Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:15 |
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| Enthalten in: |
Molecular neurodegeneration - 15(2020), 1 vom: 09. Sept., Seite 51 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Konopka, Anna [VerfasserIn] |
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| Links: |
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| Themen: |
DNA damage |
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| Anmerkungen: |
Date Completed 26.08.2021 Date Revised 26.08.2021 published: Electronic Citation Status MEDLINE |
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| doi: |
10.1186/s13024-020-00386-4 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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NLM31479879X |
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| 245 | 1 | 0 | |a Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
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| 500 | |a Date Revised 26.08.2021 | ||
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| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a DNA damage | |
| 650 | 4 | |a NHEJ | |
| 650 | 4 | |a Super-resolution microscopy | |
| 650 | 4 | |a TDP-43 mutations | |
| 650 | 7 | |a DNA-Binding Proteins |2 NLM | |
| 650 | 7 | |a TARDBP protein, human |2 NLM | |
| 650 | 7 | |a TDP-43 protein, mouse |2 NLM | |
| 700 | 1 | |a Whelan, Donna R |e verfasserin |4 aut | |
| 700 | 1 | |a Jamali, Md Shafi |e verfasserin |4 aut | |
| 700 | 1 | |a Perri, Emma |e verfasserin |4 aut | |
| 700 | 1 | |a Shahheydari, Hamideh |e verfasserin |4 aut | |
| 700 | 1 | |a Toth, Reka P |e verfasserin |4 aut | |
| 700 | 1 | |a Parakh, Sonam |e verfasserin |4 aut | |
| 700 | 1 | |a Robinson, Tina |e verfasserin |4 aut | |
| 700 | 1 | |a Cheong, Alison |e verfasserin |4 aut | |
| 700 | 1 | |a Mehta, Prachi |e verfasserin |4 aut | |
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| 700 | 1 | |a Ragagnin, Audrey M G |e verfasserin |4 aut | |
| 700 | 1 | |a Khizhnyak, Ivan |e verfasserin |4 aut | |
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| 700 | 1 | |a Shadfar, Sina |e verfasserin |4 aut | |
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