Abstract
RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein (VCP/p97) cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in VCP lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying VCP 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 VCP 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 VCP 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 VCP 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 VCP-related ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is an invariably fatal neurological disease in which there is selective and progressive degeneration of motor neurons (MNs)
We have utilized our established and robust differentiation of human induced pluripotent stem cells (iPSCs) into highly enriched and characterized spinal cord MNs, which were positive for choline acetyltransferase (ChAT), SMI-32 and bIII-tubulin (TUJ1) (Supplementary Fig. 2)
Single-cell analysis of the nuclear-to-cytoplasmic ratio of >70,000 neurons revealed a decrease in TDP-43 and SFPQ in VCP mutant human MNs (Fig. 1A, B, D, E), which builds on our recent report of reduced FUS nuclear-to-cytoplasmic ratio.[16]
Summary
Amyotrophic lateral sclerosis (ALS) is an invariably fatal neurological disease in which there is selective and progressive degeneration of motor neurons (MNs). Deregulated RNA metabolism, and in particular RNA binding protein (RBP) subcellular localization and function, play a pivotal role in ALS pathogenesis. Subcellular mislocalization of RBPs is a pathological hallmark of ALS, with TDP-43 mislocalized from the nucleus to the cytoplasm in 97% of ALS cases.[1] More recently, widespread splicing factor proline and glutamine rich (SFPQ) and FUS mislocalization across different ALS models and sporadic ALS post-mortem tissue has been reported.[2,3] Accumulation of RBPs in the cytoplasm likely contributes to the formation of RBP oligomers and fibrillar pathological cytoplasmic inclusions seen in ALS.[4,5] As each RBP can bind to thousands of RNA targets, a disturbance in even one of these proteins potentially has a broad and diverse impact on RNA metabolism.[6]
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