NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

RALY participates in nerve trauma-induced nociceptive hypersensitivity through triggering Eif4g2 gene expression in primary sensory neurons.

BACKGROUND AND PURPOSE: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. METHODS AND RESULTS: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (Eif4g2) mRNA and Eif4g2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA up-regulated the expression of Eif4g2 mRNA and Eif4g2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of Eif4g2 gene and triggered its transcriptional activity. CONCLUSION AND IMPLICATIONS: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering Eif4g2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Transcription factor EBF1 mitigates neuropathic pain by rescuing Kv1.2 expression in primary sensory neurons.

Nerve injury-induced alternations of gene expression in primary sensory neurons of the dorsal root ganglion (DRG) are molecular basis of neuropathic pain genesis. Transcription factors regulate gene expression. In this study, we examined whether early B cell factor 1 (EBF1), a transcription factor, in the DRG, participated in neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. EBF1 was distributed exclusively in the neuronal nucleus and coexpressed with cytoplasmic/membrane Kv1.2 in individual DRG neurons. The expression of Ebf1 mRNA and protein was time-dependently downregulated in the ipsilateral lumbar (L) 3/4 DRGs after unilateral CCI. Rescuing this downregulation through microinjection of the adeno-associated virus 5 expressing full-length Ebf1 mRNA into the ipsilateral L3/4 DRGs reversed the CCI-induced decrease of DRG Kv1.2 expression and alleviated the development and maintenance of mechanical, heat and cold hypersensitivities. Conversely, mimicking the downregulation of DRG EBF1 through microinjection of AAV5-expressing Ebf1 shRNA into unilateral L3/4 DRGs produced a reduction of Kv1.2 expression in the ipsilateral L3/4 DRGs, spontaneous pain, and the enhanced responses to mechanical, heat and cold stimuli in naive mice. Mechanistically, EBF1 not only bound to the Kcna2 gene (encoding Kv1.2) promoter but also directly activated its activity. CCI decreased the EBF1 binding to the Kcna2 promoter in the ipsilateral L3/4 DRGs. Our findings suggest that DRG EBF1 downregulation contributes to neuropathic pain likely by losing its binding to Kcna2 promoter and subsequently silencing Kv1.2 expression in primary sensory neurons. Exogenous EBF1 administration may mitigate neuropathic pain by rescuing DRG Kv1.2 expression.

Systemic administration of NIS-lncRNA antisense oligonucleotide alleviates neuropathic pain.

OBJECTIVE: The antisense oligonucleotide (ASO) is an FDA-approved strategy in the treatment of neurological diseases. We have shown the viability of using intrathecal ASO to suppress nerve injury-specific long noncoding RNA (NIS-lncRNA) in dorsal root ganglion (DRG), resulting in a stable and long-lasting antinociceptive effect on NP. This study examined whether systemic administration of NIS-lncRNA ASO relieved the chronic constriction injury (CCI)-induced nociceptive hypersensitivity. METHODS: A single subcutaneous injection of NIS-lncRNA ASO at a dose of 1,000 µg was carried out 7 days after CCI or sham surgery in male mice. Behavioral tests were performed one day before surgery and at different days after surgery. DRG and spinal cord were finally collected for quantitative real-time RT-PCR and Western blot assays. RESULTS: NIS-lncRNA ASO significantly alleviated CCI-induced mechanical allodynia, heat hyperalgesia, and cold hyperalgesia starting on day 14 or 21 post-ASO injection and lasting for at least 7 days on the ipsilateral side. Additionally, CCI-induced spontaneous pain and ipsilateral dorsal horn neuronal and astrocyte hyperactivation were blocked on day 28 after NIS-lncRNA ASO injection. As predicted, the CCI-induced increases in the levels of NIS-lncRNA and its downstream target C-C motif chemokine ligand 2 in the ipsilateral lumbar 3 and 4 DRGs were attenuated on day 28 following NIS-lncRNA ASO injection. CONCLUSION: Our findings indicate that systemic administration of NIS-lncRNA ASO also produces a stable and long-lasting antinociceptive effect on neuropathic pain. NIS-lncRNA ASO may have potential clinical application in the treatment of this disorder.

