Dipeptide repeats (DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). Within these DPRs, the most abundant glycine-alanine (GA) DPRs form insoluble inclusions in C9orf72-ALS patients. While GA DPRs aggregates have been considered as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers generated during the amyloidogenesis has not yet been explored due to its unstable and fast equilibrium nature. In this study,

more »

... we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows to monitor the oligomerization process of GA DPRs in cells in nanoscale. By combining lifetime-based and super-resolution fluorescence microscopies with biophysical tools, we thoroughly examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provide direct ex vivo and in vitro evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane integrity. In addition, we found GA DPRs sabotage Ran protein gradient, hamper nucleocytoplasmic shuttling in neurons, and cause the mislocalization of TAR DNA-binding protein 43 in primary cortical neurons. Our results highlight the nanoscopic properties and toxicity of GA DPRs oligomers in living cells, which is a key step toward elucidating the pathological roles of C9orf72 DPRs in disease.