Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway

To investigate the role and possible mechanisms of transactive response DNA binding protein 43 (TDP-43) in mediating neuronal injury induced by oxidative stress in mouse neuro-2a (N2a) cells and mouse pain sensitization.

① To evaluate the optimal induction concentration, N2a cells were treated with different concentrations of H₂O₂, and the cells were divided into control group, 200, 400 and 800 μmol/L H₂O₂ groups. ②To assess the optimal induction duration, N2a cells were treated with 400 μmol/L H₂O₂, and the cells were divided into control group, and the cell groups treated for 6, 12 and 24 h, respectively. ③To validate the mitochondrial DNA (mtDNA) release pathway, cyclosporin A (CsA) was used to inhibit the mitochondrial permeability transition pore (mPTP), and the cells were divided into control group, 24 h H₂O₂ group and 24 h H₂O₂+CsA group. ④To validate TDP-43-mediated cellular damage, the cells were divided into control group, 24 h H₂O₂ group and 24 h H₂O₂+siTDP-43 group. ⑤ Cell viability was assessed using CCK-8 assay, while cell proliferation was determined using EdU assay. Western blot analysis was employed to examine the expression levels of TDP-43, neuronal nuclei (NeuN), cyclic GMP-AMP synthase (cGAS), and stimulator of interferon genes (STING). qPCR was utilized to measure the release of mtDNA. Immunostaining was conducted to observe intracellular expression of TDP-43, and Calcein AM staining was employed to evaluate mPTP opening status. ⑥To elucidate the role of TDP-43 in neuropathic pain (NP), 24 healthy SPF male C57BL/6J mice (6~8 weeks old, 25~30 g) were randomly divided into control group, chronic constriction injury (CCI) group, and CCI+siTDP-43 group. In 1 d before and 7, 14 and 21 d after surgery, intrathecal injections of siTDP-43 were administered. Mechanical and thermal pain thresholds of the mice were assessed using von Frey filaments and radiant heat, respectively, on 1 preoperatively and 1, 3, 5, 7, 14 and 21 d postoperatively. Immunofluorescence assay was conducted on 21 d postoperatively to examine the changes in TDP-43 and NeuN in the lumbar spinal dorsal horn (L5-L6).

Oxidative stress induced a significant increase in TDP-43 protein level in N2a cells, prompted mtDNA release through mPTP, markedly up-regulated the expression of cGAS and STING, and consequently impacted the viability of N2a cells (P<0.05). CsA treatment inhibited mPTP channel opening and thus effectively blocked mtDNA release (P<0.05), down-regulated TDP-43 and thus significantly reduced mtDNA release, suppressed the expression of cGAS and STING, and finally restored the proliferation ability of N2a cells (P<0.05). The mechanical and thermal pain thresholds exhibited a significant decrease since 5 d after CCI, which then persisted until 21 d (P<0.05). The expression of TDP-43 in spinal cord dorsal horn neurons was increased in the mice in 21 d after CCI (P<0.05), and intrathecal injection of siRNA inhibited TDP-43 expression and effectively increased the mechanical and thermal pain thresholds in the CCI mice (P<0.05).

Oxidative stress induces an up-regulation in TDP-43 protein in neurons, which stimulates the release of mtDNA into the cytoplasm through mPTP, and subsequently activates the cGAS/STING pathway, and finally, results in neuronal injury and pain sensitization in CCI mice.