Sensory neuron-specific long noncoding RNA in small non-peptidergic dorsal root ganglion neurons selectively impairs nerve injury-induced mechanical hypersensitivity.

AIMS: Nerve injury-induced mechanical hypersensitivity is one of major clinical symptoms in neuropathic pain patients. Understanding molecular mechanisms underlying this symptom is crucial for developing effective therapies. The present study was to investigate whether sensory neuron-specific long noncoding RNA (SS-lncRNA) predominantly expressed in small non-peptidergic dorsal root ganglion (DRG) neurons repaired nerve injury-induced mechanical hypersensitivity. MATERIALS AND METHODS: SS-lncRNA downregulation in the mas-related G protein-coupled receptor member D (Mrgprd)-expressed DRG neurons was rescued and mimicked by crossbreeding MrgprdCreERT2/+ lines with Rosa26SS-lncRNA knock-in mice and SS-lncRNAfl/fl mice, respectively, followed by tamoxifen injection. KEY FINDINGS: Rescuing SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons significantly reversed the spinal nerve ligation (SNL)-induced reduction of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in these DRG neurons and alleviated the SNL-induced mechanical hypersensitivity, without affecting the SNL-induced heat and cold nociceptive hypersensitivities, on the ipsilateral side. Conversely, mimicking SS-lncRNA downregulation in the Mrgprd-expressed DRG neurons reduced basal KCNN1 expression in these DRG neurons and produced the enhanced response to mechanical stimulation, but not thermal and cold stimuli, on bilateral sides. Mechanistically, SS-lncRNA downregulation caused a reduction in its binding to lysine-specific demethylase 6B (KDM6B) and consequent recruitment of less KDM6B to Kcnn1 promoter and an increase of H3K27me3 enrichment in this promoter in injured DRG. SIGNIFICANCE: Our findings suggest that SS-lncRNA downregulation in small non-peptidergic sensory neurons is required specifically for nerve injury-induced mechanical hypersensitivity likely through silencing KCNN1 expression caused by KDM6B-gated increase of H3K27me3 enrichment in Kcnn1 promoter in these neurons.

A sensory neuron-specific long non-coding RNA reduces neuropathic pain by rescuing KCNN1 expression.

Nerve injury to peripheral somatosensory system causes refractory neuropathic pain. Maladaptive changes of gene expression in primary sensory neurons are considered molecular basis of this disorder. Long non-coding RNAs (lncRNAs) are key regulators of gene transcription; however, their significance in neuropathic pain remains largely elusive.Here, we reported a novel lncRNA, named sensory neuron-specific lncRNA (SS-lncRNA), for its expression exclusively in dorsal root ganglion (DRG) and trigeminal ganglion. SS-lncRNA was predominantly expressed in small DRG neurons and significantly downregulated due to a reduction of early B cell transcription factor 1 in injured DRG after nerve injury. Rescuing this downregulation reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivity. Conversely, DRG downregulation of SS-lncRNA reduced the expression of KCNN1, decreased total potassium currents and afterhyperpolarization currents and increased excitability in DRG neurons and produced neuropathic pain symptoms.Mechanistically, downregulated SS-lncRNA resulted in the reductions of its binding to Kcnn1 promoter and heterogeneous nuclear ribonucleoprotein M (hnRNPM), consequent recruitment of less hnRNPM to the Kcnn1 promoter and silence of Kcnn1 gene transcription in injured DRG.These findings indicate that SS-lncRNA may relieve neuropathic pain through hnRNPM-mediated KCNN1 rescue in injured DRG and offer a novel therapeutic strategy specific for this disorder.

FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons.

