Ribosome inhibition by<i>C9ORF72</i>-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Loveland, A.B.; Svidritskiy, Egor; Susorov, Denis; Lee, Soojin; Park, Alexander; Demo, Gabriel; Gao, Fen‐Biao; Коростелев, А.A.

doi:10.1101/2020.08.30.274597

Cited by 4 publications

(6 citation statements)

References 100 publications

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“…Early studies in drosophila suggested that toxicity arises not from the expanded RNA transcripts but from dipeptides, and particularly those containing arginine 19 . Other reports have also pointed to directly neurotoxic effects of these peptides 23,[53][54][55][56][57][58][59] , although directly toxic effects of the expanded RNA transcript have also been recently described 60 . Our findings strongly encourage the view that suppressing the expression of the mutant alleles of C9ORF72 may be clinically beneficial, regardless of whether the primary benefit is mediated by reduction in mutant transcripts or polydipeptide levels.…”

Section: Aso5-2 Is Non-toxic In Large Animalsmentioning

confidence: 99%

“…Both sense and antisense transcripts encompassing the HRE in V1 and V3 generate RNA foci and undergo translation into atypical, aggregation-prone dipeptide repeat (DPR) proteins in all open reading frames [14][15][16][17] . These unusual DPRs are toxic in several experimental model systems [18][19][20][21][22][23] Despite important advances in elucidating the molecular pathology of the expanded hexanucleotide repeats, there are no meaningful therapies for C9ORF72-related ALS or FTD 24,25 .…”

Section: Introduction 422 Wordsmentioning

confidence: 99%

See 1 more Smart Citation

Potent Mixed Backbone Antisense Oligonucleotide Safety Suppressed Expression of Mutant C9ORF72 Transcripts and Polypeptides: First in Human Pilot Study

Brown

Moazami

Yang

et al. 2021

Preprint

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Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Proposed disease mechanisms include a gain of toxic functions of the G4C2 repeats, implying that selective reduction in levels of the repeat-containing transcripts would represent a treatment strategy for this disorder. In the present study, using C9-ALS/FTD patient derived cells and C9ORF72 BAC transgenic mice, we have generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat containing transcripts in both the sense and anti-sense strands of C9ORF72 and effectively suppress tissue levels of polyGP dipeptides. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expressions, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of CSF polyGP.

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Section: Aso5-2 Is Non-toxic In Large Animalsmentioning

confidence: 99%

Section: Introduction 422 Wordsmentioning

confidence: 99%

Potent Mixed Backbone Antisense Oligonucleotide Safety Suppressed Expression of Mutant C9ORF72 Transcripts and Polypeptides: First in Human Pilot Study

Brown

Moazami

Yang

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…25% or less). Such lengths would be sufficient to fill the ribosome exit tunnel and stall through an electrostatic jamming mechanism as previously proposed (17–19). However, the most effective stalling required longer dipeptide lengths (>30 repeats for poly-GR (60 amino acids) and >50 repeats for poly-PR (100 amino acids)) to stall more than 50%, and indeed did so in a length-dependent manner, suggesting that emergent DPR chain is involved in stalling.…”

Section: Discussionmentioning

confidence: 59%

“…However, the most effective stalling required longer dipeptide lengths (>30 repeats for poly-GR (60 amino acids) and >50 repeats for poly-PR (100 amino acids)) to stall more than 50%, and indeed did so in a length-dependent manner, suggesting that emergent DPR chain is involved in stalling. A preprint study suggested that the addition of short synthetic 20×PR and 20×GR polypeptides strongly inhibited global translation in vitro and occluded the exit tunnel of eukaryotic 80S ribosomes (19). Accordingly, it is reasonable to predict that during R-rich DPR translation emergent poly-GR or poly-PR nascent chains could bind and block the exit tunnels of trailing ribosomes on the mRNA leading to obstruction of translation in trans, and a collateral aggregation of translational machinery.…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies from us and others showed that expression of mRNA encoding long repeats of poly-GR and poly-PR causes severe ribosomal stalling (17,18) and it was proposed that the positively charged R-rich nascent polypeptide electrostatically jams the ribosome exit tunnel (19). However, the mechanisms by which stalling occurs and whether it contributes to toxicity remain unclear.…”

