. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat Med. 2018 Aug;24(8):1136-1142. Epub 2018 Jun 25 PubMed.


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  1. This new paper reports the first mouse model expressing specifically poly(GR), which is an exciting step forward for the field. This was achieved by expressing 100 GFP-tagged poly(GR) repeats in the mouse brain using AAV. The mice show diffuse cytoplasmic GR staining and profound neurodegeneration accompanied by behavioral and motor deficits.

    A really nice aspect of the paper is the ability to compare to both GGGGCC-repeat mice and polyGA mice. For instance, poly(GR) alone was not able to induce the TDP-43 inclusions observed in the GGGGCC-repeat mice (and neither was poly(GA)). Therefore it seems that TDP-43 pathology in the GGGGCC-AAV mice is induced by either:

    1.  poly(GR) inclusions rather than the diffuse poly(GR) observed in the model,
    2. a distinct DPR (poly(GP), poly(PR), and poly(AP) are left to examine in this AAV model),
    3. a synergistic effect of two or more different DPRs,
    4. a synergistic effect requiring both DPRs and repeat RNA,
    5. repeat RNA alone, or
    6. lower DPR expression is required to tease out TDP-43 pathology, whereas higher expression may lead to direct toxicity without time to invoke TDP-43 changes.

    These are important questions to now figure out.

    A central message of the paper is that poly(GR) but not poly(GA) associated with ribosomal subunits and inhibited translation, which is consistent with previous data. Whether translation inhibition is due to direct ribosome binding, the altered ribosomal gene expression also reported, or previously suggested mechanisms such as RNA binding, remains an open question.

    Either way, these data now strongly implicate altered translation as an important factor in C9ORF72 pathogenesis. A critical next step will be to determine whether it is possible to reduce this impaired translation and if so whether such an intervention can rescue poly(GR)-induced neurodegeneration.

    View all comments by Adrian Isaacs
  2. This paper places another important puzzle piece. Arginine-containing poly-dipeptides (PR and GR) produced in C9-ALS/FTD are exquisitely toxic in a wide variety of model systems, including HeLa cells (Kwon et al., 2014), cultured neurons (Wen et al., 2014), and Drosophila (Mizielinska et al., 2014), whereas the other polypeptides produced in C9-ALS/FTD are either innocuous (PA and PG) or only mildly toxic at high levels (GA).

    One important basis for PR and GR toxicity in these systems is infiltration and disturbance of structures that arise by phase transitions. This disturbance includes stress granules and impairs translation, but is not limited to these organelles (Lee et al., 2016). However, it has been unclear how the quantities of poly-dipeptides expressed in these model systems relate to the quantities found in human C9-ALS/FTD. An important recent human study found that regional levels of GR in brains of C9-ALS-FTD patients correlates with neurodegeneration and burden of TDP-43 pathology (Saberi et al., 2018); this further implicates GR in disease but causation is not easily inferred from correlative postmortem studies.

    This paper adds important new insight to this emerging story by showing that, in transgenic mice, expressing poly(GR) at levels comparable to, or lower than, levels observed in human C9-ALS/FTD is sufficient to drive neurodegeneration.

    It is important to note, however, that these animals did not develop significant TDP-43 pathology, a hallmark of C9-ALS/FTD and most other sporadic and familial forms of ALS-FTD. Perhaps a second factor is needed to initiate this aspect of the disease phenotype, or a longer disease duration is required for significant accrual of TDP-43 pathology.


    . Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 2014 Sep 5;345(6201):1139-45. Epub 2014 Jul 31 PubMed.

    . Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron. 2014 Dec 17;84(6):1213-25. PubMed.

    . C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science. 2014 Sep 5;345(6201):1192-1194. Epub 2014 Aug 7 PubMed.

    . C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell. 2016 Oct 20;167(3):774-788.e17. PubMed.

    . Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol. 2018 Mar;135(3):459-474. Epub 2017 Dec 1 PubMed.

    View all comments by J. Paul Taylor
  3. Zhang et al. report neurodegeneration and behavioral changes in the first poly(GR) mouse model using their AAV-based expression system. Strikingly, these mice show no TDP-43 pathology, suggesting that DPRs also trigger TDP-43-independent pathways of neurotoxicity.

