Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations
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...
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| Format: | Article |
| Language: | English |
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BMC
2020-09-01
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| Series: | Molecular Neurodegeneration |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13024-020-00386-4 |
| _version_ | 1828917972661436416 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Anna Konopka |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T21:01:09Z |
| format | Article |
| id | doaj.art-3003b649896947f29c746f302d459637 |
| institution | Directory Open Access Journal |
| issn | 1750-1326 |
| language | English |
| last_indexed | 2024-12-13T21:01:09Z |
| publishDate | 2020-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Molecular Neurodegeneration |
| spelling | doaj.art-3003b649896947f29c746f302d4596372022-12-21T23:31:35ZengBMCMolecular Neurodegeneration1750-13262020-09-0115112810.1186/s13024-020-00386-4Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutationsAnna Konopka0Donna R. Whelan1Md Shafi Jamali2Emma Perri3Hamideh Shahheydari4Reka P. Toth5Sonam Parakh6Tina Robinson7Alison Cheong8Prachi Mehta9Marta Vidal10Audrey M. G. Ragagnin11Ivan Khizhnyak12Cyril J. Jagaraj13Jasmin Galper14Natalie Grima15Anand Deva16Sina Shadfar17Garth A. Nicholson18Shu Yang19Suzanne M. Cutts20Zuzana Horejsi21Toby D. M. Bell22Adam K. Walker23Ian P. Blair24Julie D. Atkin25Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityDepartment of Pharmacy and Biomedical Sciences, La Trobe Institute for Molecular Science, La Trobe UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityBrain and Mind Centre, Central Clinical School, Faculty of Medicine and Health, University of SydneyCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityDepartment of Plastic and Reconstructive Surgery, Macquarie University, and The Integrated Specialist Healthcare Education and Research FoundationCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceBarts Cancer Institute, Queen Mary University of LondonSchool of Chemistry, Monash UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityCentre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie UniversityAbstract 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.http://link.springer.com/article/10.1186/s13024-020-00386-4DNA damageTDP-43 mutationsNHEJSuper-resolution microscopy |
| spellingShingle | 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 Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations Molecular Neurodegeneration DNA damage TDP-43 mutations NHEJ Super-resolution microscopy |
| title | Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
| title_full | Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
| title_fullStr | Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
| title_full_unstemmed | Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
| title_short | Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations |
| title_sort | impaired nhej repair in amyotrophic lateral sclerosis is associated with tdp 43 mutations |
| topic | DNA damage TDP-43 mutations NHEJ Super-resolution microscopy |
| url | http://link.springer.com/article/10.1186/s13024-020-00386-4 |
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