The C9Orf72 hexanucleotide repeat expansion aggregates by means of multimolecular G-quadruplex formation

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases genetically characterised by the expansion of the hexanucleotide repeat (GGGGCC)n. Here we investigated the formation of nucleic-acid secondary structures in the expansion repeats, and their role in generating aggregates that are characteristic of ALS/FDT. We observed significant aggregation of DNA (GGGGCC)n, which we ascribe to the formation of multimolecular G-quadruplexes (G4s), as confirmed with G4-selective fluorescent staining. Exposing the aggregates to G4-unfolding conditions, such as photo-oxidation of guanines, led to prompt disassembly, suggesting an essential role of G4s in the aggregation process. We then characterised aggregation in RNA (GGGGCC)n repeats, which are directly relevant to ALS/FTD pathology. We observed that the RNA repeats aggregate at significantly lower concentrations compared to DNA, strengthening the hypothesis that nucleic acids may play a structural role in pathological aggregation at physiological concentrations. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free aggregates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for ALS and FTD.