A panel of TDP-43-regulated splicing events verify loss of TDP-43 function in amyotrophic lateral sclerosis brain tissue

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and exon changes are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database () together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a ‘differential exon usage panel’ with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons and changed exon usage discriminate ALS brain tissue from controls, verifying TDP-43 loss of function as a pathomechanism in ALS. We propose that TDP-43-regulated splicing markers with most predictive value for therapeutic intervention will be those based on splicing events that occur both in tissues/biofluids amenable to sampling, and in brain tissue susceptible to disease. ### Competing Interest Statement The authors have declared no competing interest. * ALS : Amyotrophic lateral sclerosis BBB : Blood-brain barrier BSCB : Blood-spinal cord barrier cDNA : Complementary DNA Ct : Cycle number threshold DEU : Differential exon usage FTD : Frontotemporal dementia LOF : Loss of function log2FC : Log2 fold change MC : Motor cortex mRNA : Messenger RNA MTG : Middle temporal gyrus PBS : Phosphate-buffered saline PCA : Principal component analysis qPCR : Quantitative polymerase chain reaction RIN : RNA integrity number RRM : RNA recognition motif Scr : Scrambled siRNA SD : Standard deviation siRNA : Small interfering RNA siTDP : siRNA against TARDBP SNP : Single nucleotide polymorphism TDP-43 : TAR DNA binding protein of 43 kDa UTR : Untranslated region