TDP-43 and FUS mislocalization in VCP mutant motor neurons is reversed by pharmacological inhibition of the VCP D2 ATPase domain.

RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein () cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying mutations. We first demonstrate reduced nuclear-to-cytoplasmic ratios of transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS) and splicing factor proline and glutamine rich (SFPQ) in mutant motor neurons. Upon closer analysis, we also find these RBPs are mislocalized to motor neuron neurites themselves. To address the hypothesis that altered function of the D2 ATPase domain of VCP causes RBP mislocalization, we used pharmacological inhibition of this domain in control motor neurons and found this does not recapitulate RBP mislocalization phenotypes. However, D2 domain inhibition in mutant motor neurons was able to robustly reverse mislocalization of both TDP-43 and FUS, in addition to partially relocalizing SFPQ from the neurites. Together these results argue for a gain-of-function of D2 ATPase in mutant human motor neurons driving the mislocalization of TDP-43 and FUS. Our data raise the intriguing possibility of harnessing VCP D2 ATPase inhibitors in the treatment of -related ALS.