Unmasking cryptic changes to RNA targets in ALS and FTD

Abstract

A hallmark pathological feature of ALS and FTD is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. We have been systematically identifying cryptic splicing targets regulated by TDP-43 in human brain. We discovered that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we found that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function. We are currently exploring the function of UNC13A in ALS/FTD and characterizing several other novel cryptic splicing targets. Some of these represent powerful biomarkers and other ones might be therapeutic targets. We are also using genome wide approaches to identify genes that work with TDP-43 to regulate cryptic splicing. Many cryptic splicing events caused by TDP-43 loss lead to cryptic transcripts degraded by the RNA surveillance mechanism nonsense-mediated decay (NMD). Standard RNA-sequencing approaches miss many of these and we recently found a way to unmask them (by inhibiting NMD).  In addition to cryptic splicing, we have also discovered loss of TDP-43 in FTD/ALS leads to widespread alternative polyadenylation changes, impacting expression of disease-relevant genes and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.