Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalisation in ALS motor neurons and is restored by VCP inhibition

ABSTRACT: Although the pathological hallmark of amyotrophic lateral sclerosis (ALS) is the nucleocytoplasmic mislocalisation of RNA binding proteins (RBPs), such as TDP-43 and FUS, the nucleocytoplasmic distribution of mRNA remains uncharacterised. Here, we used subcellular fractionation with RNA sequencing and proteomics to assess nucleocytoplasmic mRNA and protein localisation in human induced pluripotent stem cell-derived motor neurons (iPSNs) from ALS patients with TARDBP mutations, VCP mutations, and controls with VCP mutations knocked-in with CRISPR/Cas9. In each mutant group, we found substantial nucleocytoplasmic mRNA redistribution, particularly in transcripts involved in protein binding. Redistributed transcripts in ALS iPSNs were enriched in protein-coding biotypes, exhibited longer lengths, and had enhanced interactions with RBPs, including TDP-43 and FUS. Using mass spectrometry in TARDBP mutant, VCP mutant, and VCP mutant knock-in iPSNs, we reveal widespread protein mislocalisation, especially in RBPs. We find that mislocalised proteins exhibit substantially greater binding to redistributed transcripts than non-redistributed mRNAs, suggesting that RBP mislocalisation may be directly coupled to mRNA redistribution. Remarkably, treatment with the VCP D2 ATPase domain inhibitor ML240 restored both nucleocytoplasmic mRNA redistribution and protein mislocalisation not only in VCP mutants but also in TARDBP mutant iPSNs. Functional assays in VCP mutant iPSNs further confirmed that ML240 restores lysosomal localisation from the perinuclear region towards the neurites and reduces oxidative stress. Our findings demonstrate that ALS motor neurons exhibit concomitant nucleocytoplasmic mRNA redistribution and RBP

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Stem Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Mark Skehel  

LAB HEAD: Rickie Patani

PROVIDER: PXD037107 | Pride | 2023-10-13

REPOSITORIES: Pride