. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):129-33. Epub 2015 Aug 26 PubMed.


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  1. These three studies are remarkable for their convergence on nucleocytoplasmic transport. Rarely does it happen that multiple groups, using multiple complementary approaches, from flies to yeast to neuronal cell culture models to human tissue, arrive at the same conclusion. There are still pressing questions—foremost, in my opinion, is why there is no correlation between dipeptide-repeat aggregates and neurodegeneration in human tissue. However, these studies clearly show that nucleocytoplasmic transport defects are common to many experimental models of C9ORF72 toxicity, even those that are not based on artificial overexpression. Finally, these studies add to the evidence that abnormal RNA pathways are a key feature of ALS and FTD, be it due to abnormal nucleocytoplasmic transport, nucleolar stress pathways, or dysregulation of TDP-43.

    View all comments by Edward B. Lee
  2. These papers are an exciting advance for the field. An important question is whether impaired nucleocytoplasmic transport plays a role in FTD and ALS without C9ORF72 repeat expansion, particularly as the majority of FTD and ALS cases are characterized by mislocalization of nuclear TDP-43.

    The Gitler paper shows dipeptide repeats are sufficient to impair nucleocytoplasmic transport, so they would be my first bet for the culprit, but the repeat RNA-sequestering factors such as RanGAP could independently play a role. Both these possibilities require further investigation. For sequestration of RanGAP, soluble cytoplasmicrepeat RNA would most likely mediate the effect rather than RNA foci, which are generally nuclear and not in the appropriate location to sequester RanGAP, which is cytoplasmic.

    View all comments by Adrian Isaacs
  3. In a fascinating convergence, three papers have been published all implicating nucleocytoplasmic transport in the pathogenesis of disease associated with GGGGCC-repeat expansion of the C9ORF72 gene. This genetic variant has become extremely important in the field in recent years, as it is the most common genetic variant of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Indeed, it occurs in as many as 10 percent of all ALS patients and up to 40 percent of those with a positive family history of ALS and 25 percent of those with a positive family history of FTD (Majounie et al., 2012). Moreover, C9ORF72 disease resembles the more common sporadic form both clinically and pathologically, leading us to hope that therapies developed against C9ORF72 might be more broadly applicable (Cooper-Knock et al., 2012). Since the discovery of this genetic variant we have witnessed a number of step changes in our understanding: the discovery of RNA foci transcribed from the DNA repeat sequence in both a sense (DeJesus-Hernandez et al., 2011) and an antisense (Mizielinska et al., 2013) direction; then the discovery of dipeptide repeat proteins (DPR) translated from the repeat sequence (Mori et al., 2013). Both RNA foci and DPR proteins have been suggested as important mediators of toxicity in disease pathogenesis. The other prominent proposed pathogenic mechanism is haploinsufficiency. (Toxicity mechanisms are reviewed in Cooper-Knock et al., 2015). Now, again, we appear to have another step change in our understanding.

    A particularly interesting aspect of the focus on nucleocytoplasmic transport comes from properties of the nuclear pore complex highlighted by these studies. Nuclear pore proteins are long-lived, and the integrity of the nuclear pore complex is known to be affected by aging (D'Angelo et al., 2009); this fits well with an age-dependent disease caused by a genetic defect that is present from birth—perhaps aging exacerbates a phenotype present from early life, until a threshold is crossed that determines disease onset.

    The three papers take a related but certainly not identical approach. Broadly, they all focus on the identification of agents that ameliorate toxicity in a gain-of-function model of C9ORF72 disease, and show that these agents modulate genes and proteins involved in nucleocytoplasmic transport. However, there are important differences between the papers that deserve to be highlighted.

    Two of the papers (Freibaum et al. and Zhang et al.) focus on a drosophila model, whereas the third (Jovicic et al.) starts with a yeast model. Furthermore, two of the papers utilize an unbiased genetic screen for ameliorators and exaggerators of toxicity (Freibaum et al. and Jovicic et al.); analysis of these candidates showed they were enriched for proteins involved in nucleocytoplasmic transport. The other study analyzed candidates previously identified as binding partners of (and therefore potentially sequestered by) GGGCC-repeat RNA (Haeusler et al., 2014); overexpression of RanGAP, a protein involved in nucleocytoplasmic transport, was a potent suppressor of toxicity (Zhang et al.). None of the models is truly physiological in its reproduction of the human disease, although in each of the studies certain findings are validated in either human tissue or neurons derived from C9ORF72-patients. A note of caution should be sounded here—in the past year the field has witnessed a lot of excitement over the possible role of DPR proteins, particularly those which are arginine-rich (Mizielinska et al., 2014), only for other work to conclude that these species might be very rare and of uncertain significance in human tissue (Gomez-Deza et al., 2015). In addition, none of these models include expression of CCCCGG-repeat (antisense transcribed) RNA foci, and yet it has been suggested that the presence of these species is an important predictor of pathology in postmortem motor neurons from C9ORF72-ALS patients (Cooper-Knock et al., 2015).

