D (CAMK1D), which shows increased proximal and decreased distal PAS usage in c9ALS (c), and for SH3 and multiple ankyrin repeat domains 2 (SHANK2), in which a decreased usage of distal PAS is observed in sALS (d). (e,f) Scatter plots representing differential PAS usage index (PDUI) in frontal cortex of c9ALS (e) or sALS (f) compared to controls. PAS shifts are color coded to indicate significant distal (green) and proximal (purple) PAS usage (FDR < 0.05, |ΔPDUI| ≥ 0.2, |dPDUI| ≥ 0.2).