G₄C₂repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

Abstract

Nucleocytoplasmic transport, controlled by the nuclear pore complex, has recently emerged as a pathomechanism underlying neurodegenerative diseases including C9orf72 ALS/FTD. However, little is known about the underlying molecular events and the underlying biology in human neurons. Using super resolution structured illumination microscopy of twenty three nucleoporins in nuclei from C9orf72 iPSC derived neurons and postmortem human tissue we identify a unique subset of eight nucleoporins lost from human neuronal nuclei. POM121, an integral transmembrane nucleoporin, appears to coordinate the composition of the nucleoporins within human neuronal nuclei ultimately impacting nucleocytoplasmic transport, and subsequent cellular toxicity in C9orf72 iPSNs. These data suggest that POM121 is a critical nucleoporin in the maintenance of the nuclear localization of specific nucleoporins in human neurons. Moreover, loss of nuclear POM121, as a result of expanded C9orf72 ALS/FTD repeat RNA, initiates a pathological cascade affecting nucleoporin composition within neuronal nuclei, nuclear pore complex function, and overall downstream neuronal survival.

An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

“…Nuclear preparations and SIM are required to identify changes in nuclear pore proteins; these are not readily observed through proteomic analysis. In comparison controls, we observed a significant reduction in the nuclear expression and localization of Nup98 in C9orf72 iMNs ( Figure 1e ) similar to previous pathological observations in iPSNs and postmortem tissue 26 .…”

“…4 ). We used a modified motor neuron differentiation protocol termed direct iPSC-derived Motor Neurons 18 d diMNs 26, 58 ( Supplementary Fig. 2 ) comprised of three main stages to allow us to investigate the robustness of the original findings.…”

“…Impaired nucleocytoplasmic transport and alterations in the expression and localization of specific nucleoporins which make up the nuclear pore complex to govern functional nucleocytoplasmic transport have emerged as a prominent pathologic hallmark of multiple neurodegenerative diseases including C9orf72 ALS/FTD. To verify that nucleoporin components are altered, we performed super resolution structured illumination microscopy (SIM) on nuclei isolated from control and C9orf72 iMNs immunostained for Nup98 as previously described 26 . Nuclear preparations and SIM are required to identify changes in nuclear pore proteins; these are not readily observed through proteomic analysis.…”

“…4). We used a modified motor neuron differentiation protocol termed direct iPSC-derived Motor Neurons 18 d diMNs 26,58 ( Supplementary Fig. 2 Supplementary Fig.…”

An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

“…We next asked whether the data integration could be validated using a replication cohort and tested our results using a different cellular model and with six patients with C9ORF72 and seven controls, none of which were in common with the original set of samples (Figure S4). To investigate the robustness of the original findings, we used a modified motor neuron differentiation protocol termed direct iPSC-derived Motor Neurons diMNs at 18 days of differentiation (Coyne et al, 2020a;Sances et al, 2016;Ho et al, 2020) (Figure S2) that comprised three main stages. In stage 1, neural induction and hindbrain specification of iPSCs is achieved by inhibition of dual SMAD and GSK3b pathways.…”