S-nitrosylation in Neuropathic pain and Autophagy

Abstract

Neuropathic pain is a maladaptive form of chronic pain caused by a primary injury or lesions in the central or peripheral nervous systems. Recently nitric oxide (NO) has emerged as important pro-nociceptive signaling molecule in pain signaling and processing. Chemical inhibiton or deletion of Nitric oxide synthase (NOS), as well and inhibition of the NOS-coenzyme tetrahydrobiopterin is known to reduce or inhibit neuropathic pain. NO exert its influence through two major pathways: by stimulation of sGC and by direct S-nitrosylation (SNO) of target proteins. This study assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24 h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NAME. At 24h after nerve injury, SNO-DIGE revealed 30 proteins with increased S-nitrosylation and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 out of these 53 proteins. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7 days after nerve injury showed that SNO modifications of protein disulfide isomerase (PDI), glutathione synthase (GSS) and peroxiredoxin-6 (Prdx6) had returned to baseline within 7 days whereas S-nitrosylation of mitochondrial aconitase 2 (Aco2) was further increased. The identified SNO modified proteins are primarily involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. Several targets, including PDI, Heat shock cognate 71 kDa protein, and Serpin B6, indicated that NO might play a role in protein quality control, metabolism, and folding. Subsequently an investigation into the potential role of NO and SNO in proteasomal degradation and autophagy was performed. Autophagy is a basic catabolic mechanism involving the degradation of unnecessary or dysfunctional cellular components through the lysosomal machinery and is an important factor in the recovery of neurons after injury. A cellular model of neuronal nitric oxide synthase (nNOS) over-expressing neuroblastoma cell culture stimulated with rapamycin to induce autophagy was used. The effects of nNOS overexpression on autophagic processes were evaluated by western blotting with antibodies for known markers of autophagy. S-nitrosylation was evaluated using a combination of SNO-DIGE, SNOSID, SNO-SILAC, and SNO-ELISA methods. Increased (+ 144%, p < 0.05) LC3-I / LC3-II ratio in the nNOS over-expressing cells compared to the wild type after stimulation with rapamycin suggested that the autophagic activity may be impaired by the increase of NO and possibly by an increase of direct protein S-nitrosylation. In the nNOS over-expressing cells the total ubiquitination was increased (+100%, p < 0.01) while it was decreased (-30%, p < 0.01)) in the wild type cells after rapamycin stimulation. This indicates that the targeting or degradation processes may be impaired by the increase in S-nitrosylation of the E1, E2 or E3 ubiquitin ligases. The results suggest that S-nitrosylation may regulate protein folding, ubiquitination, and possibly de novo protein generation. In this study several proteins have emerged as major candidates for being targets of S-nitrosylation and consequentially may affect the autophagic processes, namely heat shock cognate 71 kDa protein and pyruvate kinase isozymes M1/M2, calreticulin, ubiquitin-conjugating enzyme E2 D1, and elongation factor 2. The results suggest that NO, in addition to its known regulation of cGMP signaling, may contribute to the fine tuning of protein folding and degradation which are key mechanisms for allowing neurons to recover stability after axonal injury.