    Unfortunately, this leaves the mechanistic link between the C9ORF72 repeat expansion and TDP-43 pathology found in patients still unresolved. Consistent with our recent report in primary neurons (Hartmann et al., 2018), Zhang et al. conclude that poly(GR) mainly affect ribosomal function in mice and find no evidence for impaired nucleocytoplasmic transport in vivo. Unfortunately, they did not analyze splicing changes in their RNAseq data (compare (Kwon et al., 2014). 

    They extend previous observations that cytoplasmic poly(GR) aggregates co-localize with stress granule markers in mice, but do not provide data for C9ORF72 patient tissue. In our attempt to validate neuronal poly-GR/PR interactors in C9ORF72 brains, we could not detect classical stress granule markers such as TIAR and G3BP2 in patients, and apart from ribosomes only detected co-localization with STAU2, which has also been linked to stress granules.

    Taken together, chronic impairment of translation by poly(GR) may contribute to neurodegeneration in C9ORF72 patients. It will be interesting to determine whether boosting translation would prevent neuronal death or even worsen toxicity through enhanced DPR production.


    . Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity. Life Science Alliance. 16 May 2018. DOI: 10.26508/lsa.201800070

    . Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 2014 Sep 5;345(6201):1139-45. Epub 2014 Jul 31 PubMed.

    View all comments by Dieter Edbauer
  4. The manuscript by Zhang et al. provides a new model for the study of C9ORF72, and in the process provides useful insight into common pathways that appear to mediate neurodegeneration in ALS and related disorders. Previously, Petrucelli’s group showed that strong expression of (G4C2)66 in the mouse brain produces a model with abundant dipeptide repeats, phospho-TDP-43 inclusions, and neurodegeneration. DPRs, though, come in six flavors, with poly(GR) being best correlated with neurodegeneration. In the current manuscript, Petrucelli’s group has specifically created mice expressing GFP-poly(GR)100 delivered by AAV1, which preferentially infects neurons. They observe that the mouse brains exhibit abundant cytoplasmic poly(GR) as well as exhibiting neurodegeneration and inflammation.

    The nature of the pathology is highly informative, and provides key insights into pathways that contribute to the neurodegeneration. The fact that poly(GR) ​correlates with neurodegeneration is important, since poly(GA) doesn’t correlate with neurodegeneration, and supports accumulating data that dipeptides with arginines are particularly toxic. Arginines are notable for being one of the classic amino acids that are abundant in low-complexity domains of proteins (which promote liquid-liquid phase separation). However, poly(GA) does appear to play a part in the aggregation of poly(GR), which the group shows is recruited to poly(GA) inclusions and co-localizes with poly(GA) inclusions in mice expressing poly(G4C2)100. These data indicate that the type of DPR is critical for toxicity, and that toxicity is quite distinct from aggregation. Another moderate surprise is that toxicity is also separate from aggregation of TDP-43. A fraction of DPR inclusions in poly(G4C2)100 mice co-localize with aggregated phospho-TDP-43; the presence of aggregated phospho-TDP-43 raises the possibility that it plays some role in the ensuing neurodegeneration. However, the absence of TDP-43 inclusions in the poly(GR)100 mice shows that poly(GR)100 is sufficient to cause neurodegeneration without the involvement of TDP-43.

    One of the other pathologies that is surprisingly missing is dysfunction of the nuclear pore and nucleus generally. Since poly(G4C2) pathology is associated with dysfunction of nuclear and nuclear pore biology in other animal models, the absence of such dysfunction in the poly(GR) models once again points to the mechanistic specificity by which poly(GR) causes damage, and suggests that nuclear pore pathology does not inevitably occur during neurodegeneration.

    The next set of insights comes when the group considers which types of proteins co-localize with the poly(GR) inclusions (in the poly(GR)100 mice). They find that the poly(GR) inclusions co-localize with classic markers of stress granules, including TIA1 and EIF3η. This provides support for the hypothesis that the pathophysiology of the DPR inclusions involves stress granules or a related RNA granule. Cell-culture studies show that the poly(GR)100 exerts a particularly strong effect on delaying dispersal of stress granules, which is consistent with a growing body of evidence in the field suggesting that the main effect of mutations in RNA-binding proteins is to slow dispersal/removal of stress granules. 

