TDP-43 represses cryptic exon inclusion in the FTD\u2013ALS gene UNC13A

X Rosa ,David W Wyatt,Sarah Pickles,Eric M Green,Björn Oskarsson,Shila Mekhoubad,Caitlin M Rodriguez,Tetsuya Akiyama,Fred Harbinski,James Shorter,David S Knopman,William W Seeley,G Miller ,Casey Cook ,Jack D Rubien,Beryl B Cummings,Yasuhiro Koike ,Laura R Ganser ,Neill R Graff‐Radford ,Hermann Broder Schmidt ,Katherine Kurylo,Adam Briner,Nathan A Sallee ,Mercedes Prudencio,Karen Jansen-West,Garam Kım ,Bradley F Boeve,Ronald C Petersen,Caiwei Guo,Leonard Petrucelli,Sua Myong,Gemechu Mekonnen,Dennis W Dickson,Sarat C Vatsavayai,Aaron D Gitler

doi:10.1038/s41586-022-04424-7

Abstract

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2–4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show 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 motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show 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 and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Highlights

TDP-43, encoded by the TARDBP gene, is an abundant, ubiquitously expressed RNA-binding protein that normally localizes to the nucleus
To discover cryptic splicing targets regulated by TDP-43 that may have a role in disease pathogenesis, we used a recently generated RNA sequencing (RNA-seq) dataset[12]
TDP-43 is known as TARDBP. d, e, g, h, Immunoblotting for UNC13A and TDP-43 protein levels in SH-SY5Y cells (d; quantified in e) and iPSC-MNs (g; quantified in h) treated with scramble or TDP-43 short hairpin RNA (shRNA) (n = 3 independent cell culture experiments for each condition)

Summary

FTD or FTD–ALS neocortices

To compare the transcriptomic profiles of TDP-43-positive and TDP-43-negative neuronal nuclei. Expression of full-length TDP-43 rescued the splicing defects, whereas an RNA binding-deficient mutant did not (Extended Data Fig. 9) Together, these results provide direct functional evidence that TDP-43 regulates splicing of UNC13A intron 20–21 and that genetic variants associated with ALS and FTD susceptibility in humans potentiate cryptic exon inclusion when TDP-43 is dysfunctional. Future studies will be required to explore how TDP-43 regulates the cryptic splicing of UNC13A and other splicing targets and the effect of different genetic variations on TDP-43 binding in vivo To examine whether these SNPs affect survival in patients with FTLD-TDP, we evaluated the association of the risk haplotype with survival time after disease onset using data from the Mayo Clinic Brain Bank (n = 205). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Methods

Findings

Code availability