Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2α-dependent ISR in C9ORF72 FTD/ALS

  1. Janani Parameswaran
  2. Nancy Zhang
  3. Elke Braems
  4. Kedamawit Tilahun
  5. Devesh C Pant
  6. Keena Yin
  7. Seneshaw Asress
  8. Kara Heeren
  9. Anwesha Banerjee
  10. Emma Davis
  11. Samantha L Schwartz
  12. Graeme L Conn
  13. Gary J Bassell
  14. Ludo Van Den Bosch
  15. Jie Jiang

  • Curated by eLife

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    eLife assessment

    The current study provides important, mechanistic insight into the potential contribution of antisense C4G2 expanded RNA to disease in C9orf72-associated ALS/FTD. The authors convincingly demonstrate that expression of this RNA species activates the PKR/eIF2α-dependent integrated stress response. They further provide evidence that this can contribute to disease phenotypes using multiple models and post-mortem patient samples.

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Abstract

GGGGCC (G 4 C 2 ) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C 4 G 2 ) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C 4 G 2 repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C 4 G 2 RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C 4 G 2 , but not sense G 4 C 2 , repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C 4 G 2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

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  1. Version published to 10.7554/elife.85902 on eLife
  2. eLife

    eLife assessment

    The current study provides important, mechanistic insight into the potential contribution of antisense C4G2 expanded RNA to disease in C9orf72-associated ALS/FTD. The authors convincingly demonstrate that expression of this RNA species activates the PKR/eIF2α-dependent integrated stress response. They further provide evidence that this can contribute to disease phenotypes using multiple models and post-mortem patient samples.

  3. eLife

    Reviewer #1 (Public Review):

    The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion within the first intron of the C9ORF72 gene. However, how this repeat contributes to disease pathology is still an active area of research. This study takes a targeted approach to analyzing specifically how the C9ORF72 antisense transcript (C4G2) may be contributing to FTD/ALS.

    Using an artificial (C4G2)75 antisense cassette, the authors show in both HEK293T cells and cultured neurons that the C4G2 antisense transcript leads to elevated levels of activated PKR and increased phosphorylated eIF2alpha. This then leads to a decreased level of translation, the formation of stress granules, and decreased survival, phenotypes that can be suppressed through the knockdown of PKR. The authors nicely demonstrate that PKR activation upon transfection with their antisense cassette is independent of toxic dipeptide repeat proteins by using reporter constructs that do not create these dipeptides but are still able to activate PKR. Furthermore, using a construct that expresses both sense and antisense transcripts, the authors show that knockdown of the antisense, but not the sense transcript, abrogates the PKR response (demonstrating the specificity of this stress pathway for the antisense RNA). The authors additionally show the relevance of PKR activation in FTD/ALS through the presence of activated PKR and elevated eIF2alpha in ALS postmortem brain tissue.

    This paper shows that, at least in model systems, the C4G2 transcript can have cytotoxic effects through the stimulation of PKR. The experiments are well-controlled and fairly comprehensive. The claim that PKR activation occurs via the antisense RNA, and not the sense, is well supported by the data. However, some limitations exist, some of which the authors explicitly recognize. They are as follows:
    1. It is not clear how the results from these reporter constructs inform on the repeat expansion RNAs produced in disease, which can be significantly longer, and might be expressed at different levels. Perhaps if the C4G2 repeat used in this work were expressed at levels comparable to what the antisense transcript is expressed in an actual disease, or in a similar RNA context, PKR would not be activated. This is important to keep in mind.
    2. It is still unclear how PKR is being activated in the presence of C4G2 (it could be direct or indirect). The authors list a variety of explanations in the discussion. A prior study has shown that a similar repeat expansion leads to the accumulation of cytoplasmic dsRNA inclusions marked by TDP-43 (Rodriguez et al., 2021). It would be interesting to see if these inclusions are present upon expression of the antisense construct.
    3. In the context of C9ORF72 FTD/ALS disease, it is still difficult to say how much of the disease pathology is on account of antisense triggered stress responses as opposed to dipeptide repeat, RBP titration, etc. This study nevertheless provides a new perspective to consider for how the C9ORF72 repeat expansion contributes to the diseased state.

  4. eLife

    Reviewer #2 (Public Review):

    The underlying toxic species in C9ORF72 FTD/ALS is debated, with evidence for the contribution of both loss of function and gain of function of sense G4C2 repeat-expanded mRNAs and DRPS has been shown. The authors ask what the role - if any - of the antisense C4G2 repeat expanded mRNAs, which are equally abundant in patient brains, in producing toxicity. They convincingly show a role for these, independent of DRP expression, and distinct from sense G4C2repeat-expanded in toxicity in cell lines, neurons, and zebrafish, mediated via PKR activation. The latter is shown through increased p-eIF2alpha and reduced protein synthesis rates, associated with toxic phenotypes, rescued by PKR knockdown. The authors have achieved their aims, where the excellent data strongly support their conclusions.

    The mechanism for PKR activation by antisense but sense repeat-expanded mRNAs is not examined, but the authors reasonably propose secondary structure differences in PKR activation. This could be tested in future work.

    The work adds to our understanding of mechanisms of toxicity in repeat disorders, and this particular mechanism has implications for therapy via ISR modulation to reverse the effects of PKR activation.

    The human data adds to the spectrum of protein-misfolding neurodegenerative diseases that show UPR/ISR activation, again with implications for therapy via ISR modulation.

    Interestingly, PKR knockdown only partially rescues cell toxicity in neuronal cells, possibly reflecting other toxic mechanisms at play.

  5. Version published to 10.1101/2022.06.06.495030v1 on bioRxiv