Blocking the Spinal Fbxo3/CARM1/K+ Channel Epigenetic Silencing Pathway as a Strategy for Neuropathic Pain Relief

Abstract

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K + channel promoter, thereby causing K + channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K + channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K + channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.

Phosphorylated Upstream Frameshift 1–dependent Nonsense-mediated μ-Opioid Receptor mRNA Decay in the Spinal Cord Contributes to the Development of Neuropathic Allodynia–like Behavior in Rats

“…Western blot analysis of dorsal horn was performed as described in our previous study with modifications. 23 The membranes were incubated overnight with primary antibodies, including total UPF1 (mouse, 1:500, Santa Cruz Biotechnology, USA), phosphorylated UPF1 (rabbit, 1:1000, Millipore, USA), μ-opioid receptor (rabbit, 1:500, Abcam, USA), anti-SMG7 (rabbit, 1:1,000, Genetex, USA), SMG1 (rabbit, 1:500, Cell Signaling, USA), eIF4A3 (rabbit, 1:1,000, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (rabbit, 1:5,000, Genetex), followed by incubation with specific secondary antibodies at dilutions of 1:5,000 to 1:10,000 (Supplementary Content 2, https://links.lww.com/ALN/D105).…”

“…The protocol for spinal nerve ligation operation was as described in our previous study. 23 The surgical plane of anesthesia was verified by areflexia, toe pinch, and normal undisturbed respiratory rate and pattern. The body temperature of the rats was maintained by heating pad.…”

“…Total RNA isolation and cDNA synthesis were as described in our previous study. 23 Briefly, 1 μg RNA was applied to the reaction of reverse transcription, and 2 μl of the cDNA products was used for quantitative polymerase chain reaction amplification with primers specific for Oprm1 (5'-AACTTTGGTGGCCAGAGATG-3' and 5'-TGGGAAGAGAGAACTGGACAA-3') and Gapdh (5'-ATGACTCTACCCACGGCAAG-3' and 5'-GATCTCGCTCCTGGAAGATG-3'). Quantitative polymerase chain reaction was monitored on the QuantStudio 3 Real-Time polymerase chain reaction device (Applied Biosystems).…”

“…Detailed cDNA preparation and quantitative polymerase chain reaction protocol were described in our previous study. 23…”

“…Section preparation and immunostaining were done as previously described with modifications. 23 The sections were incubated with the primary antibodies overnight, including rabbit anti-phosphorylated UPF1 (1:200, Millipore), rabbit anti-SMG7 (1:1,000, Genetex), rabbit anti-SMG1 (1:500, Cell Signaling), and rabbit anti-eIF4A3 (1:200, Abcam), and then the Alexa Fluor secondary antibodies were applied (1:500 to 1:1,000, Invitrogen, USA). To measure immunofluorescence intensity, cell numbers in the lamina II dorsal horn were determined using a microscope with a 40× objective.…”