The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (c9FTD/ALS). Recently, it was reported that an unconventional mechanism of repeat-associated non-ATG (RAN) translation arises from C9orf72 expansion. Sense and anti-sense transcripts of the expanded C9orf72 repeat, i.e. the dipeptide repeat protein (DRP) of glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA), are deposited in the brains of patients with c9FTD/ALS. However, the pathological significance of RAN-translated peptides remains unknown. We generated synthetic cDNAs encoding 100 repeats of DRP without a GGGGCC repeat and evaluated the effects of these proteins on cultured cells and cortical neurons in vivo. Our results revealed that the poly-GA protein formed highly aggregated ubiquitin/p62-positive inclusion bodies in neuronal cells. In contrast, the highly basic proteins poly-GR and PR also formed unique ubiquitin/p62-negative cytoplasmic inclusions, which co-localized with the components of RNA granules. The evaluation of cytotoxicity revealed that overexpressed poly-GA, poly-GP and poly-GR increased the substrates of the ubiquitin-proteasome system (UPS), including TDP-43, and enhanced the sensitivity to a proteasome inhibitor, indicating that these DRPs are cytotoxic, possibly via UPS dysfunction. The present data indicate that a gain-of-function mechanism of toxic DRPs possibly contributes to pathogenesis in c9FTD/ALS and that DRPs may serve as novel therapeutic targets in c9FTD/ALS.
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