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 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.

ALS monocyte-derived microglia reveal cytoplasmic TDP-43 accumulation, DNA damage, and cell-specific impairment of phagocytosis associated with disease progression

“…We show that MDMi from ALS patients displayed increased γH2AX foci and pan-nuclear γH2AX phosphorylation in ALS MDMi compared to HC MDMi under basal conditions. This result is in line with other reports showing that ALS tissues exhibit increased DNA damage foci, likely mediated by oxidative and replication stress, as recently substantiated in studies of different ALS cell models (Konopka et al, 2020; Mitra et al, 2019; Riancho et al, 2020). Although it has been shown that TDP-43 is recruited less efficiently to DNA damage foci leading to unresolved DDR (Mitra et al, 2019), further studies are needed to examine DDR signalling pathways in ALS MDMi.…”

“…Given that TDP-43 mislocalisation is involved in the accumulation of DNA damage (Konopka et al, 2020), we evaluated DNA damage in ALS MDMi using an antibody recognising phosphorylated H2AX (γH2AX), which labels DNA double-strand breaks (DSBs). We found significantly increased γH2AX foci positive cells in ALS (30%) compared to HC MDMi (4%) (Fig.…”

“…Taken together, the interplay between TDP-43 mislocalisation, neuroinflammation and DNA-damage is a complex process. For instance, (1) dysregulation of TDP-43 can lead to impaired DNA damage (Mitra et al, 2019), DDR, (Konopka et al, 2020), and (2) inflammation (Zhu, Cynader, & Jia, 2015). (3) Unresolved DNA damage and aberrant cytosolic DNA leading to impaired DDR signalling contributes to neuroinflammation (Quek et al, 2017).…”

Emerging Evidence Highlighting the Importance of Redox Dysregulation in the Pathogenesis of Amyotrophic Lateral Sclerosis (ALS)

“…However, ultimately it is imperative to elucidate the primary, upstream mechanisms responsible for neurodegeneration in ALS so that effective therapeutics can be designed. These mechanisms include impaired axonal transport (Collard et al, 1995 ; Williamson and Cleveland, 1999 ), neurofilament aggregation (Al-Chalabi et al, 1995 ; Xiao et al, 2006 ; Xu Z. et al, 2016 ), protein misfolding (Kopito, 2000 ; Basso et al, 2006 ), abnormal RNA processing (Chen et al, 2010 ; Dejesus-Hernandez et al, 2011 ; Parisi et al, 2013 ; Droppelmann et al, 2014 ), lipid peroxidation (Shibata et al, 2001 ) and cholesterol esterification (Cutler et al, 2002 ; Chaves-Filho et al, 2019 ), defects in nucleocytoplasmic transport (Boeynaems et al, 2016 ), induction of DNA damage (Konopka and Atkin, 2018 ; Naumann et al, 2018 ; Konopka et al, 2020 ), cytoplasmic mislocalization of nuclear proteins (Neumann et al, 2006 ), mitochondrial dysfunction (Albers and Beal, 2000 ), glutamate excitotoxicity (Shaw and Ince, 1997 ), proteasomal and autophagic dysfunction (Chen et al, 2012 ), ER stress (Nagata et al, 2007 ; Walker et al, 2010 ), mitochondrial associated membrane (MAM) dysfunction (Watanabe et al, 2016 ), ER-Golgi transport defects (Atkin et al, 2014 ; Soo et al, 2015 ), autophagy dysregulation and apoptosis (Ravits et al, 2013 ; Robberecht and Philips, 2013 ; Gao et al, 2017 ; Mandrioli et al, 2020 ). For a detailed discussion of these mechanisms, please see several excellent reviews (Zarei et al, 2015 ; Taylor et al, 2016 ; Weishaupt et al, 2016 ; Mejzini et al, 2019 ).…”

“…This implies the presence of defects in DNA nick ligation and oxidative damage, and DNA repair mechanisms in FUS-associated ALS (Wang H. et al, 2018 ). Recently, TDP-43 was also shown to have a role in DNA repair (Mitra et al, 2019 ; Konopka et al, 2020 ), which is linked to oxidative stress (Guerrero et al, 2019 ). Collectively, these studies suggest that defects in DNA damage are a component of dysregulated redox homeostasis in ALS.…”