The C9Orf72 hexanucleotide repeat expansion transcript forms insoluble aggregates facilitated by multimolecular G-quadruplex structures

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are just two of the neurodegenerative diseases characterised by the presence of pathological aggregates[1][1]. 40% of familiar cases in ALS and FTD have been correlated to expansion of the hexanucleotide repeat (GGGGCC) n , which has been previously shown to assemble into G-quadruplexes (G4s) structures[2][2]. In this study we investigated the role of nucleic acids and secondary structure formation in the generation of ALS/FTD aggregates, something that has often been relegated as a peripheral effect in the protein-led aggregation[3][3]. We showed a correlation between the emergence of multimolecular G4s (mG4s) formed by the DNA (GGGGCC) n repeats and the formation of protein free insoluble aggregates. We revealed that the aggregation is dependent on K+ concentration and repeat-length, suggesting that G4-formation plays a role in the the formation of aggregates. G4-structures were detected in the aggregates by staining with the G4-specific fluorescent dye NMM. G4-unfolding promoted by NMM-mediated guanine photooxidation led to prompt disassembly of the insoluble aggregates, further confirming a G4-based mechanism. To reinforce the physiological relevance of our observations, we characterised the aggregation of RNA (GGGGCC) n , which is thought to contribute to pathological aggregation in ALS/FTD. We observed that RNA repeats can aggregate at significant lower concentrations compared to DNA, suggesting that under physiological conditions RNA repeats can aggregate in the absence of any protein. Our findings constitute the first evidence supporting the formation of mG4-structures to drive protein-free aggregation, highlighting the potential of mG4s as therapeutic target to for the treatment of ALS and FTD. ![Figure][4] ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: pending:yes