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

Molecular neurodegeneration (2020-09-11)
Anna Konopka, Donna R Whelan, Md Shafi Jamali, Emma Perri, Hamideh 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, Suzanne M Cutts, Zuzana Horejsi, Toby D M Bell, Adam K Walker, Ian P Blair, Julie D Atkin
ABSTRACT

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. 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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Etoposide, synthetic, 98.0-105.0%, powder
Sigma-Aldrich
Anti-GAPDH antibody, Mouse monoclonal, clone GAPDH-71.1, purified from hybridoma cell culture
Sigma-Aldrich
Goat Anti-Rabbit IgG Antibody, Peroxidase Conjugated, 1 mg/mL (after reconstitution), Chemicon®
Sigma-Aldrich
Goat Anti-Mouse IgG & IgM Antibody, HRP conjugate, 0.8 mg/mL, Chemicon®
Sigma-Aldrich
Anti-phospho-TDP-43 (pSer410) antibody produced in rabbit, ~1.0 mg/mL, affinity isolated antibody
Sigma-Aldrich
MISSION® esiRNA, targeting human TARDBP

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