ALS Gene Repeats Obstruct Traffic Between Nucleus and Cytoplasm

Could a molecular traffic jam at the nuclear membrane explain one kind of motor neuron disease? Extensive hexanucleotide repeats in the C9ORF72 gene, which are transcribed into RNAs and translated into dipeptide oligomers, cause amyotrophic lateral sclerosis and frontotemporal dementia. Now, two papers published August 26 in Nature, and one in Nature Neuroscience, reveal that those repeats snarl traffic through pores in the nuclear envelope. “All three papers converge on the same cellular pathway,” said Fen-Biao Gao of the University of Massachusetts Medical School in Worcester, co-senior author on one of the Nature papers. “Nuclear-cytoplasmic transport is affected in C9 ALS-FTD.” However, the findings do not settle whether the repeat RNAs or the dipeptide repeats are responsible for the defect.

Fractured envelopes.

The nuclear envelope is normally smooth and round, but looks irregular in cells expressing C9ORF72 repeats. [Courtesy of Freibaum et al., 2015.]

“These three studies are remarkable for their convergence on nucleocytoplasmic transport,” commented Edward Lee of the University of Pennsylvania Perelman School of Medicine in Philadelphia, who was not involved in the work. “They clearly show that transport deficits are common to many experimental models of C9ORF72 toxicity, even those that are not based on artificial overexpression.”

All Arrows Point to the Nuclear Pores
C9ORF72’s hexanucleotide repeats—hundreds or thousands of them in some cases of ALS and FTD—are transcribed into repetitive RNAs, which are then translated into dipeptide repeats. Scientists believe either the RNA or peptides might be toxic to neurons (see Jan 2013 news; Feb 2013 news). The authors of each of the three new papers started with different assumptions about which does the dirty work. At Johns Hopkins University in Baltimore, Jeffrey Rothstein and colleagues focused on the RNAs. Aaron Gitler at Stanford University in Palo Alto, California, went after toxic effects of the dipeptides. Gao and co-senior author Paul Taylor of St. Jude Children’s Research Hospital in Memphis, Tennessee, studied fruit flies that make both, screening for genetic modifiers of repeat toxicity.

For the latter, co-first author Brian Freibaum at St. Jude expressed a construct containing 58 G4C2 repeats in Drosophila eyes. Flies have no C9ORF72 gene, and the construct included only the repeats, not the full gene. The result was a “rough eye” phenotype, in which the facets of the compound eye become disorganized.

Freibaum and co-first author Yubing Lu at UMass Medical School then crossed the hexanucleotide repeat model with fly deletion mutants, yielding four suppressors and 14 enhancers of the rough-eye phenotype. Because these 18 genes all were involved in nucleocytoplasmic shuttling, Lu examined the nuclei of fly cells expressing the repeats. He found the nuclear envelope to be in disarray, as judged by immunohistochemistry for the nuclear envelope protein Lamin C (see image above). “The nuclear membrane was frayed, like it was falling apart,” Taylor said. In addition, the protein Nup107, a component of nuclear pores, formed inclusions adjacent to the nuclear envelope. Something was clearly wrong with the gateway between nucleus and cytosol.

At the same time Freibaum and Lu were conducting these experiments, scientists led by co-senior authors Rothstein and Thomas Lloyd at Johns Hopkins were taking a different tack by examining the RNAs produced by the repeat. They also report their findings in Nature. Rothstein and colleagues had previously identified more than 400 human proteins that might bind C9ORF72 repeat RNA (see Oct 2013 news). They used that list to come up with candidate modifiers for their own screen, in this case using fruit flies that had abnormal eyes due to a 30-hexanucleotide repeat (Xu et al., 2013). From 385 of the repeat RNA-binding proteins that had orthologs in Drosophila, Zhang came up with 35 genetic modifiers of the rough-eye phenotype. Based on the prior RNA-binding study in human cells and the fly screen, one of the strongest hits was RanGAP1, activator of Ran GTPase, an enzyme that cycles between the nucleus and cytoplasm to direct traffic (Joseph, 2006).

Co-author Aaron Haeusler, in the laboratory of Jiou Wang, confirmed by gel shift assay that RanGAP1 binds specifically to G4C2-repeat RNA. If overexpressed, RanGAP1 suppressed the repeat’s eye phenotype, while knocking down the activator worsened it.

Once the researchers were interested in nuclear transport, Zhang tested mutations in other nucleocytoplasmic transport proteins, such as RanGEF and importin-α. These genes, as well, affected repeat toxicity in flies. Mutations that enhanced nuclear import of proteins or blocked their export were beneficial, suggesting the C9ORF72 repeat prevents uptake of proteins into the nucleus. If this is the case, blocking export might help by keeping useful proteins inside the nucleus.

Meanwhile, the third research group, led by Gitler at Stanford, was also pursuing the effects of C9ORF72 repeats. Ana Jovičić, first author on the Nature Neuroscience paper, focused on the dipeptides that result from nonstandard translation of the hexanucleotide RNA. Because this occurs in all reading frames, five different dipeptides are created (two reading frames yield the same dipeptide). To distinguish the effects of repeat dipeptides from those of the repeat RNA, the authors constructed artificial genes that encoded an individual peptide without using the G4C2 sequence. Jovičić made constructs that generate 50-mers of each dipeptide, and expressed them in yeast. Because the most toxic ones were those containing arginine, Jovičić selected a proline-arginine-expressing yeast strain to screen for genes that would modify this toxicity. She identified 43 suppressors and 35 enhancers. When she grouped the modifiers by known function, nucleocytoplasmic transport emerged as the largest category, subsuming 11 genes.