    The yeast model utilized by Jovicic et al. is very different from the others in that it is a DPR-only model—the authors take care to express the DPR proteins in the absence of RNA foci. This is particularly important when one considers that the mechanism of toxicity suggested by Zhang et al. is based on sequestration of proteins by RNA foci—it seems counterintuitive to think that overexpression of a nucleocytoplasmic transport protein should still rescue toxicity when there are no RNA foci to sequester the protein and therefore initiate a relative loss of function. The complex interplay of factors raised by these studies remains to be resolved.

    Although all three papers agree that they are discovering an effect on nucleocytoplasmic transport, they raise quite different interpretations of the consequence of this effect. Freibaum et al. highlight an increase in the ratio of nuclear cytoplasmic RNA; they suggest the key to toxicity might be a compromise of nuclear export of RNA. In contrast, Zhang et al. suggest the key problem might be in transport of proteins carrying a nuclear import signal; notably, this includes TDP-43, whose mislocalization is perhaps the molecular hallmark of not only C9ORF72 disease, but of much of sporadic ALS and FTD.  Clearly nucleocytoplasmic transport has a broad set of functions in both RNA and protein transport and therefore a broad range of possible mechanisms need to be examined in the search for effective therapies. This is not the end, it is a new beginning.  


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    . Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy. Acta Neuropathol. 2015 Jul;130(1):63-75. Epub 2015 May 6 PubMed.

    . The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis. Neurotherapeutics. 2015 Apr;12(2):326-39. PubMed.

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    . Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012 Apr;11(4):323-30. PubMed.

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    View all comments by Johnathan Cooper-Knock
  4. A perfect storm of research identifies nucleocytoplasmic shuttling defects as a tractable therapeutic target for ALS/FTD.

    Hexanucleotide repeat expansions (HRE) in C9ORF72 are the most common genetic cause of ALS/FTD (DeJesus-Hernandez et al., 2011; Renton et al., 2011). Three mechanisms of toxicity have been proposed:

    Understanding the mechanistic basis of this toxicity is vital to the development of effective therapies. Now, in a major advancement in the field, three independent teams have shown using genetic screens that toxicity caused by expression of C9ORF72 HREs in yeast and flies is due to defects in nucleocytoplasmic transport. Toxicity was dependent on repeat length, with longer repeats giving a more aggressive phenotype, as is observed in humans, although the absolute number of maximal repeats used in the studies was 58, which may not be pathologically significant in ALS/FTD7.

    In studies from the Gitler and Taylor teams, toxicity was clearly related to expression of arginine-rich DPR proteins through use of codon-optimized constructs, preventing expression of the GGGGCC repetitive sequence, thereby excluding the possibility of RNA-mediated toxicity. In contrast, the Rothstein team identified RanGAP1 as directly interacting with GGGGCC repeats in vitro, and confirmed the role of RanGAP1 in modifying C9ORF72-HRE toxicity in fly using a candidate genetic screen. Although the Rothstein team could not discount a contribution of DPRs in their model system, toxicity could be ameliorated by disruption of HRE G-quadruplexes, to which RanGAP1 binds, supporting an RNA-mediated mode of toxicity. Nevertheless, all three studies identified components of the nucleocytoplasmic transport machinery as suppressors/enhancers of toxicity caused by expression of C9ORF72 HREs.

    In another twist to the story, a recent publication from the Robertson group has shown that C9ORF72 is localized to the nuclear membrane of healthy motor neurons, but this is lost in diseased motor neurons in ALS, where C9ORF72 is mislocalized to the plasma membrane. Intriguingly, C9ORF72 was shown to interact with RanGTPase and Importin-β, again implicating defects of nucleocytoplasmic shuttling as a disease mechanism in ALS/FTD8.

    This perfect storm of findings clearly shows that defective nucleocytoplasmic transport has a key role in the disease mechanism causing ALS/FTD. The question remains as to what cargo, protein and/or RNA, either retained within or aberrantly exported from the nucleus, is responsible for toxicity. Is it a specific species, or multiple? In this regard, the Rothstein and Robertson teams showed that TDP-43 mislocalization from the nucleus to the cytoplasm correlated with defects of nucleocytoplasmic shuttling and/or loss of C9ORF72 from the nuclear membrane, respectively. This provides a direct link with the pathology pathognomonic of ALS/FTD.

    Whether mislocalization of TDP-43 is the major contributor to disease pathogenesis remains to be determined. However, restoring normal activity of nucleocytoplasmic shuttling is a tractable target for therapeutic intervention, and holds great promise as a treatment strategy for ALS/FTD.  


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    View all comments by Ekaterina Rogaeva

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