Jia-You Wei

Up-regulation of CX3CL1 via Nuclear Factor-κB-dependent Histone Acetylation Is Involved in Paclitaxel-induced Peripheral Neuropathy.

Background: Up-regulation of CX3CL1 has been revealed to be involved in the neuropathic pain induced by nerve injury. However, whether CX3CL1 participates in the paclitaxel-induced painful peripheral neuropathy remains unknown. The aim of the current study was to elucidate the involvement of transcriptional factors nuclear factor-&kgr;B (NF-&kgr;B) and its causal interaction with CX3CL1 signaling in the paclitaxel-induced painful peripheral neuropathy. Methods: Painful peripheral neuropathy induced by paclitaxel treatment was established in adult male Sprague-Dawley rats. The von Frey test were performed to evaluate neuropathic pain behavior, and real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, Western blot, immunohistochemistry, and small interfering RNA were performed to understand the molecular mechanisms. Results: The application of paclitaxel induced an up-regulation of CX3CL1 expression in the spinal neurons, which is reduced significantly by NF-&kgr;B inhibitor ammonium pyrrolidinedithiocarbamate or p65 small interfering RNA. Blockade of either CX3CL1 (n = 12 each) or NF-&kgr;B (n = 12 each) signaling pathway attenuated mechanical allodynia induced by paclitaxel. Chromatin immunoprecipitation further found that paclitaxel induced an increased recruitment of nuclear factor-&kgr;B (NF-&kgr;B)p65 to the Cx3cl1 promoter region. Furthermore, an increased acetylation level of H4, but not H3, in Cx3cl1 promoter region in spinal neurons was detected after paclitaxel treatment, which was reversed by inhibition of NF-&kgr;B with ammonium pyrrolidinedithiocarbamate or p65 small interfering RNA. Conclusions: These findings suggest that up-regulation of CX3CL1 via NF-&kgr;B–dependent H4 acetylation might be critical for paclitaxel-induced mechanical allodynia.

TNF-α-mediated JNK activation in the dorsal root ganglion neurons contributes to Bortezomib-induced peripheral neuropathy

Bortezomib (BTZ) is a frequently used chemotherapeutic drug for the treatment of refractory multiple myeloma and hematological neoplasms. The mechanism by which the administration of BTZ leads to painful peripheral neuropathy remains unclear. In the present study, we first determined that the administration of BTZ upregulated the expression of TNF-α and phosphorylated JNK1/2 in the dorsal root ganglion (DRG) of rat. Furthermore, the TNF-α synthesis inhibitor thalidomide significantly blocked the activation of both isoforms JNK1 and JNK2 in the DRG and attenuated mechanical allodynia following BTZ treatment. Knockout of the expression of TNF-α receptor TNFR1 (TNFR1 KO mice) or TNFR2 (TNFR2 KO mice) inhibited JNK1 and JNK2 activation and decreased mechanical allodynia induced by BTZ. These results suggest that upregulated TNF-α expression may activate JNK signaling via TNFR1 or TNFR2 to mediate mechanical allodynia following BTZ treatment.

Epigenetic upregulation of Cxcl12 expression mediates antitubulin chemotherapeutics-induced neuropathic pain.

