Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C‐terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC‐derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single‐molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self‐association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain‐of‐function as an underlying disease mechanism in KIF5A‐associated ALS.

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. In this study, we performed a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis and premature death. Our results suggest gain of function as an underlying disease mechanism in KIF5A-associated ALS.

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 (C4G2) antisense 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. 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 can activate the PKR/eIF2α pathway. These results provide a mechanism by which antisense repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9orf72 repeat expansions.

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.

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