Role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain

Abstract

Using the chronic constriction injury (CCI) model of neuropathic pain, we profiled gene expression in the rat spinal cord, and identified SIP30 as a gene whose expression was elevated after CCI. SIP30 was previously shown to interact with SNAP25, but whose function was otherwise unknown. We now show that in the spinal cord, SIP30 was present in dorsal horn laminae where peripheral nociceptive inputs first synapse, colocalizing with nociception-related neuropeptides CGRP and substance P. With the onset of neuropathic pain after CCI surgery, SIP30 mRNA and protein levels increased in the ipsilateral side of the spinal cord, suggesting a potential association between SIP30 and neuropathic pain. When CCI-upregulated SIP30 was inhibited by intrathecal antisense oligonucleotide administration, neuropathic pain was attenuated. This neuropathic pain-reducing effect was observed both during neuropathic pain onset following CCI, and after neuropathic pain was fully established, implicating SIP30 involvement in the development and maintenance phases of neuropathic pain. Using a secretion assay in PC12 cells, anti-SIP30 siRNA decreased the total pool of synaptic vesicles available for exocytosis, pointing to a potential function for SIP30. These results suggest a role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain.

SIP30 Is Regulated by ERK in Peripheral Nerve Injury-induced Neuropathic Pain

“…We found that SIP30 was up-regulated during both the development and the maintenance phases of CCI and that knocking down SIP30 has similar effect to ERK inhibition on neuropathic pain behavior (Fig. 1B) (22). Although basal expression of SIP30 was not affected by inhibition of the ERK pathway, the stimulus-induced SIP30 expression was regulated by ERK in both in vivo and in vitro models (Figs.…”

“…Through a differential screening of a rat brain cDNA library, we found that sip30 was among the genes that were differentially expressed in the spinal cord CCI rats (21). We further showed that sip30 mRNA and protein levels were up-regulated in the spinal cord of CCI-treated rats and that administration of SIP30 antisense oligonucleotides significantly suppressed CCI-induced pain hypersensitivity in both the onset and the continuous manifestation phases, suggesting that SIP30 may be functionally involved in the development and maintenance of chronic neuropathic pain (22). However, the mechanism underlying the regulation of sip30 gene expression in this pathological process is unknown.…”

“…Briefly, the cells or tissues were homogenized and harvested in 100 l of lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 5 mM EDTA, 0.25 mM phenylmethanesulfonyl fluoride, and a mixture of protease inhibitors. The lysates were subjected to immunoblotting with the following primary antibodies: anti-phospho-ERK1/2 (1:2000, Cell Signaling Technology), anti-ERK (1:1000, Santa Cruz Biotechnology), and anti-SIP30 (22). For each blot, either the mouse monoclonal anti-␣-tubulin (1:10,000, Sigma) or the anti-␤-actin (1:7000, Sigma) was applied to serve as an internal control.…”

“…Three micrograms of RNA was used as the template for first strand cDNA synthesis. Real time PCR was performed using an Eppendorf Realplex Cycler, as described previously (22). Briefly, the program was run in a final reaction volume of 20 l containing the following reagents: 5.5 l of PCR-grade water, 10 l of 2ϫ Taq mixture (Sigma), 0.5 l of EvaGreen, 3 l of cDNA template, and 0.5 l of forward and reverse primer (20 M).…”

“…sistent pain hypersensitivity (23, 29). Previously, we found that SIP30 is functionally involved in CCI-induced neuropathic pain (22). To understand how SIP30 expression is regulated in neuropathic pain, we examined spinal cord SIP30 expression in response to CCI by immunostaining.…”