Melatonin relieves neuropathic allodynia through spinal MT2‐enhanced PP2Ac and downstream HDAC4 shuttling‐dependent epigenetic modification of hmgb1 transcription

Abstract

Melatonin (MLT; N-acetyl-5-methoxytryptamine) exhibits analgesic properties in chronic pain conditions. While researches linking MLT to epigenetic mechanisms have grown exponentially over recent years, very few studies have investigated the contribution of MLT-associated epigenetic modification to pain states. Here, we report that together with behavioral allodynia, spinal nerve ligation (SNL) induced a decrease in the expression of catalytic subunit of phosphatase 2A (PP2Ac) and enhanced histone deacetylase 4 (HDAC4) phosphorylation and cytoplasmic accumulation, which epigenetically alleviated HDAC4-suppressed hmgb1 gene transcription, resulting in increased high-mobility group protein B1 (HMGB1) expression selectively in the ipsilateral dorsal horn of rats. Focal knock-down of spinal PP2Ac expression also resulted in behavioral allodynia in association with similar protein expression as observed with SNL. Notably, intrathecal administration with MLT increased PP2Ac expression, HDAC4 dephosphorylation and nuclear accumulation, restored HDAC4-mediated hmgb1 suppression and relieved SNL-sensitized behavioral pain; these effects were all inhibited by spinal injection of 4P-PDOT (a MT2 receptor antagonist, 30 minutes before MLT) and okadaic acid (OA, a PP2A inhibitor, 3 hr after MLT). Our findings demonstrate a novel mechanism by which MLT ameliorates neuropathic allodynia via epigenetic modification. This MLT-exhibited anti-allodynia is mediated by MT2-enhanced PP2Ac expression that couples PP2Ac with HDAC4 to induce HDAC4 dephosphorylation and nuclear import, herein increases HDAC4 binding to the promoter of hmgb1 gene and upregulates HMGB1 expression in dorsal horn neurons.

Spinal Fbxo3-Dependent Fbxl2 Ubiquitination of Active Zone Protein RIM1α Mediates Neuropathic Allodynia through CaV2.2 Activation

“…A previous study demonstrated that SNL-enhanced spinal Fbxo3 expression underlies the development of neuropathic pain by affecting postsynaptic components of the spinal neural circuitry (Lin et al, 2015b). Moreover, the above results showed that pharmacological antagonism of spinal Fbxo3 alleviated SNL-induced allodynia by modifying presynaptic RIM1␣-dependent machinery.…”

“…Ligations of spinal nerves in rats were performed under isoflurane anesthesia (induction 5%, maintenance 2% in oxygen), following the methods of a previous study (Lin et al, 2016). After isoflurane anesthesia, rats were placed in a prone position.…”

“…A recent study has found that Fbxl2, a synapse-localized E3 ubiquitin ligase, directly binds to and ubiquitinates RIM1 to regulate synaptic vesicle release (Yao et al, 2007). As our previous study showed that Fbxo3, another E3-ubiquitin ligase subunit, contributes to the development of neuropathic pain by destabilizing Fbxl2 (Lin et al, 2015a), we speculated that presynaptic RIM1␣ contributes to neuropathic pain through Fbxo3-abating Fbxl2-dependent RIM1␣ ubiquitination in the SPM of the dorsal horn. To address this issue, we first assayed spinal Fbxo3 and Fbxl2 expression in the SPM following nerve ligation.…”

“…Next, we examined whether the intrathecal application of BC-1215, a novel activity inhibitor of Fbxo3, could modify SNL-induced behavioral allodynia. The results of the von Frey test demonstrated that a single injection bolus of BC-1215 (100 nM, 10 l), but not the vehicle solution (10 l), significantly ameliorated SNL-induced behavioral allodynia by 3 h after injection (from 0.58 Ϯ 0.15 to 6.28 Ϯ 0.52 g, n ϭ 7), a time point at which BC-1215 was shown to exhibit maximal effect on withdrawal threshold of SNL rats (Lin et al, 2015b). Nevertheless, application of neither the vehicle solution nor BC-1215 (100 nM, 10 l) affected the withdrawal threshold of the contralateral hindpaw at this time point (Fig.…”

“…Together, these studies link the Fbxo3/Fbxl2 cascade to RIM1␣ unubiquitination and activation. As RIM1␣ is known to be essential for Ca V 2.2-associated vesicle exocytosis (Schoch et al, 2006) and our laboratory has demonstrated that Fbxo3/Fbxl2 signaling contributes to the spinal plasticity underlying neuropathic pain (Lin et al, 2015b), we wondered whether the Fbxo3/ Fbl2 cascade contributes to the spinal plasticity underlying neuropathic pain development by affecting RIM1␣ and the subsequent Ca V 2.2-dependent neurotransmission.…”