Abstract Clinically, Microtubule-targeted agents–induced neuropathic pain hampers chemotherapeutics for patients with cancer. Here, we found that application of paclitaxel or vincristine increased the protein and mRNA expression of CXCL12 and frequency and amplitude of miniature excitatory post synaptic currents (mEPSCs) in spinal dorsal horn neurons. Spinal local application of CXCL12 induced the long-term potentiation of nociceptive synaptic transmission and increased the amplitude of mEPSCs. Inhibition of CXCL12 using the transgenic mice (CXCL12−/+) or neutralizing antibody or siRNA ameliorated the mEPSCs enhancement and mechanical allodynia. In addition, paclitaxel and vincristine both could increase the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the acetylation of histone H4 in the CXCL12-expressing neurons. Immunoprecipitation and chromatin immunoprecipitation assays demonstrated that antitubulin chemotherapeutics increased the binding of STAT3 to the CXCL12 gene promoter and the interaction between STAT3 and p300, and contributed to the enhanced transcription of CXCL12 by increasing the acetylation of histone H4 in CXCL12 gene promoter. Inhibition of STAT3 by intrathecal injection of adeno-associated virus encoding Cre and green fluorescent protein into STAT3flox/flox mice or inhibitor S3I-201 into rats suppressed the CXCL12 upsurge by decreasing the acetylation of histone H4. Finally, blockade of CXCR4 but not CXCR7 ameliorated the paclitaxel- or vincristine-induced mechanical allodynia. Together, these results suggested that enhanced interaction between STAT3 and p300 mediated the epigenetic upregulation of CXCL12 in dorsal horn neurons, which contributed to the antitubulin chemotherapeutics–induced persistent pain.

Cerebrospinal Fluid Oxaliplatin Contributes to the Acute Pain Induced by Systemic Administration of Oxaliplatin

Background:Systemic administration of oxaliplatin has no effect on the tumors in the central nervous system (CNS) due to the limited concentration of oxaliplatin in the cerebrospinal fluid (CSF), while it was clinically reported that oxaliplatin can induce acute encephalopathy. Currently, the impairment of neuronal functions in the CNS after systemic administration of oxaliplatin remains uninvestigated. Methods:The von Frey test and the plantar test were performed to evaluate neuropathic pain behavior after a single intraperitoneal administration of oxaliplatin (4 mg/kg) in rats. Inductively coupled plasma–mass spectrometry, electrophysiologic recording, real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, Western blot, immunohistochemistry, and small interfering RNA were applied to understand the mechanisms. Results:Concentration of oxaliplatin in CSF showed a time-dependent increase after a single administration of oxaliplatin. Spinal application of oxaliplatin at the detected concentration (6.6 nM) significantly increased the field potentials in the dorsal horn, induced acute mechanical allodynia (n = 12 each) and thermal hyperalgesia (n = 12 each), and enhanced the evoked excitatory postsynaptic currents and spontaneous excitatory postsynaptic currents in the projection neurokinin 1 receptor–expressing lamina I to II neurons. The authors further found that oxaliplatin significantly increased the nuclear factor-&kgr;B p65 binding and histone H4 acetylation in cx3cl1 promoter region. Thus, the upregulated spinal CX3CL1 markedly mediated the induction of central sensitization and acute pain behavior after oxaliplatin administration. Conclusions:The findings of this study suggested that oxaliplatin in CSF may directly impair the normal function of central neurons and contribute to the rapid development of CNS-related side effects during chemotherapy. This provides novel targets to prevent oxaliplatin-induced acute painful neuropathy and encephalopathy.

Upregulation of CCL2 via ATF3/c-Jun interaction mediated the Bortezomib-induced peripheral neuropathy

Bortezomib (BTZ) is a frequently used chemotherapeutic drug for the treatment of refractory multiple myeloma and hematological neoplasms. The mechanism by which the administration of BTZ leads to painful peripheral neuropathy remains unclear. In present study, we found that application of BTZ at 0.4 mg/kg for consecutive 5 days significantly increased the expression of CCL2 in DRG, and intrathecal administration of neutralizing antibody against CCL2 inhibited the mechanical allodynia induced by BTZ. We also found an increased expression of c-Jun in DRG, and that inhibition of c-Jun signaling prevented the CCL2 upregulation and mechanical allodynia in the rats treated with BTZ. Furthermore, the results with luciferase assay in vitro and ChIP assay in vivo showed that c-Jun might be essential for BTZ-induced CCL2 upregulation via binding directly to the specific position of the ccl2 promoter. In addition, the present results showed that an upregulated expression of ATF3 was co-expressed with c-Jun in the DRG neurons, and the enhanced interaction between c-Jun and ATF3 was observed in DRG in the rats treated with BTZ. Importantly, pretreatment with ATF3 siRNA significantly inhibited the recruitment of c-Jun to the ccl2 promoter in the rats treated with BTZ. Taken together, these findings suggested that upregulation of CCL2 resulting from the enhanced interaction between c-Jun and ATF3 in DRG contributed to BTZ-induced mechanical allodynia.

