Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and inCaenorhabditis elegansmutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression.

No abstract

HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.