Section: Introductionmentioning

confidence: 99%

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Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate

Kriachkov

McWilliam

Mintern

et al. 2022

Preprint

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Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of DPR-induced stalling and examined whether known regulatory mechanisms of ribosome quality control (RQC) are involved to sense and resolve the stalls. We demonstrate that arginine-containing DPRs lead to stalling in a length dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40xGly-Xxx DPRs shows that stalling is most pronounced where Xxx are positively charged amino acids (Arg or Lys). Through a genome-wide knockout screen we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, respond differently to the readthrough of arginine-rich DPRs. Indeed, we find evidence that DPR-mediated stalling has no natural regulatory responses even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as posing a particular danger to cellular health and viability.

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Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

Tran

Moazami

Yang

et al. 2021

Nat Med

101

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GGGGCC (G 4 C 2 ) hexanucleotide repeat expansion (HRE) in the first intron of the C9ORF72 (C9) gene is the most common genetic cause of ALS and FTD, two devastating adult-onset neurodegenerative disorders 1,2 . Proposed disease mechanisms include a partial loss of the C9ORF72 protein function (C9ORF72 haploinsufficiency) and acquired toxicity of the repeat expansion 3 . Transcription of the C9ORF72 gene generates three transcript variants: V1, V2 and V3 (Fig. 1a). V1 is translated to produce a short protein isoform (222 amino acids), whereas V2 and V3 generate the most predominant C9ORF72 protein (481 amino acids), which functions in vesicular trafficking 4 . Located adjacent to the promoter region of the most abundant V2 transcript variant, the G 4 C 2 repeat expansion impairs its transcription, leading to C9ORF72 protein haploinsufficiency 5,6 , impaired function of myeloid cells 7,8 and diminished neuronal viability 9 . Both sense and antisense transcripts encompassing the HRE in V1 and V3 generate RNA foci and undergo translation into atypical, aggregation-prone dipeptide repeat (DPR) proteins in all open reading frames 10,11 . These unusual DPRs are toxic in several experimental model systems [12][13][14][15] . Despite important advances in elucidating the molecular pathology of the expanded hexanucleotide repeats, there are no meaningful therapies for C9ORF72-related ALS or FTD.ASOs can drive therapeutic effects by mechanisms that include splice-modulation or, if the ASO contains DNA, activation of endogenous RNase H 16 to degrade the target RNA. The broad bioavailability of ASOs in the central nervous system (CNS), including both neurons and glial cells 17 , has prompted development of ASOs as therapy for dominantly transmitted genetic disorders of the CNS (for example, ALS caused by mutations in the SOD1 gene).Here we report development of ASOs targeting C9ORF72 to treat ALS and FTD. Using different C9-related model systems, including patient-derived samples and two C9BAC transgenic mouse models 18,19 , we generated ASOs that specifically reduce levels of the transcripts harboring the HRE as well as their DPR products, with minimal effects on the most abundant V2 isoform, which does not contain the HRE. We show that modification of a subset of the phosphodiester internucleoside linkages significantly improves ASO tolerability without impairing potency. We demonstrate that, in a single patient harboring mutant C9ORF72 with the G 4 C 2 repeat expressions, repeated intrathecal dosing of the optimal ASO was well tolerated and led to significant and durable reduction in levels of cerebrospinal fluid (CSF) poly(GP). Results ASO suppresses C9ORF72 in fibroblasts and mouse neurons.Because haploinsufficiency of C9ORF72 is thought to be adverse, we developed ASOs that target only the 5′ end of transcripts V1 and V3 that bear the G 4 C 2 repeat expansion, sparing transcript V2. As it is not fully clear whether the repeat-containing intron is retained or spliced out, we focused our effort on ASO sequences targeting ...

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Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Cited by 4 publications

References 100 publications

Potent Mixed Backbone Antisense Oligonucleotide Safety Suppressed Expression of Mutant C9ORF72 Transcripts and Polypeptides: First in Human Pilot Study

Potent Mixed Backbone Antisense Oligonucleotide Safety Suppressed Expression of Mutant C9ORF72 Transcripts and Polypeptides: First in Human Pilot Study

Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate

Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide

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