mir-500-Mediated GAD67 Downregulation Contributes to Neuropathic Pain

Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the interactions between synaptic dysfunction and the genes that are involved in persistent pain remain elusive. In the present study, we found that neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection significantly impaired the function of GABAergic synapses of spinal dorsal horn neurons via the reduction of the GAD67 expression. We also found that mir-500 expression was significantly increased and involved in the modulation of GAD67 expression via targeting the specific site of Gad1 gene in the dorsal horn. In addition, knock-out of mir-500 or using mir-500 antagomir rescued the GABAergic synapses in the spinal dorsal horn neurons and attenuated the sensitized pain behavior in the rats with neuropathic pain. To our knowledge, this is the first study to investigate the function significance and the underlying molecular mechanisms of mir-500 in the process of neuropathic pain, which sheds light on the development of novel therapeutic options for neuropathic pain. SIGNIFICANCE STATEMENT Neuropathic pain is a common neurobiological disease involving multifaceted maladaptations ranging from gene modulation to synaptic dysfunction, but the underlying molecular mechanisms remain elusive. The present study illustrates for the first time a mir-500-mediated mechanism underlying spinal GABAergic dysfunction and sensitized pain behavior in neuropathic pain induced by the chemotherapeutic drug paclitaxel or L5 ventral root transection, which sheds light on the development of novel therapeutic options for neuropathic pain.

The inhibition of spinal synaptic plasticity mediated by activation of AMP-activated protein kinase signaling alleviates the acute pain induced by oxaliplatin

ABSTRACT Our recent findings demonstrated that oxaliplatin entering CNS may directly induce spinal central sensitization, and contribute to the rapid development of CNS‐related side effects including acute pain during chemotherapy. However, the mechanism is largely unclear. In the current study, we found that the amplitude of C‐fiber‐evoked field potentials was significantly increased and the expression of phosphorylated mammalian AMP‐activated protein kinase &agr; (AMPK&agr;) was markedly decreased following high frequency stimulation (HFS) or single intraperitoneal injection of oxaliplatin (4 mg/kg). Spinal local application of AMPK agonist metformin (25 &mgr;g) prevented the long term potentiation (LTP) induction and the activation of mTOR/p70S6K signal pathway, and significantly attenuated the acute thermal hyperalgesia and mechanical allodynia following single oxaliplatin treatment. Importantly, we found that incubation of low concentration oxaliplatin at dose of 6.6 nM (the detected concentration in CSF following a single intraperitoneal injection of oxaliplatin) also significantly inhibited the AMPK&agr; activation and increased the amplitude of sEPSCs, the number of action potential, and the expression of p‐mTOR and p‐p70S6K in spinal cord slices. Metformin (25 &mgr;g) or rapamycin (2 &mgr;g) inhibited the increased excitability of dorsal horn neurons and the decrease of p‐AMPK&agr; expression induced by low concentration oxaliplatin incubation. Furthermore, spinal application of AMPK inhibitor compound C (5 &mgr;g) induced the spinal LTP, thermal hyperalgesia and mechanical allodynia, and rapamycin attenuated the spinal LTP, the thermal hyperalgesia and mechanical allodynia following oxaliplatin treatment (i.p.). Local application of metformin significantly decreased the mTOR and p70S6K activation induced by tetanus stimulation or oxaliplatin (i.p.). These results suggested that the decreased AMPK&agr; activity via negatively regulating mTOR/p70S6K signal pathway enhanced the synaptic plasticity and contributed to acute pain induced by low concentration of oxaliplatin entering CNS. HighlightsThe decreased AMPK&agr; via enhancing spinal synaptic plasticity contributed to the acute pain following single intraperitoneal injection of oxaliplatin.Activation of mTOR/p70S6K signal pathway contributed to acute pain induced by low concentration of oxaliplatin entering CNS.The decreased AMPK&agr; via negatively regulating the mTOR/p70S6K signal pathway mediated the acute pain induced by oxaliplatin.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib

&NA; Bortezomib is a first‐line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib‐induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5 days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p‐STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p‐STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I‐201 attenuated bortezomib‐induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.

