Antisense, but not sense, repeat expanded RNAs activate PKR/eIF2a-dependent ISR in C9ORF72 FTD/ALS

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/eIF2a-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/eIF2a 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/eIF2a 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.