Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD

Abstract

Background: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. Methods and results: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. Conclusions: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Crosstalk between Different DNA Repair Pathways Contributes to Neurodegenerative Diseases

“…A non-canonical translation mechanism leads to the production of five dipeptide repeat proteins (DPRs) from the hexanucleotide repeat expanded C9ORF72 gene. A recent study indicated that, among the five DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and decrease the efficiency of NHEJ, single-strand annealing and microhomology-mediated end joining (MMEJ) [ 118 ]. Consistently, increased levels of several DDR markers (γH2AX, phosphorylated ATM, cleaved PARP1 and 53BP1) were observed in spinal cord motor neurons of C9ORF72 ALS patients [ 119 ].…”

Emerging Perspectives on Dipeptide Repeat Proteins in C9ORF72 ALS/FTD

“…Besides protein sequestration and proteasomal inhibition, poly-GA expression was found to decrease the efficiency of DNA double-strand break repair mechanisms, specifically impacting non-homologous end joining, single-strand annealing, and microhomology-mediated end joining processes ( Andrade et al, 2020 ). Furthermore, mobile poly-GA aggregates can be found in the axons and dendrites of primary cortical and motor neurons overexpressing the poly-GA DPR ( Jensen et al, 2020 ).…”

Emerging Technologies for Genome-Wide Profiling of DNA Breakage

“…Neurons are nonetheless capable of utilizing other repair pathways including single strand annealing (SSA) and microhomology-mediated end joining (MMEJ) that require small stretches of sequence homology (<25 nucleotides) to direct repair (Bhargava et al, 2016 ). Homology-directed DNA DSB repair pathways appear to be particularly relevant for the repair of transcribed DNA and may play a role in neurodegenerative disease (Keskin et al, 2014 ; McDevitt et al, 2018 ; Welty et al, 2018 ; Yasuhara et al, 2018 ; Andrade et al, 2020 ). Overall, accumulating DNA damage is characteristic of many neurological disorders, including aging and age-related neurodegenerative disease [for review consider (McKinnon, 2017 )].…”