Palmitoylation of δ-catenin promotes kinesin-mediated membrane trafficking of Nav1.6 in sensory neurons to promote neuropathic pain

Blocking the palmitoylation of the cell adhesion protein δ-catenin or its subsequent interactions may be therapeutic in patients with chronic pain. Palmitoylation and pain Chronic pain is associated with inflammation and increased synaptic activity in sensory neurons. Zhang et al. found that induction of the inflammatory cytokine TNF-α after chemotherapy or nerve injury in rats promoted the formation of a complex between the cell adhesion protein δ-catenin, the voltage-gated sodium channel Nav1.6, and the kinesin motor protein KIF3A, which increased the trafficking of the sodium channel to the cell membrane. Formation of this complex was dependent on the palmitoylation of δ-catenin, and inhibiting the activity of palmitoyl acyltransferases prevented the increase in both Nav1.6 surface abundance in sensory neurons and pain sensitivity in rats. These findings reveal potential therapeutic targets for treating chronic, neuropathic pain in patients. Palmitoylation of δ-catenin is critical to synapse plasticity and memory formation. We found that δ-catenin palmitoylation is also instrumental in the development of neuropathic pain. The abundances of palmitoylated δ-catenin and the palmitoyl acyltransferase DHHC3 were increased in dorsal root ganglion (DRG) sensory neurons in rat models of neuropathic pain. Inhibiting palmitoyl acyltransferases or decreasing δ-catenin abundance in the DRG by intrathecal injection of 2-bromopalmitate or shRNA, respectively, alleviated oxaliplatin or nerve injury–induced neuropathic pain in the rats. The palmitoylation of δ-catenin, which was induced by the inflammatory cytokine TNF-α, facilitated its interaction with the voltage-gated sodium channel Nav1.6 and the kinesin motor protein KIF3A, which promoted the trafficking of Nav1.6 to the plasma membrane in DRG neurons and contributed to mechanical hypersensitivity and allodynia in rats. These findings suggest that a palmitoylation-mediated KIF3A/δ-catenin/Nav1.6 complex enhances the transmission of mechanical and nociceptive signals; thus, blocking this mechanism may be therapeutic in patients with neuropathic pain.

Epigenetic upregulation of CXCL12 expression contributes to the acquisition and maintenance of morphine-induced conditioned place preference

ABSTRACT Addiction and rewarding effect is a primary side effect of morphine, which is commonly used to relieve the acute or chronic pain. Several lines of evidence have suggested that inflammation response in the VTA contributes to morphine‐induced reward (conditioned place preference, CPP), while the mechanism are poorly understood. The present study showed that repeated morphine conditioning persistently increased the expression of CXCL12 mRNA and protein in VTA. Furthermore, inhibition of CXCL12 prevented the acquisition and maintenance, but not the expression, of morphine‐induced CPP in rodent. In addition, molecular analysis revealed that morphine conditioning increased the occupancy of p‐STAT3 in the specific binding site (‐1667/‐1685) of CXCL12 promoter regions, and enhanced the interaction between acetyltransferase p300 and STAT3, and, hence, induced the histone H4 hyperacetylation in the promoter region and facilitated the transcription and expression of CXCL12 in VTA. Collectively, these results, for the first time, provided the evidence that persisted increase of VTA CXCL12 via epigenetic mechanism mediated the acquisition and maintenance, but not the expression, of morphine CPP. HIGHLIGHTSPersistently increased CXCL12 expression in the VTA contributed to the acquisition and maintenance of morphine CPP.p‐STAT3 bound to special sites in the CXCL12 promoter region and regulated CXCL12 expression.Interaction between p‐STAT3 and P300 induced epigenetic upregulation of CXCL12 after morphine conditioning.