Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations

Anna Konopka, Donna R. Whelan, Md Shafi Jamali, Emma Perri, Hami Shahheydari, Reka P. Toth, Sonam Parakh, Tina Robinson, Alison Cheong, Prachi Mehta, Marta Vidal, Audrey M. G. Ragagnin, Ivan Khizhnyak, Cyril J. Jagaraj, Jasmin Galper, Natalie Grima, Anand Deva, Sina Shadfar, Garth A. Nicholson, Shu Yang & 6 others Suzanne M. Cutts, Zuzana Horejsi, Toby D. M. Bell, Adam K. Walker, Ian P. Blair, Julie D. Atkin*

*Corresponding author for this work

Research output: Contribution to journalArticle

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Abstract

Background: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response.

Methods: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage.

Results: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm.

Conclusions: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.
Original languageEnglish
Article number51
Pages (from-to)1-28
Number of pages28
JournalMolecular Neurodegeneration
Volume15
Issue number1
DOIs
Publication statusPublished - 9 Sep 2020

Bibliographical note

Copyright the Author(s) 2020. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • DNA damage
  • TDP-43 mutations
  • NHEJ
  • Super-resolution microscopy

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