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Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway

Abstract

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis–fused in sarcoma (ALS–FUS). Expression of ALS–FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS–Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.

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Fig. 1: The Ran gradient is perturbed in motor neurons expressing ALS-linked FUS.
Fig. 2: Diminished nuclear pore signal and NCT in ALS–FUS human neurons.
Fig. 3: WT and mutant FUS interact with NUP62 in iPSCs.
Fig. 4: NUP62 and FUS alter the phase-separation properties of each other in a manner that is modulated by RNA in vitro.
Fig. 5: Mutant FUS interacts with F/G Nup in the cytoplasm of human neurons.
Fig. 6: Nuclear abnormalities associated with ALS-linked FUS are rescued by knockdown of Nup62 in vivo.
Fig. 7: Modulation of Nup expression rescues FUS-induced toxicity in vivo.

Data availability

No datasets that require mandatory deposition into a public database were generated during the current study. Any data generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The code used for the FRAP analysis of recombinant proteins is provided in the Supplementary Software (Supplemental_Script_FRAP.py).

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Acknowledgements

We thank C. Fallini and S. Parekh for helpful discussion on the manuscript. We are thankful to the following investigators and facilities for providing iPSC lines: R. Baloh and the Induced Pluripotent Stem Cell Core at Cedars Sinai; K. Eggan (Harvard Medical School); F.-B. Gao (University of Massachusetts Medical School); N. Maragakis and the Ansari ALS Stem Cell Core at Johns Hopkins; L. Van Den Bosch (Vlaams Instituut voor Biotechnologie); and Target ALS and Rutgers University-based core facility (RUCDR). We gratefully acknowledge the contribution of the Cellular Engineering Service at The Jackson Laboratory for expert assistance in the gene editing of the iPSCs described in this manuscript. We thank J. McKeon for assistance with cell culture, A. Murthy for advice on the recombinant MBP–FUS protein, and M. Hammer for help with microscopy. We are grateful to the US National Institutes of Health (NIH), National Science Foundation (NSF) and other funding sources as follows to D.A.B: NIH R01 NS078145, R01 NS108769 (to D.A.B. and D.G.) and R21 NS091860; to U.B.P: NIH R01 NS081303, R21 NS101661, NS111768, AG064940 and NS100055, the Muscular Dystrophy Association, the ALS Association, the Robert Packard Center for ALS at Johns Hopkins; to N.L.F.: NIH R01 GM118530, the Human Frontier Science Program RGP0045/2018, NSF 1845734; to D.G.: NIH U01 DA047733, NIH GM123541, NSF 1917206; to E.B.L.: NIH P30 AG072979 and P01 AG066597; and to J.E.L.: NIH R01 NS073873 and R56 NS073873.

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Y.-C.L., M.S.K., N.R., U.B.P. and D.A.B. conceptualized and designed the project. Y.-C.L., M.S.K., N.R., E.N.A., A.T.N., B.K., S.C., J.A.M. and W.C.S. conducted the experiments. Y.-C.L., M.S.K., N.R., E.N.A., A.T.N., B.K., S.C., J.A.M., W.C.S., I.R.A.M., E.B.L., J.A.N., D.G., U.B.P. and D.A.B. analyzed the data. J.E.L. and N.L.F. provided critical intellectual input. R.L.-G. reprogrammed the control iPSC line designated as C1. D.A.B. wrote the paper with the co-first authors and contributions from all co-authors.

Corresponding author

Correspondence to Daryl A. Bosco.

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Peer review information Nature Neuroscience thanks Ludo Van Den Bosch, Liesbeth Veenhoff, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Figures 1–19, Supplementary Table 1, Supplementary References.

Reporting Summary

Supplementary Software

A python script to normalize in vitro FRAP intensities

Supplementary Data 1

Unprocessed western blots for Supplementary Fig. 4

Supplementary Data 2

Unprocessed western blots for Supplementary Fig. 6

Supplementary Data 3

Unprocessed western blots and gels for Supplementary Fig. 7

Supplementary Video 1

Video of time lapse fluorescence images of the FRAP experiment on MBP-FUS droplets

Supplementary Video 2

Video of time lapse fluorescence images of the FRAP experiment on MBP-FUS/Nup62 assemblies

Source data

Source Data Fig. 1

Unprocessed western blots for Fig. 1

Source Data Fig. 3

Unprocessed western blots for Fig. 3

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Lin, YC., Kumar, M.S., Ramesh, N. et al. Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway. Nat Neurosci (2021). https://doi.org/10.1038/s41593-021-00859-9

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