Analysis of the splicing landscape of the frontal cortex in FTLD-TDP reveals subtype specific patterns and cryptic splicing

Abstract

Dysregulation of TDP-43 as seen in TDP-43 proteinopathies leads to specific RNA splicing dysfunction. While discovery studies have explored novel TDP-43-driven splicing events in induced pluripotent stem cell (iPSC)-derived neurons and TDP-43 negative neuronal nuclei, transcriptome-wide investigations in frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP) brains remain unexplored. Such studies hold promise for identifying widespread novel and relevant splicing alterations in FTLD-TDP patient brains. We conducted the largest differential splicing analysis (DSA) using bulk short-read RNAseq data from frontal cortex (FCX) tissue of 127 FTLD-TDP (A, B, C, GRN and C9orf72 carriers) and 22 control subjects (Mayo Clinic Brain Bank), using Leafcutter. In addition, long-read bulk cDNA sequencing data were generated from FCX of 9 FTLD-TDP and 7 controls and human TARDBP wildtype and knock-down iPSC-derived neurons. Publicly available RNAseq data (MayoRNAseq, MSBB and ROSMAP studies) from Alzheimer's disease patients (AD) was also analyzed. Our DSA revealed extensive splicing alterations in FTLD-TDP patients with 1881 differentially spliced events, in 892 unique genes. When evaluating differences between FTLD-TDP subtypes, we found that C9orf72 repeat expansion carriers carried the most splicing alterations after accounting for differences in cell-type proportions. Focusing on cryptic splicing events, we identified STMN2 and ARHGAP32 as genes with the most abundant and differentially expressed cryptic exons between FTLD-TDP patients and controls in the brain, and we uncovered a set of 17 cryptic events consistently observed across studies, highlighting their potential relevance as biomarkers for TDP-43 proteinopathies. We also identified 16 cryptic events shared between FTLD-TDP and AD brains, suggesting potential common splicing dysregulation pathways in neurodegenerative diseases. Overall, this study provides a comprehensive map of splicing alterations in FTLD-TDP brains, revealing subtype-specific differences and identifying promising candidates for biomarker development and potential common pathogenic mechanisms between FTLD-TDP and AD.

Keywords: Frontotemporal dementia; Splicing; TDP-43; Transcriptomics.

Fig. 1

Brain splicing alterations landscape in FTLD-TDP. a Workflow of the differential splicing analysis in bulk short-read sequencing data. Figure created with BioRender.com. b Volcano plot of the differentially spliced events in FTLD-TDP vs controls without adjusting by cell type proportions. In green, events within a significant cluster (FDR < 0.05) and a│Δ PSI│ > 0.1. c. Network of pathways enriched in differentially spliced genes, with the blue module representing pathways involved in dendrite and cell projections, and the pink module with pathways involved in synapse dysfunction. d Relative proportions of the major cell types in the brains of FTLD-TDP and controls. Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e Volcano plot of the differentially spliced events in FTLD-TDP vs controls after adjusting by cell-type proportions. Ast: astrocytes, Mic: microglia, Oli: oligodendrocytes, Neu: neurons, End: endothelial cells

Fig. 2

FTLD-TDP subtypes show distinct splicing profiles. a Upset plot of the differentially spliced clusters (FDR < 0.05) among FTLD-TDP subtypes, without adjusting by differences in cell proportions. b Relative proportions of the major cell types in the FTLD-TDP subtypes and controls. Mann–Whitney U test, adjusted by Bonferroni. c Upset plot of the differentially spliced clusters (FDR < 0.05) among FTLD-TDP subtypes, adjusting by differences in cell proportions. d Protein–protein interaction network between connected genes that are commonly spliced in C9orf72 repeat expansion carriers and FTLD-TDP type C. e. Sashimi plot for NOTCH1 splicing cluster. The numbers represent the PSI of that splice junction f. Violin plot of the PSI of the splicing event in NOTCH1 in controls, C9orf72 repeat expansion carriers and FTLD-TDP type C. Ast: astrocytes, Mic: microglia, Oli: oligodendrocytes, Neu: neurons, End: endothelial cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Cryptic splicing in FTLD-TDP. a Venn diagram representing genes with perfect splicing matches from the three examined studies. The lower panels represent b in-frame exons, c. out-of-frame exons, d terminal exons, e alternative splice sites, f exon skipping, and g initial exons of those splicing events with perfect matches in the present study and any of the other datasets. For each gene, the upper diagram represents the annotated canonical transcript (MANE selected transcript) and the lower diagram represents the cryptic transcript. Sizes of the cryptic exons are representative to the real size of the exon, but size of the annotated exons are equal in all genes. Next to each gene name, the total number of exons of the canonical transcript is shown. The red lines in the cryptic exons represent premature termination codons (PTC), and the green lines represent novel start codons. The star next to the exon name indicates that part of the exon is the same as the canonical one. Figure created with BioRender.com