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Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells

Abstract

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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Fig. 1: Design and schematic structures of chemically oligomerizable TDP-43.
Fig. 2: AP20187 induces oligomerization of TDP-43.
Fig. 3: AP20187 treatment induces oligomer formation of TDP43 followed by its cytosolic distribution.
Fig. 4: The chemically oligomerizable TDP-43 incorporate wt-TDP43 via multiple domains.
Fig. 5: The chemically oligomerized TDP-43 contains mobile fractions.
Fig. 6: The chemically oligomerized TDP-43 mimics the ALS pathology.

Data availability

All data are available in the main text. Further information and requests for resources and reagents should be addressed by Kohsuke Kanekura (kanekura@tokyo-med.ac.jp).

References

  1. 1.

    Logroscino G, Piccininni M, Marin B, Nichols E, Abd-Allah F, Abdelalim A, et al. Global, regional, and national burden of motor neuron diseases 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2018;17:1083–97.

    Article  Google Scholar 

  2. 2.

    Maurel C, Dangoumau A, Marouillat S, Brulard C, Chami A, Hergesheimer R, et al. Causative Genes in Amyotrophic Lateral Sclerosis and Protein Degradation Pathways: a Link to Neurodegeneration. Mol. Neurobiol. 2018;55:6480–99.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008;319:1668–72.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006;314:130–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 2006;351:602–11.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Morera AA, Ahmed NS, Schwartz JC. TDP-43 regulates transcription at protein-coding genes and Alu retrotransposons. Biochim Biophys Acta Gene Regul Mech. 2019;1862:194434.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Afroz T, Hock EM, Ernst P, Foglieni C, Jambeau M, Gilhespy LAB, et al. Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nat Commun. 2017;8:45.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Arnold ES, Ling SC, Huelga SC, Lagier-Tourenne C, Polymenidou M, Ditsworth D, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43. Proc Natl Acad Sci U S A. 2013;110:E736–745.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Highley JR, Kirby J, Jansweijer JA, Webb PS, Hewamadduma CA, Heath PR, et al. Loss of nuclear TDP-43 in amyotrophic lateral sclerosis (ALS) causes altered expression of splicing machinery and widespread dysregulation of RNA splicing in motor neurones. Neuropathol. Appl. Neurobiol. 2014;40:670–85.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Quinones-Valdez G, Tran SS, Jun HI, Bahn JH, Yang EW, Zhan L, et al. Regulation of RNA editing by RNA-binding proteins in human cells. Commun Biol. 2019;2:19.

    PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Wang C, Duan Y, Duan G, Wang Q, Zhang K, Deng X, et al. Stress induces dynamic, cytotoxicity-antagonizing TDP-43 nuclear bodies via paraspeckle LncRNA NEAT1-mediated liquid-liquid phase separation. Mol. Cell. 2020;79:443–58.e447.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Khalfallah Y, Kuta R, Grasmuck C, Prat A, Durham HD, Vande Velde C. TDP-43 regulation of stress granule dynamics in neurodegenerative disease-relevant cell types. Sci. Rep. 2018;8:7551.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Konopka A, Whelan DR, Jamali MS, Perri E, Shahheydari H, Toth RP, et al. Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations. Mol. Neurodegener. 2020;15:51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Mitra J, Guerrero EN, Hegde PM, Liachko NF, Wang H, Vasquez V, et al. Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proc Natl Acad Sci U S A. 2019;116:4696–705.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Iguchi Y, Katsuno M, Niwa J, Takagi S, Ishigaki S, Ikenaka K, et al. Loss of TDP-43 causes age-dependent progressive motor neuron degeneration. Brain. 2013;136:1371–82.

    PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

    Babinchak WM, Haider R, Dumm BK, Sarkar P, Surewicz K, Choi JK, et al. The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain. J. Biol. Chem. 2019;294:6306–17.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Wang, A, Conicella, AE, Schmidt, HB, Martin, EW, Rhoads, SN, Reeb, AN et al. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. EMBO J. 2018;37:e97452.

  18. 18.

    Jiang LL, Xue W, Hong JY, Zhang JT, Li MJ, Yu SN, et al. The N-terminal dimerization is required for TDP-43 splicing activity. Sci. Rep. 2017;7:6196.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. 19.

    Wang L, Kang J, Lim L, Wei Y, Song J. TDP-43 NTD can be induced while CTD is significantly enhanced by ssDNA to undergo liquid-liquid phase separation. Biochem. Biophys. Res. Commun. 2018;499:189–95.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Taslimi A, Vrana JD, Chen D, Borinskaya S, Mayer BJ, Kennedy MJ, et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat Commun. 2014;5:4925.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Mann JR, Gleixner AM, Mauna JC, Gomes E, DeChellis-Marks MR, Needham PG, et al. RNA binding antagonizes neurotoxic phase transitions of TDP-43. Neuron. 2019;102:321–38.e328.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Asakawa K, Handa H, Kawakami K. Optogenetic modulation of TDP-43 oligomerization accelerates ALS-related pathologies in the spinal motor neurons. Nat Commun. 2020;11:1004.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Polesskaya O, Baranova A, Bui S, Kondratev N, Kananykhina E, Nazarenko O, et al. Optogenetic regulation of transcription. BMC Neurosci. 2018;19:12.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. 24.