Dysregulation of pain-associated genes in the dorsal root ganglion (DRG) is considered to be a molecular basis of neuropathic pain genesis. Fused in sarcoma (FUS), a DNA/RNA-binding protein, is a critical regulator of gene expression. However, whether it contributes to neuropathic pain is unknown. This study showed that peripheral nerve injury caused by the fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of the sciatic nerve produced a marked increase in the expression of FUS protein in injured DRG neurons. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5-expressing Fus shRNA into the ipsilateral L4 DRG mitigated the SNL-induced nociceptive hypersensitivities in both male and female mice. This microinjection also alleviated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in the ipsilateral L4 dorsal horn. Furthermore, mimicking this increase through microinjection of AAV5 expressing full-length Fus mRNA into unilateral L3/4 DRGs produced the elevations in the levels of p-ERK1/2 and GFAP in the dorsal horn, enhanced responses to mechanical, heat and cold stimuli, and induced the spontaneous pain on the ipsilateral side of both male and female mice in the absence of SNL. Mechanistically, the increased FUS activated the NF-κB signaling pathway by promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Our results indicate that DRG FUS contributes to neuropathic pain likely through the activation of NF-κB in primary sensory neurons.SIGNIFICANCE STATEMENT In the present study, we reported that fused in sarcoma (FUS), a DNA/RNA-binding protein, is upregulated in injured dorsal root ganglion (DRG) following peripheral nerve injury. This upregulation is responsible for nerve injury-induced translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Because blocking this upregulation alleviates nerve injury-induced nociceptive hypersensitivity, DRG FUS participates in neuropathic pain likely through the activation of NF-κB in primary sensory neurons. FUS may be a potential target for neuropathic pain management.

Effect of intrathecal NIS-lncRNA antisense oligonucleotides on neuropathic pain caused by nerve trauma, chemotherapy, or diabetes mellitus.

BACKGROUND: Blocking increased expression of nerve injury-specific long non-coding RNA (NIS-lncRNA) in injured dorsal root ganglia (DRG) through DRG microinjection of NIS-lncRNA small hairpin interfering RNA or generation of NIS-lncRNA knockdown mice mitigates neuropathic pain. However, these strategies are impractical in the clinic. This study employed a Food and Drug Administration (FDA)-approved antisense oligonucleotides strategy to examine the effect of NIS-lncRNA ASOs on neuropathic pain. METHODS: Effects of intrathecal injection of NIS-lncRNA antisense oligonucleotides on day 7 or 14 after chronic constriction injury (CCI) of the sciatic nerve, fourth lumbar (L4) spinal nerve ligation, or intraperitoneal injection of paclitaxel or streptozotocin on the expression of DRG NIS-lncRNA and C-C chemokine ligand 2 (CCL2, an NIS-lncRNA downstream target) and nociceptive hypersensitivity were examined. We also assessed whether NIS-lncRNA antisense oligonucleotides produced cellular toxicity. RESULTS: Intrathecal NIS-lncRNA antisense oligonucleotides attenuated CCI-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and ongoing nociceptive responses, without changing basal or acute nociceptive responses and locomotor function. Intrathecal NIS-lncRNA antisense oligonucleotides also blocked CCI-induced increases in NIS-lncRNA and CCL2 in the ipsilateral L3 and L4 DRG and hyperactivities of neurones and astrocytes in the ipsilateral L3 and L4 spinal cord dorsal horn. Similar results were found in antisense oligonucleotides-treated mice after spinal nerve ligation or intraperitoneal injection of paclitaxel or streptozotocin. Normal morphologic structure and no cell loss were observed in the DRG and spinal cord of antisense oligonucleotides-treated mice. CONCLUSION: These findings further validate the role of NIS-lncRNA in trauma-, chemotherapy-, or diabetes-induced neuropathic pain and demonstrate potential clinical application of NIS-lncRNA antisense oligonucleotides for neuropathic pain management.

E74-like factor 1 contributes to nerve trauma-induced nociceptive hypersensitivity through transcriptionally activating matrix metalloprotein-9 in dorsal root ganglion neurons.

ABSTRACT: Nerve trauma-induced alternations of gene expression in the neurons of dorsal root ganglion (DRG) participate in nerve trauma-caused nociceptive hypersensitivity. Transcription factors regulate gene expression. Whether the transcription factor E74-like factor 1 (ELF1) in the DRG contributes to neuropathic pain is unknown. We report here that peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve or unilateral fourth lumbar spinal nerve ligation led to the time-dependent increases in the levels of Elf1 mRNA and ELF1 protein in injured DRG, but not in the spinal cord. Preventing this increase through DRG microinjection of adeno-associated virus 5 expressing Elf1 shRNA attenuated the CCI-induced upregulation of matrix metallopeptidase 9 (MMP9) in injured DRG and induction and maintenance of nociceptive hypersensitivities, without changing locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking this increase through DRG microinjection of AAV5 expressing full-length Elf1 upregulated DRG MMP9 and produced enhanced responses to mechanical, heat, and cold stimuli in naive mice. Mechanistically, more ELF1 directly bond to and activated Mmp9 promoter in injured DRG neurons after CCI. Our data indicate that ELF1 participates in nerve trauma-caused nociceptive hypersensitivity likely through upregulating MMP9 in injured DRG. E74-like factor 1 may be a new target for management of neuropathic pain.

Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes.

Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury-associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury-specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury-induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury-induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

TET1 overexpression attenuates paclitaxel-induced neuropathic pain through rescuing K2p1.1 expression in primary sensory neurons of male rats.

AIMS: Paclitaxel-induced downregulation of two-pore domain K+ channel 1.1 (K2p1.1) caused by increasing DNA methylation within its gene promoter in the dorsal root ganglion (DRG) contributes to neuropathic pain. Given that ten-eleven translocation methylcytosine dioxygenase 1 (TET1) promotes DNA demethylation and gene transcription, the present study investigated whether DRG overexpression of TET1 produces an antinociceptive effect on the paclitaxel-induced nociceptive hypersensitivity. MAIN METHODS: TET1 was overexpressed in the DRG through unilateral microinjection of the herpes simplex virus expressing full-length Tet1 mRNA into the fourth and fifth lumbar DRGs of male rats. Behavioral tests were carried out to examine the effect of this overexpression on the paclitaxel-induced nociceptive hypersensitivity. Western blot analysis, chromatin immunoprecipitation assay and 5-hydroxymethylcytosine detection assay were performed to assess the levels of TET1/K2p1.1, 5-methylcytosine and 5-hydroxymethylcytosine, respectively. KEY FINDINGS: DRG overexpression of TET1 mitigated the paclitaxel-induced mechanical allodynia, heat hyperalgesia and cold hyperalgesia on the ipsilateral side during the development and maintenance periods. Locomotor function or basal (acute) responses to mechanical, heat or cold stimuli were not affected. Mechanistically, DRG overexpression of TET1 rescued the expression of K2p1.1 by blocking the paclitaxel-induced increase in the level of 5-methylcytosine and correspondingly reversing the paclitaxel-induced decreases in the amount of 5-hydroxymethylcytosine within the K2p1.1 promoter region in the microinjected DRGs of male rats. SIGNIFICANCE: Our findings suggest that DRG overexpression of TET1 alleviated chemotherapy-induced neuropathic pain likely through rescuing DRG K2p1.1 expression. Our findings may provide a potential avenue for the management of this disorder.

Orai1 is a crucial downstream partner of group I metabotropic glutamate receptor signaling in dorsal horn neurons.

ABSTRACT: Group I metabotropic glutamate receptors (group I mGluRs) have been implicated in several central nervous system diseases including chronic pain. It is known that activation of group I mGluRs results in the production of inositol triphosphate (IP3) and diacylglycerol that leads to activation of extracellular signal-regulated kinases (ERKs) and an increase in neuronal excitability, but how group I mGluRs mediate this process remains unclear. We previously reported that Orai1 is responsible for store-operated calcium entry and plays a key role in central sensitization. However, how Orai1 is activated under physiological conditions is unknown. Here, we tested the hypothesis that group I mGluRs recruit Orai1 as part of its downstream signaling pathway in dorsal horn neurons. We demonstrate that neurotransmitter glutamate induces STIM1 puncta formation, which is not mediated by N-Methyl-D-aspartate (NMDA) or α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Glutamate-induced Ca2+ entry in the presence of NMDA or AMPA receptor antagonists is eliminated in Orai1-deficient neurons. Dihydroxyphenylglycine (DHPG) (an agonist of group I mGluRs)-induced Ca2+ entry is abolished by Orai1 deficiency, but not affected by knocking down of transient receptor potential cation channel 1 (TRPC1) or TRPC3. Dihydroxyphenylglycine-induced activation of ERKs and modulation of neuronal excitability are abolished in cultured Orai1-deficient neurons. Moreover, DHPG-induced nociceptive behavior is markedly reduced in Orai1-deficient mice. Our findings reveal previously unknown functional coupling between Orai1 and group I mGluRs and shed light on the mechanism underlying group I mGluRs-mediated neuronal plasticity.

A Compound Mitigates Cancer Pain and Chemotherapy-Induced Neuropathic Pain by Dually Targeting nNOS-PSD-95 Interaction and GABAA Receptor.

Metastatic bone pain and chemotherapy-induced peripheral neuropathic pain are the most common clinical symptoms in cancer patients. The current clinical management of these two disorders is ineffective and/or produces severe side effects. The present study employed a dual-target compound named as ZL006-05 and examined the effect of systemic administration of ZL006-05 on RM-1-induced bone cancer pain and paclitaxel-induced neuropathic pain. Intravenous injection of ZL006-05 dose-dependently alleviated RM-1-induced mechanical allodynia, heat hyperalgesia, cold hyperalgesia, and spontaneously ongoing nociceptive responses during both induction and maintenance periods, without analgesic tolerance, affecting basal/acute pain and locomotor function. Similar behavioral results were observed in paclitaxel-induced neuropathic pain. This injection also decreased neuronal and astrocyte hyperactivities in the lumbar dorsal horn after RM-1 tibial inoculation or paclitaxel intraperitoneal injection. Mechanistically, intravenous injection of ZL006-05 potentiated the GABAA receptor agonist-evoked currents in the neurons of the dorsal horn and anterior cingulate cortex and also blocked the paclitaxel-induced increase in postsynaptic density-95-neuronal nitric oxide synthase interaction in dorsal horn. Our findings strongly suggest that ZL006-05 may be a new candidate for the management of cancer pain and chemotherapy-induced peripheral neuropathic pain.

Alternative Pre-mRNA Splicing of the Mu Opioid Receptor Gene, OPRM1: Insight into Complex Mu Opioid Actions.

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating multiple splice variants or isoforms via a variety of alternative splicing events. These OPRM1 splice variants can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (TM) C-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating the distinct actions of various mu opioids. More importantly, the OPRM1 variants can be targeted for development of novel opioid analgesics that are potent against multiple types of pain, but devoid of many side-effects associated with traditional opiates. In this review, we provide an overview of OPRM1 alternative splicing and its functional relevance in opioid pharmacology.

Downregulation of a Dorsal Root Ganglion-Specifically Enriched Long Noncoding RNA is Required for Neuropathic Pain by Negatively Regulating RALY-Triggered Ehmt2 Expression.

Nerve injury-induced maladaptive changes of gene expression in dorsal root ganglion (DRG) neurons contribute to neuropathic pain. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression. Here, a conserved lncRNA is reported, named DRG-specifically enriched lncRNA (DS-lncRNA) for its high expression in DRG neurons. Peripheral nerve injury downregulates DS-lncRNA in injured DRG due, in part, to silencing of POU domain, class 4, transcription factor 3, a transcription factor that interacts with the DS-lncRNA gene promoter. Rescuing DS-lncRNA downregulation blocks nerve injury-induced increases in the transcriptional cofactor RALY-triggered DRG Ehmt2 mRNA and its encoding G9a protein, reverses the G9a-controlled downregulation of opioid receptors and Kcna2 in injured DRG, and attenuates nerve injury-induced pain hypersensitivities in male mice. Conversely, DS-lncRNA downregulation increases RALY-triggered Ehmt2/G9a expression and correspondingly decreases opioid receptor and Kcna2 expression in DRG, leading to neuropathic pain symptoms in male mice in the absence of nerve injury. Mechanistically, downregulated DS-lncRNA promotes more binding of increased RALY to RNA polymerase II and the Ehmt2 gene promoter and enhances Ehmt2 transcription in injured DRG. Thus, downregulation of DS-lncRNA likely contributes to neuropathic pain by negatively regulating the expression of RALY-triggered Ehmt2/G9a, a key neuropathic pain player, in DRG neurons.

FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons.

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6-methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Methods: Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Ftofl/fl mice on the Coll IV microinjection­induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5-Fto) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6-methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) were observed. Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Ftofl/fl mouse thalamus attenuated the Coll IV microinjection­induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N6-methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

Endocannabinoid signaling in the lateral habenula regulates pain and alcohol consumption.

Hyperalgesia, which often occurs in people suffering from alcohol use disorder, may drive excessive drinking and relapse. Emerging evidence suggests that the lateral habenula (LHb) may play a significant role in this condition. Previous research suggests that endocannabinoid signaling (eCBs) is involved in drug addiction and pain, and that the LHb contains core components of the eCBs machinery. We report here our findings in rats subjected to chronic ethanol vapor exposure. We detected a substantial increase in endocannabinoid-related genes, including Mgll and Daglb mRNA levels, as well as monoacylglycerol lipase (MAGL) protein levels, as well as a decrease in Cnr1 mRNA and type-1 cannabinoid receptor (CB1R) protein levels, in the LHb of ethanol-exposed rats. Also, rats withdrawing from ethanol exposure displayed hypersensitivity to mechanical and thermal nociceptive stimuli. Conversely, intra-LHb injection of the MAGL inhibitor JZL184, the fatty acid amide hydrolase inhibitor URB597, or the CB1R agonist WIN55,212-2 produced an analgesic effect, regardless of ethanol or air exposure history, implying that alcohol exposure does not change eCB pain responses. Intra-LHb infusion of the CB1R inverse agonist rimonabant eliminated the analgesic effect of these chemicals. Rimonabant alone elicited hyperalgesia in the air-, but not ethanol-exposed animals. Moreover, intra-LHb JZL184, URB597, or WIN55,212-2 reduced ethanol consumption in both homecages and operant chambers in rats exposed to ethanol vapor but not air. These findings suggest that LHb eCBs play a pivotal role in nociception and facilitating LHb eCBs may attenuate pain in drinkers.

CREB Participates in Paclitaxel-Induced Neuropathic Pain Genesis Through Transcriptional Activation of Dnmt3a in Primary Sensory Neurons.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) often occurs in cancer patients treated with antineoplastic drugs. Therapeutic management of CIPNP is very limited, at least in part due to the largely unknown mechanisms that underlie CIPNP genesis. Here, we showed that systemic administration of the chemotherapeutic drug paclitaxel significantly and time-dependently increased the levels of cyclic AMP response element-binding protein (CREB) in dorsal root ganglion (DRG) neurons. Blocking this increase through DRG microinjection of Creb siRNA attenuated paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of the adeno-associated virus 5 expressing full-length Creb mRNA led to enhanced responses to basal mechanical, heat, and cold stimuli in mice in absence of paclitaxel treatment. Mechanically, paclitaxel-induced increase of DRG CREB protein augmented Dnmt3a promoter activity and participated in the paclitaxel-induced upregulation of DNMT3a protein in the DRG. CREB overexpression also elevated the expression of DNMT3a in in vivo and in vitro DRG neurons of naïve mice. Given that DNMT3a is an endogenous instigator of CIPNP and that CREB co-expresses with DNMT3a in DRG neurons, CREB may be a key player in CIPNP through transcriptional activation of the Dnmt3a gene in primary sensory neurons. CREB is thus a likely potential target for the therapeutic management of this disorder.

Nerve trauma-caused downregulation of opioid receptors in primary afferent neurons: Molecular mechanisms and potential managements.

Neuropathic pain is the most common clinical disorder destroying the quality of patient life and leading to a marked economic and social burden. Opioids are still last option for pharmacological treatment of this disorder, but their antinociceptive effects are limited in part due to the downregulation of opioid receptors in the primary afferent neurons after peripheral nerve trauma. How this downregulation occurs is not completely understood, but recent studies have demonstrated that peripheral nerve trauma drives the alterations in epigenetic modifications (including DNA methylation, histone methylation and mciroRNAs), expression of transcription factors, post-transcriptional modifications (e.g., RNA methylation) and protein translation initiation in the neurons of nerve trauma-related dorsal root ganglion (DRG) and that these alternations may be associated with nerve trauma-caused downregulation of DRG opioid receptors. This review presents how opioid receptors are downregulated in the DRG after peripheral nerve trauma, specifically focusing on distinct molecular mechanisms underlying transcriptional and translational processes. This review also discusses how this downregulation contributes to the induction and maintenance of neuropathic pain. A deeper understanding of these molecular mechanisms likely provides a novel avenue for prevention and/or treatment of neuropathic pain.