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Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro

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Abstract

FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.

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Acknowledgments

This work was supported by research grants from the Japan Society for the Promotion of Science (#25461267) and Japan ALS Association (JALSA).

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Authors and Affiliations

  1. Department of Neurobiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Sakiko Fujii, Keisuke Takanashi, Keiko Kitajo & Atsushi Yamaguchi

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  1. Sakiko Fujii
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  2. Keisuke Takanashi
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  3. Keiko Kitajo
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  4. Atsushi Yamaguchi
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Correspondence to Atsushi Yamaguchi.

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Electronic supplementary material

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11064_2015_1758_MOESM1_ESM.mp4

HEK293 cells, transfected with GFP-FUS P525L plasmid, were treated with 60 μM AdOx for 24 h initiating at 12 h after transfection. Then cells were fixed, stained with DAPI, and observed under confocal laser scanning microscopy (Olympus FV10i). 3D animation of the nucleus was reconstructed using Olympus FV10i software (MP4 675 kb)

Supplementary Fig. 1

(A) HEK293 or SH-SY5Y cells, transfected with plasmid encoding HA-tagged FUS wt, FUS R521G, FUS P525L, or FUS dC, were fixed at 24 after transfection. Cells were immunostained with anti-HA antibody, and then incubated with DAPI. Images were obtained using fluorescence microscopy. Bars, 5 μm. (B) HEK293 cells, transfected with plasmids encoding GFP-FUS wt or each mutant, were lysed for Western blot analyses with anti-FUS and -Actin antibody at 24 h after transfection (upper panels). HEK293 cells, transfected with plasmid encoding GFP-FUS P525L, were lysed for Western blot analyses with anti-FUS and –Actin antibody at the indicated time points (lower panels). The bar graph indicates the relative fold of the band intensity for the ratio of ectopic GFP-FUS/endogenous FUS protein (n = 3, mean ± SE). (C) HEK293 cells, transiently transfected with GFP-FUS wt or HA-FUS wt plasmid, were treated with 60 μM AdOx for 24 h and then fixed with DAPI. Bars, 2 μm. Arrows indicate the intranuclear aggregates (PDF 506 kb)

Supplementary Fig. 2

(A) (B) HEK293 cells, transiently transfected with GFP-control (A) or HA-tagged FUS P525L (B), were treated with (+) or without (−) 20 μM AdOx for 24 h initiating at 12 h after transfection. Cells were fixed and then stained with DAPI. Images were obtained using fluorescence microscopy. Bars, 5 μm. (C) HEK293 and SH-SY5Y cells, transiently transfected with GFP-FUS P525L plasmid, were treated with AMI-1(0, 100, 400 μM) for 24 h initiating at 12 h after transfection. Cells were fixed and then stained with DAPI. Bars, 5 μm. (D) HEK293 cells were treated with Mock, 5 or 20 μM AdOx, or 800 μM MTA for 24 h (upper panel). HEK293 cells were also treated with Mock, 20 μM AdOx or 400 μM AMI-1for 24 h (lower panel). Then FUS proteins, obtained by the immunoprecipitation with anti-FUS antibody in lysates of HEK293 cells, were then resolved by SDS-PAGE. The asymmetric arginine-methylation levels were measured by the immunoblot with anti-dimethyl-Arginine, asymmetric (ASYM24) Antibody (Millipore, #07-414). IgG HC, IgG heavy chain. “Relative ratio” means the relative levels of methylation compared with those of Mock sample (value = 1) (PDF 276 kb)

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Fujii, S., Takanashi, K., Kitajo, K. et al. Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro. Neurochem Res 41, 826–835 (2016). https://doi.org/10.1007/s11064-015-1758-z

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  • DOI: https://doi.org/10.1007/s11064-015-1758-z

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