What Does This Mean for ALS?
Because three separate screens pointed to nucleocytoplasmic transport as a central defect caused by G4C2 repeats in these disease models, the authors looked for direct evidence of faulty transport in human induced neurons made from the cells of C9ORF72 carriers and controls. Nucleocytoplasmic transport shepherds RNAs out of the nucleus, and proteins both in and out. The Gao/Taylor group focused on RNA trafficking, while the others probed protein shuttling.

Tracking fluorescently labeled RNA, Freibaum saw that neurons made from C9ORF72 carriers accumulated more RNA in the nucleus, and less in the cytoplasm, than control lines, suggesting a defect in RNA export.

To study protein trafficking, Gitler and colleagues stained cells for RCC1, a normally nuclear Ran guanine exchange factor and homolog of one of the enhancers of C9ORF72 toxicity identified in their yeast screen. While RCC1 inhabited the nucleus of induced neurons from controls, it was mostly cytoplasmic in induced neurons from expansion carriers. This suggests that nucleocytoplasmic protein transfer was altered in the human C9ORF72 lines, Jovičić concluded.

In Rothstein’s lab, co-first author Christopher Donnelly evaluated nuclear shuttling with a red fluorescent protein hooked to both a nuclear localization sequence and nuclear export signal so it would travel back and forth between the nucleus and the cytoplasm. He photobleached the reporter in induced neuron nuclei, then measured recovery of the fluorescence. It came back slowly in the C9ORF72 expansion neurons, indicating sluggish nuclear import.

Donnelly, who now runs his own lab at the University of Pittsburgh, further found that traffic of another ALS- and FTD-related protein, TDP-43, was perturbed in the human neurons. Cells derived from C9ORF72 mutation carriers tended to have more TDP-43 in the cytoplasm, and less in the nucleus. Donnelly suggested that defective nucleocytoplasmic transport might explain why other researchers have seen TDP-43 vacating the nucleus and aggregating in the cytoplasm in both C9ORF72-based disease and sporadic ALS (see Jan 2010 news). “We think that nuclear trafficking deficits might … promote [TDP-43] accumulation and TDP-43 pathology,” Donnelly said.

If nuclear trafficking was disrupted, the key players in that process, Ran and RANGAP1, might also be altered. Rothstein and colleagues confirmed this was the case. While RanGAP1 normally coats the nucleus of induced neurons smoothly, in the repeat-carrying cells it joined the RNA repeats in large, abnormal foci around the nucleus. This disrupted the localization of Ran, with less in the nucleus and more in the cytoplasm than normal. The authors found similar RANGAP1 defects in brain tissue from C9ORF72 expansion carriers who died of ALS. Other proteins of the nuclear pore complex also mislocalized in the ALS brain tissue. Based on these data, Rothstein said the C9ORF72 repeats cause two problems: disruption of transport by binding RanGAP1, and aggregation of nuclear pores. He said he does not know how those two defects are related.

Multiple Mechanisms?
Taylor noted that whether researchers figured the RNAs were toxic, or blamed the dipeptides, or went in without preconception as his group did, they all came to the same conclusion—that the repeats alter nucleocytoplasmic transport.

Even so, the scientists are still left wondering which repeat molecule—RNA or peptide—interferes with nucleocytoplasmic shuttling in human disease. Rothstein and Donnelly believe the RNA blocks the transport but could not rule out the possibility that dipeptides contributed in their models. Gitler and colleagues specifically expressed only dipeptide repeats in yeast, but do not know if those same peptides reach toxic levels in human neurons. Most scientists who spoke with Alzforum suggested that both RNAs and dipeptides might affect nucleocytoplasmic traffic.

None of the work offers any clues to C9ORF72’s normal function, since all groups studied just the repeats. However, in a recent study, researchers led by Janice Robertson of the University of Toronto reported that wild-type C9ORF72, without extra repeats, localizes to the nuclear envelope (see Aug 2015 news). It appeared to bind nuclear pore components there, though Robertson did not determine if C9ORF72 plays an active role in nucleocytoplasmic transport. Donnelly and Rothstein noted that they, too, detect full-length protein on the nuclear envelope. Researchers who spoke with Alzforum could not explain why the repeats and the normal protein seem to land in the same place, or if they perform similar functions once there. What they can conclude, Robertson said, is that nucleocytoplasmic transport clearly has a share in ALS pathology.

Could nucleocytoplasmic trafficking offer a new therapeutic target for FTD and ALS? Rothstein and colleagues gave their C9 repeat flies KPT-276, a small molecule that inhibits export from the nucleus. It rescued both transport deficit and eye phenotype. The authors surmised that blocking export with KPT-276 compensated for reduced import due to the repeats, normalizing protein levels inside and outside the nucleus. Donnelly plans to research related molecules further. Antisense oligonucleotides against the C9ORF72-repeat RNA also rescued both the fly-eye phenotype and the defective transport in induced neurons.

“These papers are an exciting advance for the field,” commented Adrian Isaacs of University College London, who did not participate in the work. “An important question is whether impaired nucleocytoplasmic transport plays a role in FTD and ALS without the C9ORF72 repeat expansion.” Rothstein told Alzforum his group will try to answer that.


Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, Badders N, Valentine M, Miller BL, Wong PC, Petrucelli L, Kim HJ, Gao FB, Taylor JP. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Aug 26; PubMed.

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 Aug 26; PubMed.

Jovičić A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, Paul JW 3rd, Sun S, Herdy JR, Bieri G, Kramer NJ, Gage FH, Van Den Bosch L, Robberecht W, Gitler AD. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. 2015 Aug 26;18(9):1226-9. PubMed.

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