Ho Chia-Yi, Chang Yu-Jen, Yang Chih-Wen, Shih Orion, Jeng U-Ser, Hwang Ing-Shouh, Huang Wan-Chen, Chen Yun-Ru
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan; Department of Biochemical Science & Technology, National Taiwan University, Taipei 106, Taiwan.
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
J Mol Biol. 2025 Sep 1;437(17):169296. doi: 10.1016/j.jmb.2025.169296. Epub 2025 Jun 11.
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.
C9ORF72六核苷酸重复扩增是肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）中最常见的基因突变。这种扩增可通过重复相关非ATG（RAN）翻译转化为二肽重复序列（DPRs）。富含精氨酸的DPRs，即聚甘氨酸精氨酸（poly-GR）和聚脯氨酸精氨酸（poly-PR），被认为是五种DPRs中毒性最强的。我们最近发现poly-GR形成螺旋构象并能够穿透细胞膜，导致细胞毒性，但其机制仍不清楚。在这里，我们研究了与poly-GR序列和膜组成相关的膜破坏机制。为了验证这一点，我们通过插入几个脯氨酸残基来中断其连续重复序列，以破坏其螺旋结构。我们发现这种修饰降低了其细胞毒性和膜破坏能力。接下来，我们使用各种脂质体研究了脂质组成对poly-GR膜破坏能力的影响。与中性或带正电荷的脂质体相比，poly-GR在带负电荷的脂质体中引起更高的渗漏。胆固醇含量影响破坏程度，而神经节苷脂没有显著影响。使用小角X射线散射（SAXS）、全内反射荧光（TIRF）显微镜和原子力显微镜（AFM），我们观察了poly-GR在脂质膜上的行为。最后，我们直接处理小鼠神经母细胞瘤以调节胆固醇含量，发现胆固醇耗竭抑制了poly-GR进入细胞的内化并降低了细胞毒性。这些发现揭示了poly-GR的构象和脂质组成影响其膜穿透，为C9ORF72相关疾病的潜在治疗策略提供了见解。
