TY - JOUR
T1 - Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations
AU - Konopka, Anna
AU - Whelan, Donna R.
AU - Jamali, Md Shafi
AU - Perri, Emma
AU - Shahheydari, Hamideh
AU - Toth, Reka P.
AU - Parakh, Sonam
AU - Robinson, Tina
AU - Cheong, Alison
AU - Mehta, Prachi
AU - Vidal, Marta
AU - Ragagnin, Audrey M.G.
AU - Khizhnyak, Ivan
AU - Jagaraj, Cyril J.
AU - Galper, Jasmin
AU - Grima, Natalie
AU - Deva, Anand
AU - Shadfar, Sina
AU - Nicholson, Garth A.
AU - Yang, Shu
AU - Cutts, Suzanne M.
AU - Horejsi, Zuzana
AU - Bell, Toby D.M.
AU - Walker, Adam K.
AU - Blair, Ian P.
AU - Atkin, Julie D.
PY - 2020/9/9
Y1 - 2020/9/9
N2 - 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.
AB - 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.
KW - DNA damage
KW - NHEJ
KW - Super-resolution microscopy
KW - TDP-43 mutations
UR - http://www.scopus.com/inward/record.url?scp=85090820933&partnerID=8YFLogxK
U2 - 10.1186/s13024-020-00386-4
DO - 10.1186/s13024-020-00386-4
M3 - Article
C2 - 32907630
AN - SCOPUS:85090820933
SN - 1750-1326
VL - 15
JO - Molecular Neurodegeneration
JF - Molecular Neurodegeneration
IS - 1
M1 - 51
ER -