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

GGGGCC (GC) hexanucleotide repeat expansion in the 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 (CG) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that antisense CG 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 CG RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of FTD/ALS patients. Finally, only antisense CG, but not sense GC, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense CG repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by repeat expansions.