DNA damage mediates changes in neuronal sensitivity induced by the inflammatory mediators, MCP-1 and LPS, and can be reversed by enhancing the DNA repair function of APE1

Abstract

Although inflammation-induced peripheral sensitization oftentimes resolves as an injury heals, this sensitization can be pathologically maintained and contribute to chronic inflammatory pain. Numerous inflammatory mediators increase the production of reactive oxygen (ROS) and nitrogen species (RNS) during inflammation and in animal models of chronic neuropathic pain. Our previous studies demonstrate that ROS/RNS and subsequent DNA damage mediate changes in neuronal sensitivity induced by anticancer drugs and by ionizing radiation in sensory neurons, thus we investigated whether inflammation and inflammatory mediators also could cause DNA damage in sensory neurons and whether that DNA damage alters neuronal sensitivity. DNA damage was assessed by pH2A.X expression and the release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Peripheral inflammation or exposure of cultured sensory neurons to the inflammatory mediators, LPS and MCP-1, elicited DNA damage. Moreover, exposure of sensory neuronal cultures to LPS or MCP-1 resulted in changes in the stimulated release of CGRP, without altering resting release or CGRP content. Genetically enhancing the expression of the DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE1) or treatment with a small-molecule modulator of APE1 DNA repair activity, both which enhance DNA repair, attenuated DNA damage and the changes in neuronal sensitivity elicited by LPS or MCP-1. In conclusion, our studies demonstrate that inflammation or exposure to inflammatory mediators elicits DNA damage in sensory neurons. By enhancing DNA repair, we demonstrate that this DNA damage mediates the alteration of neuronal function induced by inflammatory mediators in peptidergic sensory neurons.

Keywords: DNA damage; E3330; TRPV1; apurinic/apyrimidinic endonuclease 1/redox effector factor 1; dorsal root ganglia; inflammation.

Figure 1

DNA damage is enhanced in the lumbar DRG following hindpaw inflammation. A. Representative western blot of pH2A.X and vinculin (loading control) expression in contralateral and ipsilateral L4/L5 DRG 5 days following unilateral CFA injection into the rat hindpaw. B. Each column represents the mean ± SEM of the density of pH2A.X from 6 experiments normalized to the amount of vinculin. An asterisk indicates a statistically significant increase in the DRG ipsilateral to CFA injection compared to those contralateral to the injection using Student t-test. C. Photomicrographs (20X) of pH2A.X in L5 DRG from a rat 5 days after CFA injection. Green fluorescence indicates the immunoreactivity to pH2A.X.

Figure 2

DNA damage is enhanced in neuronal cultures in a time-dependent manner following exposure to inflammatory mediators. A. Representative western blots for pH2A.X and vinculin (loading control) from cultures grown in the absence or presence of LPS or MCP-1 for the indicated time periods. B. Each column represents the mean ± SEM of pH2A.X band density from 3 experiments normalized to that of vinculin following treatment with 1 μg/ml LPS (light bars) or 100 ng/ml MCP-1 (dark bars). An asterisk indicates a significant difference from expression at time zero using one-way ANOVA with Dunnett’s post-test.

Figure 3

CGRP release from neuronal cultures is altered following exposure to inflammatory mediators and the changes in DNA damage or stimulated CGRP release following exposure to LPS or MCP-1 are reversed by antagonists to the TLR4 (LPS) and CCR2 (MCP-1 and LPS). A B. Columns represent the mean ± SEM of CGRP release stimulated by a 10-minute exposure to 30 nM capsaicin following a 24 hr exposure to increasing concentrations of LPS (A) or MCP-1 (B). An asterisk indicates a significant difference from release in the absence of LPS or MCP-1 using one-way ANOVA with Dunnett’s post-test. C–D. Columns represent the mean ± SEM of the density of pH2A.X from 3–4 experiments normalized to the amount of vinculin following a 24hr exposure to LPS (C) or MCP-1 (D) in the absence or presence of LPS-RS or RS 50493, as indicated. E–F. Columns represent the mean ± SEM of CGRP release stimulated by a 10-minute exposure to 30 nM capsaicin following a 24hr exposure to LPS (E) or MCP-1 (F) in the absence or presence of LPS-RS or RS 50493, as indicated. An asterisk indicates a significant difference from expression/release in the presence of vehicle treatment in the absence of antagonists, whereas a cross indicates a significant difference from expression/release in the presence of inflammatory mediator treatment compared to inflammatory mediator ± antagonist using two-way ANOVA with Dunnett’s post-test.

Figure 4

The effects of LPS to induce DNA damage and inhibit CGRP release are reversed by increasing APE1-mediated DNA repair. A. Treatment schema. B. Representative western blots for pH2A.X, APE1, HA tag, and vinculin (loading control) from cultures grown in the absence or presence of LPS for 24hr following the indicated pretreatments. C. Each column represents the mean ± SEM of pH2A.X band density from 3 experiments normalized to vinculin induced by treatment with 3 μg/ml LPS following the indicated pretreatments in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars). An asterisk indicates a significant difference from expression compared to SCsiRNA-treated vector control using two-way ANOVA with Dunnett’s multiple comparisons post-test. D. Each column represents the mean ± SEM of CGRP release (expressed as % of total content) stimulated by capsaicin following treatment with 3 μg/ml LPS in the absence and presence of APE1 overexpression, as indicated, in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars). An asterisk indicates a significant difference from release in the absence of LPS using two-way ANOVA with Dunnett’s multiple comparisons post-test.

Figure 5

The effects of MCP-1 to induce DNA damage and augment CGRP release are reversed by increasing APE1-mediated DNA repair. A. Treatment schema. B. Representative western blots for pH2A.X, APE1, HA tag, and vinculin (loading control) from cultures grown in the absence or presence of MCP-1 for 24hr following the indicated pretreatments. C. Each column represents the mean ± SEM of pH2A.X band density from 4 experiments normalized to vinculin induced by treatment with 0.3 μg/ml MCP-1 following the indicated pretreatments in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars). An asterisk indicates a significant difference from expression compared to SCsiRNA-treated vector control using two-way ANOVA with Dunnett’s multiple comparisons post-test. D. Each column represents the mean ± SEM of CGRP release (expressed as % of total content) stimulated by capsaicin following treatment with 0.3 μg/ml MCP-1 in the absence and presence of APE1 overexpression, as indicated, in conjunction with SCsiRNA (light bars) or APE1siRNA (dark bars). An asterisk indicates a significant difference from release in the absence of MCP-1 using two-way ANOVA with Dunnett’s multiple comparisons post-test.