TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loopmediated DNA damage

TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SHSY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R-loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R-loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.Research in C.C’s lab was funded by Fondazione Regionale per la Ricerca Biomedica for TRANS–ALS (Translating Molecular Mechanisms into ALS risk and patient’s well-being: FRRB 2015- 0023) and in A.A.’s lab was funded by grants from the European Research Council (ERC2014- AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058- P), and the European Union (FEDER). A.B.F. was supported by a Juan de la Cierva-Formación fellowship (FJCI-2017-34536) from Spanish Ministry of Science and Innovation. M.G. was recipient of a scholarship provided by Erasmus+ Mobility for Traineeships from University of Pavia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript