No, TDP-43 and FUS Are Not Actively Exported From the Nucleus

In amyotrophic lateral sclerosis and frontotemporal dementia, the nuclear RNA-binding proteins TDP-43 and FUS mislocalize to cytoplasm, where they aggregate and contribute to pathology. Because both proteins contain putative nuclear export signals (NES) for the receptor exportin-1, some current therapeutic strategies focus on inhibiting this receptor in order to retain the proteins in the nucleus. However, new evidence casts doubt upon this strategy. In back-to-back papers published in the May 4 Scientific Reports, researchers led by Dorothee Dormann at Ludwig-Maximilians University, Munich, and Philip Thomas at the University of Texas Southwestern Medical Center, Dallas, independently reported that the NES in TDP-43 does not work (Ederle et al., 2018; Pinarbasi et al., 2018). Instead of active export, TDP-43 appears to exit the nucleus via passive diffusion. Dormann’s group obtained similar data for FUS. These papers follow one in the March 15 Scientific Reports from Sami Barmada and colleagues at the University of Michigan, Ann Arbor, reporting that exportin-1 blockers had no effect on TDP-43 localization (Archbold et al., 2018).

Passive aggressive? Nuclear export inhibitors (NEIs) are being developed for ALS in hopes to reduce cytoplasmic mislocalization of FUS and TDP-43. However, new evidence casts doubt upon this strategy by suggesting these RNA-binding proteins exit the nucleus by passive diffusion. [Courtesy of Sun et al., 2016 under CC BY 4.0 license.]

“This series of manuscripts … shows convincingly that nuclear export of TDP-43 and FUS are not as simple as they seem,” Barmada wrote to Alzforum. He thinks the data complicate the rational design of therapies aimed at preventing nuclear export of TDP-43. Au contraire, Wilfried Rossoll, Mayo Clinic, Jacksonville, believes targeting exportin-1 is still a viable strategy (see April 2018 news). “It appears likely that these drugs address a general defect in nuclear protein import by inhibiting nuclear export, thus restoring a balance between these processes. I don’t think that correcting FUS and TDP-43 localization has been considered the most likely mechanism for the observed therapeutic effect in ALS disease models,” he wrote.

Some groups, such as Karyopharm Therapeutics in Newton, Massachusetts, are developing exportin-1 inhibitors to treat ALS and FTD. In preclinical models, KPT-276, KPT-335, and KPT-350 modestly protected against degeneration and cell death caused by TDP-43 overexpression and C9ORF72 repeats, which lead to TDP-43 mislocalization (see April 2018 news; Zhang et al., 2015; Chou et al., 2018). Similarly, deletion of the putative NES in FUS reportedly improved survival in flies (Lanson et al., 2011). These findings seemed to support active nuclear export.

To examine how TDP-43 and FUS leave the nucleus, Dormann set out to test the proteins in classic nuclear export assays, which had not been done before. First author Helena Ederle used an interspecies heterokaryon assay, which fuses cells from two different species to measure how much of a nuclear protein from one shows up in the nucleus of the other. To her surprise, TDP-43 and FUS from human cells accumulated in mouse nucleus equally well whether or not their putative NES motifs were disrupted by mutation. This suggested the sequences were not functional. Supporting this, inhibiting or silencing exportin-1 did not slow export of either protein.

How else were the proteins getting out? Ederle and colleagues knocked down the related receptor exportin-5, and silenced part of the mRNA export machinery, which can also shuttle proteins out of the nucleus. No effect, so that wasn’t it.

The authors then considered that the proteins might escape the nucleus by simple passive diffusion. Small molecules of less than 60 kD are known to shuttle through the nuclear membrane within minutes (Popken et al., 2015). TDP-43 is 43 kD, and FUS 53; lo and behold, increasing their size by adding large domains trapped them in the nucleus.

If nuclear egress occurs through diffusion, then binding TDP-43 and FUS to long, unspliced mRNAs in the nucleus should also hold them back. To test this, Ederle and colleagues treated cells with a transcriptional inhibitor for three hours. This resulted in a gradual loss of TDP-43 from the nucleus, suggesting that RNA binding acts to anchor the proteins there. The data also imply that physiological dimerization or oligomerization of TDP-43 and FUS in the nucleus could help retain the proteins, Dormann noted (see Jan 2016 news; Afroz et al., 2017). She plans to investigate whether this oligomerization is disrupted in disease models.

