Reduction of pathological R-loop accumulation is associated with an improved microenvironment in spinal cord injury

Spinal cord injury (SCI) leads to severe neurological impairment, accompanied by a cascade of secondary pathological events, including inflammation, oxidative stress, and DNA damage. R-loops, three-stranded nucleic acid structures consisting of RNA-DNA hybrids and single-stranded DNA, have emerged as critical regulators of genome stability. However, their role in SCI pathogenesis remains underexplored. To elucidate the dynamics of R-loop formation in SCI, a mouse model was employed to analyze the spatiotemporal accumulation of R-loops through DNA-RNA immunoprecipitation followed by high-throughput sequencing (DRIPc-Seq). Primary neuronal, astrocytic, and microglial cultures were utilized to assess the functional consequences of R-loop accumulation. Lentiviral-mediated overexpression of RNase H1, a ribonuclease that resolves R-loops, was implemented both in vitro and in vivo. Behavioral testing, histopathological analysis, and molecular assays were conducted to evaluate neurological recovery, cellular viability, apoptosis, ferroptosis, inflammation, and DNA damage. SCI induced a rapid and transient accumulation of R-loops in the acute phase (3–7 days post-injury), primarily within gene body regions, which correlated with heightened neuronal apoptosis, ferroptosis, and glial activation. Overexpression of RNase H1 resulted in a marked reduction of R-loop levels, alleviating DNA damage, suppressing pro-inflammatory cytokine secretion, and promoting neuronal survival. Behavioral assessments revealed significant improvements in motor function recovery, while histological analysis indicated reduced tissue damage, less glial scar formation, and enhanced neuroprotection in SCI mice overexpressing RNase H1. Furthermore, molecular assays confirmed enhanced antioxidant capacity and decreased oxidative stress in the treatment group. Pathological R-loop accumulation is associated with aggravated neuronal injury and glial inflammatory responses after SCI. In a pre-injury overexpression model, RNase H1-mediated reduction of R-loop burden was associated with improved histological and functional outcomes, supporting a proof-of-concept for targeting R-loop homeostasis in SCI. These findings not only offer novel insights into the molecular mechanisms underlying SCI but also provide a potential avenue for the development of targeted treatments for SCI and other neurodegenerative disorders.