    Stress granules are thought to be part of the translational stress response, which adapts RNA translation to cope with stress. Co-localization experiments combined with RNAseq experiments show that poly(GR) inclusions co-localize with many ribosomal proteins, including RPS25, which is involved in non-canonical RNA translation. It is important to note that the group finds co-localization with ribosomal proteins from both the large 60S subunit (RPLs) as well as the smaller 40S subunit (RPSs). The presence of both RPL and RPS proteins is consistent with what we have seen in inclusions in brains of models of tauopathies, but differs from the classic view of stress granules, which are thought to contain only RPS subunits. This difference could reflect differences in the regulatory pathways and/or differences between the biology of non-dividing neurons vs. transformed peripheral cell lines.

    Taken together, this work highlights the specific pathophysiological mechanisms of poly-(GR)100, and demonstrates that the pathophysiology of DPRs differ by DPR type. The strong connection between poly(GR) and stress granule (or related) biology also highlights mechanisms shared in common with other neurodegenerative diseases.

    View all comments by Benjamin Wolozin
  5. In this paper the Petrucelli lab extend their previous work by generating new viral-mediated models of poly-glycine-arginine (GR) expression in mice.

    The group has previously demonstrated that overexpression of sense-direction 66 repeat RNA via intracerebroventricular injection of adeno-associated virus (AAV) leads to a strongly toxic phenotype in mice (Chew et al., 2015). These mice demonstrate sense RNA foci, dipeptide repeat proteins, and nuclear TDP-43 pathology.

    Here, the group overexpressed GFP-tagged, poly(GR)100 repeats alone and observed a strong neuronal toxicity. Interestingly, in contrast to the repeat RNA expressing model, the GR mice do not develop TDP-43 pathology, or poly(GR) aggregates, suggesting that other dipeptide proteins or repeat RNA are required to mediate this aggregative process.

    Based on previous studies that have identified ribosomal proteins as key poly(GR) interacting proteins (Lee et al., 2016; Lin et al., 2016; Boeynaems et al., 2017), the group demonstrated that poly(GR) was able to induce translational repression, and that poly(GR) co-localized with ribosomal proteins in mouse and patient brain. These findings fit well with previous reports that arginine-rich dipeptides like poly(GR) can lead to translational repression (Kanekura et al., 2016; Lee et al., 2016; Hartmann et al., 2018). However the mechanism(s) that cause this translational repression are not fully resolved and may include: binding of ribosomes by dipeptide proteins, induction of stress granules, downregulation of ribosomal proteins, or binding of RNA by arginine containing dipeptides.

    Further work will be required to distinguish these potential mechanisms, although these results strongly suggest that targeting translational repression may be a therapeutic avenue in C9ORF72 ALS/FTD.

    The authors demonstrate that, besides suppressing canonical translation, poly(GR) is capable of also suppressing repeat-associated non-AUG initiated translation (RANT). This could provide a compelling mechanism as to how a seemingly low-abundance protein can induce pathology, as poly(GR) may simultaneously induce a toxic inhibition of translation whilst also preventing its own expression.


    . Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science. 2015 Jun 5;348(6239):1151-4. Epub 2015 May 14 PubMed.

    . C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell. 2016 Oct 20;167(3):774-788.e17. PubMed.

    . Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell. 2016 Oct 20;167(3):789-802.e12. PubMed.

    . Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. Mol Cell. 2017 Mar 16;65(6):1044-1055.e5. PubMed.

    . Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum Mol Genet. 2016 May 1;25(9):1803-13. Epub 2016 Feb 29 PubMed.

    . Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity. Life Science Alliance. 16 May 2018. DOI: 10.26508/lsa.201800070

    View all comments by Thomas Moens

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This paper appears in the following:


  1. In New ALS/FTD Mouse Model, Poly(GR) Peptides Poison Ribosomes

Research Models

  1. AAV-GFP–(GR)100