Membrane Disruption Properties of Poly-Glycine Arginine Dipeptide Repeats Affected by Peptide Repeats Continuity and Membrane Composition.

C9ORF72 hexanucleotide expansion is the most common genetic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTD). This expansion can be translated into dipeptide repeats (DPRs) through repeat-associated non-ATG (RAN) translation. Arginine-rich DPRs, i.e., poly-glycine arginine (poly-GR) and poly-proline arginine (poly-PR) are considered the most toxic ones among the five types of DPRs. We recently discovered that poly-GR forms helical conformation and is able to penetrate cell membranes, leading to cytotoxicity, but the mechanism remains unclear. Here, we investigated the membrane disruption mechanism of poly-GR related to its sequence and membrane composition. To test this, we stopped its continuously repeated sequence by inserting several proline residues to disrupt its helical structure. We found that the modification reduced its cytotoxicity and membrane disruption capability. Next, we examined the influence of lipid composition on the membrane-disrupting ability of poly-GR using various liposomes. Poly-GR caused higher leakage in the negatively charged liposomes compared to the neutral or positively charged ones. Cholesterol content affected the extent of disruption, while gangliosides had no significant effect. Using small-angle x-ray scattering (SAXS), total internal reflection fluorescence (TIRF) microscopy, and atomic force microscopy (AFM), we observed the behavior of poly-GR on lipid membranes. Finally, we directly treated mouse neuroblastoma to modulate the cholesterol content and found that cholesterol depletion inhibited the internalization of poly-GR into the cells and reduced cytotoxicity. These findings reveal that the conformation of poly-GR and the lipid composition influence its membrane penetration, offering insights into potential therapeutic strategies for C9ORF72-related diseases.