Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations
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(2020) 15:51
RESEARCH ARTICLE
Open Access
Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations Anna Konopka1, Donna R. Whelan2, Md Shafi Jamali1†, Emma Perri1†, Hamideh Shahheydari1, Reka P. Toth1, Sonam Parakh1, Tina Robinson3, Alison Cheong3, Prachi Mehta1, Marta Vidal1, Audrey M. G. Ragagnin1, Ivan Khizhnyak1, Cyril J. Jagaraj1, Jasmin Galper4, Natalie Grima1, Anand Deva5, Sina Shadfar1, Garth A. Nicholson1,6, Shu Yang1, Suzanne M. Cutts3, Zuzana Horejsi7, Toby D. M. Bell8, Adam K. Walker1,9, Ian P. Blair1 and Julie D. Atkin1,3*
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 TDP43 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 coimmunoprecipitation 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. (Continued on next page)
* Correspondence: [email protected] † Md Shafi Jamali and Emma Perri contributed equally to this work. 1 Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, 75 Talavera Road NSW, North Ryde, NSW 2109, Australia 3 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, Bundoora, VIC, Australia Full list of author information is available at the end of the article © The Author(s). 2020 Open Access
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