    Pouzet, S, Banderas, A, Le Bec, M, Lautier, T, Truan, G & Hersen, P. The promise of optogenetics for bioproduction: dynamic control strategies and scale-up instruments. Bioengineering (Basel). 2020;7:151.

  25. 25.

    Stockley JH, Evans K, Matthey M, Volbracht K, Agathou S, Mukanowa J, et al. Surpassing light-induced cell damage in vitro with novel cell culture media. Sci. Rep. 2017;7:849.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. 26.

    Clackson T, Yang W, Rozamus LW, Hatada M, Amara JF, Rollins CT, et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci U S A. 1998;95:10437–42.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Fegan A, White B, Carlson JC, Wagner CR. Chemically controlled protein assembly: techniques and applications. Chem Rev. 2010;110:3315–36.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Yang W, Rozamus LW, Narula S, Rollins CT, Yuan R, Andrade LJ, et al. Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket. J. Med. Chem. 2000;43:1135–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29.

    Minaki H, Sasaki K, Honda H, Iwaki T. Prion protein oligomers in Creutzfeldt-Jakob disease detected by gel-filtration centrifuge columns. Neuropathology. 2009;29:536–42.

    PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Kanekura K, Ma X, Murphy JT, Zhu LJ, Diwan A, Urano F. IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions. Sci Signal. 2015;8:ra62.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  31. 31.

    Lin W, Lin Y, Li J, Fenstermaker AG, Way SW, Clayton B, et al. Oligodendrocyte-specific activation of PERK signaling protects mice against experimental autoimmune encephalomyelitis. J. Neurosci. 2013;33:5980–91.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Johnson BS, Snead D, Lee JJ, McCaffery JM, Shorter J, Gitler AD. TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity. J. Biol. Chem. 2009;284:20329–39.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Pinarbasi ES, Cagatay T, Fung HYJ, Li YC, Chook YM, Thomas PJ. Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization. Sci. Rep. 2018;8:7083.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 34.

    Nonaka T, Masuda-Suzukake M, Arai T, Hasegawa Y, Akatsu H, Obi T, et al. Prion-like properties of pathological TDP-43 aggregates from diseased brains. Cell Rep. 2013;4:124–34.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Shimonaka S, Nonaka T, Suzuki G, Hisanaga S, Hasegawa M. Templated Aggregation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Seeding with TDP-43 Peptide Fibrils. J. Biol. Chem. 2016;291:8896–907.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Li HR, Chiang WC, Chou PC, Wang WJ, Huang JR. TAR DNA-binding protein 43 (TDP-43) liquid-liquid phase separation is mediated by just a few aromatic residues. J. Biol. Chem. 2018;293:6090–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Maekawa S, Leigh PN, King A, Jones E, Steele JC, Bodi I, et al. TDP-43 is consistently co-localized with ubiquitinated inclusions in sporadic and Guam amyotrophic lateral sclerosis but not in familial amyotrophic lateral sclerosis with and without SOD1 mutations. Neuropathology. 2009;29:672–83.

    PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Tanji K, Zhang HX, Mori F, Kakita A, Takahashi H, Wakabayashi K. p62/sequestosome 1 binds to TDP-43 in brains with frontotemporal lobar degeneration with TDP-43 inclusions. J. Neurosci. Res. 2012;90:2034–42.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Fecto F, Yan J, Vemula SP, Liu E, Yang Y, Chen W, et al. SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch. Neurol. 2011;68:1440–6.

    PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol. Cell. Biol. 2004;24:8055–68.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Bjorkoy G, Lamark T, Johansen T. p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy. 2006;2:138–9.

    PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Watanabe S, Inami H, Oiwa K, Murata Y, Sakai S, Komine O, et al. Aggresome formation and liquid-liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43. Cell Death Dis. 2020;11:909.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;323:1208–11.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    McDonald KK, Aulas A, Destroismaisons L, Pickles S, Beleac E, Camu W, et al. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1. Hum. Mol. Genet. 2011;20:1400–10.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, et al. TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics. Neuron. 2017;95:808–16. e809.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Bosque PJ, Boyer PJ, Mishra P. A 43-kDa TDP-43 species is present in aggregates associated with frontotemporal lobar degeneration. PLoS ONE. 2013;8:e62301.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Yamashita T, Hideyama T, Hachiga K, Teramoto S, Takano J, Iwata N, et al. A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology. Nat Commun. 2012;3:1307.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

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Acknowledgements

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.

Funding

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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Y.Y., M.K. and K.K. designed the research and wrote the paper. Y.Y., T.M., Y.H. and K.K. designed and performed all the imaging studies, vector constructions, immunoblot analyses, and FRAP analyses.

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Correspondence to Masahiko Kuroda or Kohsuke Kanekura.

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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

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