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

Abstract

GGGGCC (G4C2) 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 (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C4G2 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 C4G2 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 C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.

Traumatic injury causes selective degeneration and TDP-43 mislocalization in human iPSC-derivedC9orf72-associated ALS/FTD motor neurons

“…We showed that targeting the C9-HRE with an ASO designed against the sense transcript was partially neuroprotective, implicating the gain-of-function effects of the mutation in the increased susceptibility to severe trauma. The incomplete rescue of the ASO could be related to the contribution of anti-sense C9orf72 transcript driven toxicity [93,94] or simply insufficient dosing. Nevertheless, these experiments represent one of the first examples of a pharmacological pre-treatment that can successfully prevent post-traumatic neurodegeneration in human ALS patient neurons.…”

Dual-targeting CRISPR-CasRx reducesC9orf72ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

“…This is despite a growing body of evidence suggesting the antisense repeat expansion RNA contributes to disease pathogenesis. This includes the overt toxicity of antisense-derived polyPR in vitro and in vivo 17–19; recent work showing antisense repeat-containing RNA triggers activation of the PKR/eIF2α-dependent integrated stress response independent of DPRs and sense strand-related pathology, leading to aberrant stress granule formation 21 ; and data showing that only ASOs targeting antisense repeats are able to reduce TDP-43 loss of function cryptic splicing changes in C9orf72 patient iPSC-neurons 20 . The need to target antisense repeats appears particularly compelling given the recent failures of two clinical trials targeting sense repeats, which did not show clinical efficacy despite reducing sense pathologies (https://investors.biogen.com/news-releases/news-release-details/biogen-and-ionis-announce-topline-phase-1-study-results; https://www.thepharmaletter.com/article/wave-life-sciences-ends-wve-004-program).…”

“…To date, therapeutic strategies to target C9 ALS/FTD repeat RNAs have focused on targeting the sense G 4 C 2 repeat transcripts 12–16 , however, there is a considerable and growing body of evidence that supports the contribution of the antisense strand to C9 ALS/FTD disease pathogenesis 17–21 . Additionally, recent failures in clinical trial of two antisense oligonucleotides (ASOs) targeting C9orf72 sense repeat-containing transcripts (BIIB078; clinicaltrials.gov: NCT03626012 and WVE-004; clinicaltrials.gov: NCT04931862), despite showing target engagement and lowering of the sense transcript, highlight the importance of developing therapeutic approaches that can also target C9orf72 antisense repeat RNA transcripts.…”

RNA recognition by PKR during DNA virus infection

“…12 PKR has been demonstrated to play a significant role in a variety of stresses including viral infection, tumor suppression, cell growth and proliferation, regulation of mitosis, as well as neurodegeneration and metabolic diseases. [13][14][15][16] In the context of viral infections, PKR has been shown to restrict the replication of both DNA and RNA viruses. Here, we discuss mechanisms regulating PKR activation with an emphasis on RNA recognition by PKR.…”