One of the pathological hallmarks of amyotrophic lateral sclerosis (ALS) is mislocalized, cytosolic aggregation of TAR DNA-Binding Protein-43 (TDP-43). Not only TDP-43 per se is a causative gene of ALS but also mislocalization and aggregation of TDP-43 seems to be a common pathological change in both sporadic and familial ALS. The mechanism how nuclear TDP-43 transforms into cytosolic aggregates remains elusive, but recent studies using optogenetics have proposed that aberrant liquid–liquid phase separation (LLPS) of TDP-43 links to the aggregation process, leading to cytosolic distribution. Although LLPS plays an important role in the aggregate formation, there are still several technical problems in the optogenetic technique to be solved to progress further in vivo study. Here we report a chemically oligomerizable TDP-43 system. Oligomerization of TDP-43 was achieved by a small compound AP20187, and oligomerized TDP-43 underwent aggregate formation, followed by cytosolic mislocalization and induction of cell toxicity. The mislocalized TDP-43 co-aggregated with wt-TDP-43, Fused-in-sarcoma (FUS), TIA1 and sequestosome 1 (SQSTM1)/p62, mimicking ALS pathology. The chemically oligomerizable TDP-43 also revealed the roles of the N-terminal domain, RNA-recognition motif, nuclear export signal and low complexity domain in the aggregate formation and mislocalization of TDP-43. The aggregate-prone properties of TDP-43 were enhanced by a familial ALS-causative mutation. In conclusion, the chemically oligomerizable TDP-43 system could be useful to study the mechanisms underlying the droplet-aggregation phase transition and cytosolic mislocalization of TDP-43 in ALS and further study in vivo.
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All data are available in the main text. Further information and requests for resources and reagents should be addressed by Kohsuke Kanekura (firstname.lastname@example.org).
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The authors thank Addgene and Dr. Aaron Gitler and Dr. Michael Davidson for providing us TDP-43-YFP (Addgene plasmid #84911) and mCherry-Sequestosome1 (SQSTM1)-N-18 (Addgene plasmid #55132). We also thank Dr. Masaaki Matsuoka for providing us wt-FUS cDNA.
This work was supported by grants from the JSPS KAKENHI Grant numbers (16H06247, 17H03923 and 20H03593 to K.K., 17K15671 to Y.H., and 17H04067 and 21H02706 to M.K.). This work was also supported in part by the Japan Agency for Medical Research and Development (AMED) (16ek0109180h0001 and 17ae0101016s0904), Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.K.), Takeda Science Foundation (K.K.), Japan Intractable Diseases (Nanbyo) Research Foundation (K.K.), the Tokyo Biochemistry Research Foundation (K.K.), and the Ichiro Kanehara Foundation (K.K.).
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Yamanaka, Y., Miyagi, T., Harada, Y. et al. Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells. Lab Invest (2021). https://doi.org/10.1038/s41374-021-00623-4