Thomas and colleagues also found that the putative NES in TDP-43 was not required for export. They further showed that because this motif was nestled inside the protein, it was not exposed to solvent and would not be accessible for binding to exportin-1. On the other hand, enlarging TDP-43 by adding a large domain prevented egress, again suggesting the protein gets out through diffusion.

Then why has previous research found a beneficial effect from exportin-1 inhibitors? Dormann suggested these inhibitors might act indirectly, by inhibiting export of other nuclear proteins. Ke Zhang at Johns Hopkins University, Baltimore, thinks the inhibitors may prevent formation of stress granules. These structures are believed to induce TDP-43 and FUS to undergo liquid-liquid phase separation and then aggregate. Zhang recently reported that that several stress granule factors are shuttled between the nucleus and cytoplasm (see May 2018 news; Zhang et al., 2018). If exportin-1 inhibitors interrupt nuclear export of these factors, that could prevent stress granule formation and secondarily suppress TDP-43 and FUS aggregation as well.

Featured Papers

Ederle H, Funk C, Abou-Ajram C, Hutten S, Funk EB, Kehlenbach RH, Bailer SM, Dormann D. Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1. Sci Rep. 2018 May 4;8(1):7084. PubMed.

Pinarbasi ES, Cağatay T, Fung HY, Li YC, Chook YM, Thomas PJ. Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Sci Rep. 2018 May 4;8(1):7083. PubMed.

Archbold HC, Jackson KL, Arora A, Weskamp K, Tank EM, Li X, Miguez R, Dayton RD, Tamir S, Klein RL, Barmada SJ. TDP43 nuclear export and neurodegeneration in models of amyotrophic lateral sclerosis and frontotemporal dementia. Sci Rep. 2018 Mar 15;8(1):4606. PubMed.

References

Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S, Gupta S, Thomas MA, Hong I, Chiu SL, Huganir RL, Ostrow LW, Matunis MJ, Wang J, Sattler R, Lloyd TE, Rothstein JD. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):56-61. Epub 2015 Aug 26 PubMed.

Chou CC, Zhang Y, Umoh ME, Vaughan SW, Lorenzini I, Liu F, Sayegh M, Donlin-Asp PG, Chen YH, Duong DM, Seyfried NT, Powers MA, Kukar T, Hales CM, Gearing M, Cairns NJ, Boylan KB, Dickson DW, Rademakers R, Zhang YJ, Petrucelli L, Sattler R, Zarnescu DC, Glass JD, Rossoll W. TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nat Neurosci. 2018 Feb;21(2):228-239. Epub 2018 Jan 8 PubMed.

Lanson NA, Maltare A, King H, Smith R, Kim JH, Taylor JP, Lloyd TE, Pandey UB. A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43. Hum Mol Genet. 2011 Jul 1;20(13):2510-23. PubMed.

Popken P, Ghavami A, Onck PR, Poolman B, Veenhoff LM. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol Biol Cell. 2015 Apr 1;26(7):1386-94. Epub 2015 Jan 28 PubMed.

Afroz T, Hock EM, Ernst P, Foglieni C, Jambeau M, Gilhespy LA, Laferriere F, Maniecka Z, Plückthun A, Mittl P, Paganetti P, Allain FH, Polymenidou M. Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nat Commun. 2017 Jun 29;8(1):45. PubMed.

Zhang K, Daigle JG, Cunningham KM, Coyne AN, Ruan K, Grima JC, Bowen KE, Wadhwa H, Yang P, Rigo F, Taylor JP, Gitler AD, Rothstein JD, Lloyd TE. Stress Granule Assembly Disrupts Nucleocytoplasmic Transport. Cell. 2018 May 3;173(4):958-971.e17. Epub 2018 Apr 5 PubMed.

Further Reading

To learn more about emerging strategies targeting nuclear export in ALS, check out Scientists Pore Over New Strategies to Target ALS.


To view commentaries, primary articles and linked stories, go to the original posting on Alzforum.org here.

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disease-als FUS karyopharm KPT-350 nuclear export nucleocytoplasmic transport RNA processing stress granules tdp-43 topic-preclinical XPO1
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