Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis

Abstract

Dominant mutations in the RNA/DNA-binding protein TDP-43 have been linked to amyotrophic lateral sclerosis (ALS). Here, we screened genomic DNA extracted from spinal cord specimens of sporadic ALS patients for mutations in the TARDBP gene and identified a patient specimen with previously reported Q331K mutation. The patient spinal cord tissue with Q331K mutation showed accumulation of higher levels of DNA strand breaks and the DNA double-strand break (DSB) marker γH2AX, compared to age-matched controls, suggesting a role of the Q331K mutation in genome-damage accumulation. Using conditional SH-SY5Y lines ectopically expressing wild-type (WT) or Q331K-mutant TDP-43, we confirmed the increased cytosolic sequestration of the poly-ubiquitinated and aggregated form of mutant TDP-43, which correlated with increased genomic DNA strand breaks, activation of the DNA damage response factors phospho-ataxia-telangiectasia mutated (ATM), phospho-53BP1, γH2AX and neuronal apoptosis. We recently reported the involvement of WT TDP-43 in non-homologous end joining (NHEJ)-mediated DSB repair, where it acts as a scaffold for the recruitment of XRCC4-DNA ligase 4 complex. Here, the mutant TDP-43, due to its reduced interaction and enhanced cytosolic mislocalization, prevented the nuclear translocation of XRCC4-DNA ligase 4. Consistently, the mutant cells showed significantly reduced DNA strand break sealing activity and were sensitized to DNA-damaging drugs. In addition, the mutant cells showed elevated levels of reactive oxygen species, suggesting both dominant negative and loss-of-function effects of the mutation. Together, our study uncovered an association of sporadic Q331K mutation with persistent genome damage accumulation due to both damage induction and repair defects.

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

“…In this study we also demonstrate that TDP-43 is involved in the phosphorylation of γH2AX, further implying that TDP-43 functions upstream of 53BP1. In addition, DNA damage was detected in mutant TDP-43 Q331K patient tissues, and in vitro, TDP-43 Q331K mislocalisation to the cytoplasm prevented nuclear translocation of the XRCC4-DNA Ligase 4 complex [32]. In this study we now demonstrate that expression of TDP-43 Q331K and other TDP-43 ALS mutants, M337V and A315Ts impair NHEJ repair.…”

“…This was subsequently linked to deficiencies in R loop repair and defective ubiquitylation of H2A, which impair DSB signalling [29]. TDP-43 has also been implicated in R-loop repair [30] and two recent studies link TDP-43 to DNA damage [31,32]. It has also been reported that TDP-43 is a component of NHEJ, acting as a scaffold for recruitment of the break-sealing XRCC4-DNA ligase 4 complex.…”

“…It has also been reported that TDP-43 is a component of NHEJ, acting as a scaffold for recruitment of the break-sealing XRCC4-DNA ligase 4 complex. Moreover, nuclear loss of TDP-43 is linked to defects in the repair of DSBs [31], and expression of TDP-43 mutant Q331K perturbs repair of DSBs, by inhibiting NHEJ associated DNA Ligase 4 [32].…”

The role of DNA damage response in amyotrophic lateral sclerosis

“…Pathologic TDP43 mislocalization activates the mitochondrial unfolded protein response [ 62 ], elevates ROS levels and affects cytoplasmic-nuclear trafficking, eventually leading to increased neuronal stress and subsequent cell death [ 63 , 64 ]. Associated with such stress, GIN accumulation was described in sALS and fALS patients as well as in model organisms with orthologous TDP43 loss-of-function [ 34 ].…”

“…Although initially the connection between TDP43 dysfunction and the accumulation of GIN in ALS was thought to be a secondary feature, recent evidence shows that neuronal TDP43 plays an important direct role in DDR by controlling the nuclear recruitment of the XRCC4-DNA ligase 4 (LIG4) complex, critical for DSB repair via NHEJ [ 63 ]. In ALS/FTD, TDP43 nuclear exclusion incapacitates the transport of XRCC4/LIG4 leading to abortive NHEJ with consequent accumulation of toxic DSBs.…”