Cell Stress and Chaperones

, Volume 23, Issue 6, pp 1177–1183 | Cite as

Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation

  • Akira Kitamura
  • Nodoka Iwasaki
  • Masataka KinjoEmail author
Original Paper


Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization.


Proteostasis Protein aggregation HSP FRAP ALS 



Amyotrophic lateral sclerosis


Fluorescence recovery after photobleaching


Fused in sarcoma/translated in liposarcoma


Green fluorescent protein


Heat shock cognate


Heat shock protein


Inclusion body


Red fluorescent protein


Sodium dodecyl sulfate


Transactive response element DNA/RNA-binding protein 43-kDa


25-kDa C-terminal fragment of TDP-43


Funding information

A.K. was supported by a Japan Society for Promotion of Science (JSPS) Grant-in-Aid for the Promotion of Joint International Research (Fostering Joint International Research) (16KK0156), a JSPS Grant-in-Aid for Scientific Research (C) (#26440090), a grant-in-aid from The Nakabayashi Trust for ALS Research (Tokyo, Japan), and by a grant-in-aid from the Japan Amyotrophic Lateral Sclerosis Association (JALSA, Tokyo, Japan) for ALS research. M.K. was partially supported by a grant-in-aid from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering.

Supplementary material

12192_2018_930_MOESM1_ESM.avi (34.5 mb)
ESM 1 Three-dimensional reconstruction of confocal super-resolution fluorescence image of a Neuro2A cell harboring G-TDP25 IBs and HSP70-R. Stack series were acquired using oil-immersion objective with high numerical aperture (1.4) and highly sensitive avalanche photodiode detectors. Green and magenta colors show G-TDP25 and HSP-70, respectively. Upper numerical value shows Z-stack position (μm). Scale bar, 1 μm. (AVI 35368 kb)


  1. Blokhuis AM, Groen EJ, Koppers M, van den Berg LH, Pasterkamp RJ (2013) Protein aggregation in amyotrophic lateral sclerosis. Acta Neuropathol 125:777–794CrossRefGoogle Scholar
  2. Brady OA, Meng P, Zheng Y, Mao Y, Hu F (2011) Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. J Neurochem 116:248–259CrossRefGoogle Scholar
  3. Brehme M, Voisine C, Rolland T, Wachi S, Soper JH, Zhu Y, Orton K, Villella A, Garza D, Vidal M, Ge H, Morimoto RI (2014) A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease. Cell Rep 9:1135–1150CrossRefGoogle Scholar
  4. Che MX, Jiang YJ, Xie YY, Jiang LL, Hu HY (2011) Aggregation of the 35-kDa fragment of TDP-43 causes formation of cytoplasmic inclusions and alteration of RNA processing. FASEB J: Official Publication Federation Am Societies Experimental Biology 25:2344–2353CrossRefGoogle Scholar
  5. Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding and proteostasis. Nature 475:324–332CrossRefGoogle Scholar
  6. He WT, Xue W, Gao YG, Hong JY, Yue HW, Jiang LL, Hu HY (2017) HSP90 recognizes the N-terminus of huntingtin involved in regulation of huntingtin aggregation by USP19. Sci Rep 7:14797CrossRefGoogle Scholar
  7. Ito D, Hatano M, Suzuki N (2017) RNA binding proteins and the pathological cascade in ALS/FTD neurodegeneration. Sci Transl Med 9:eaah5436CrossRefGoogle Scholar
  8. Kampinga HH, Craig EA (2010) The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 11:579–592CrossRefGoogle Scholar
  9. Kim S, Nollen EA, Kitagawa K, Bindokas VP, Morimoto RI (2002) Polyglutamine protein aggregates are dynamic. Nat Cell Biol 4:826–831CrossRefGoogle Scholar
  10. Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82:323–355CrossRefGoogle Scholar
  11. Kitamura A, Kinjo M (2018) Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy. Biophysics Physicobiology 15:1–7CrossRefGoogle Scholar
  12. Kitamura A, Kubota H, Pack CG, Matsumoto G, Hirayama S, Takahashi Y, Kimura H, Kinjo M, Morimoto RI, Nagata K (2006) Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state. Nat Cell Biol 8:1163–1170CrossRefGoogle Scholar
  13. Kitamura A, Nagata K, Kinjo M (2015) Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques. Int J Mol Sci 16:6076–6092CrossRefGoogle Scholar
  14. Kitamura A, Nakayama Y, Shibasaki A, Taki A, Yuno S, Takeda K, Yahara M, Tanabe N, Kinjo M (2016) Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity. Sci Rep 6:19230CrossRefGoogle Scholar
  15. Kitamura A, Yuno S, Muto H, Kinjo M (2017) Different aggregation states of a nuclear localization signal-tagged 25-kDa C-terminal fragment of TAR RNA/DNA-binding protein 43 kDa. Genes Cells: Devoted Molecular Cellular Mechanisms 22:521–534CrossRefGoogle Scholar
  16. Labbadia J, Morimoto RI (2015) The biology of proteostasis in aging and disease. Annu Rev Biochem 84:435–464CrossRefGoogle Scholar
  17. Ling SC, Polymenidou M, Cleveland DW (2013) Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79:416–438CrossRefGoogle Scholar
  18. Lopez T, Dalton K, Frydman J (2015) The mechanism and function of group II chaperonins. J Mol Biol 427:2919–2930CrossRefGoogle Scholar
  19. Matsuda T, Nagai T (2014) Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins. Microscopy 63:403–408CrossRefGoogle Scholar
  20. Matsumoto G, Stojanovic A, Holmberg CI, Kim S, Morimoto RI (2005) Structural properties and neuronal toxicity of amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase 1 aggregates. J Cell Biol 171:75–85CrossRefGoogle Scholar
  21. Neumann M, Sampathu DM, Kwong LK et al (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130–133CrossRefGoogle Scholar
  22. Pesiridis GS, Tripathy K, Tanik S, Trojanowski JQ, Lee VM (2011) A “two-hit” hypothesis for inclusion formation by carboxyl-terminal fragments of TDP-43 protein linked to RNA depletion and impaired microtubule-dependent transport. J Biol Chem 286:18845–18855CrossRefGoogle Scholar
  23. Renton AE, Chio A, Traynor BJ (2014) State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci 17:17–23CrossRefGoogle Scholar
  24. Schopf FH, Biebl MM, Buchner J (2017) The HSP90 chaperone machinery. Nat Rev Mol Cell Biol 18:345–360CrossRefGoogle Scholar
  25. Yahara M, Kitamura A, Kinjo M (2017) U6 snRNA expression prevents toxicity in TDP-43-knockdown cells. PLoS One 12:e0187813CrossRefGoogle Scholar
  26. Yamashita T, Hideyama T, Hachiga K, Teramoto S, Takano J, Iwata N, Saido TC, Kwak S (2012) A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology. Nat Commun 3:1307CrossRefGoogle Scholar
  27. Zhang YJ, Xu YF, Cook C et al (2009) Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci U S A 106:7607–7612CrossRefGoogle Scholar
  28. Zhang YJ, Gendron TF, Xu YF, Ko LW, Yen SH, Petrucelli L (2010) Phosphorylation regulates proteasomal-mediated degradation and solubility of TAR DNA binding protein-43 C-terminal fragments. Mol Neurodegener 5:33CrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2018

Authors and Affiliations

  • Akira Kitamura
    • 1
  • Nodoka Iwasaki
    • 1
  • Masataka Kinjo
    • 1
    Email author
  1. 1.Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life ScienceHokkaido UniversitySapporoJapan

Personalised recommendations