Mislocalization of FTD3-Associated Mutant CHMP2B to the Nucleus of Human Neurons Due to Loss of a Nuclear Export Signal

Frontotemporal dementia linked to chromosome 3 (FTD3) is caused by a splice site point mutation in CHMP2B, resulting in the production of mutant proteins CHMP2BIntron5 and CHMP2B?10. Here, we found that wildtype CHMP2B (CHMP2BWT) is mostly present in the cytoplasm, but CHMP2BIntron5 is mislocalized to the nucleus of human induced pluripotent stem cell (iPSC)-derived cortical neurons. To understand the underlying mechanism, we identified a previously unreported nuclear export signal (NES) in the C-terminus of CHMP2B. Functional assays, including CRM1 inhibition and site-directed mutagenesis of key hydrophobic residues, demonstrated that this NES motif is both necessary and sufficient for nuclear export of CHMP2BWT and ALS-associated CHMP2BQ206H, and its loss in CHMP2BIntron5 is responsible for the observed nuclear mislocalization. CHMP2B?10 remains in the cytoplasm due to the presence of an artificial NES in the C-terminus. These results reveal the presence of an NES in CHMP2B and highlight the need to dissect the gain-of-toxic nuclear functions of CHMP2BIntron5 in FTD3 pathogenesis. Overall design: ATAC-seq was performed on 3-month-old iPSC-derived cortical neurons that were either wild type at the CHMP2B locus, heterozygous for the CHMP2B G26403C mutation, or homozygous for this mutation. Two different parental wild-type iPSC lines were used to generate the mutant lines. For one parental wild-type line, only a heterozygous mutant line was generated. For the second parental wild-type line, both heterozygous and homozygous mutant lines were generated.