Intraneural delivery of CBD3A6K-RhB via PEG-PLGA nanoparticles for neuropathic pain therapy.

Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca(2+) channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca(2+) channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca(2+) currents culminated in changes in synaptic transmission. Following a brief high-K(+)-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2-EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by approximately 70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by approximately 54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca(2+) channels and in transmitter release.

The collapsin response mediator protein 2 (CRMP2) has emerged as a central node in assembling nociceptive signaling complexes involving voltage-gated ion channels. Concerted actions of post-translational modifications, phosphorylation and SUMOylation, of CRMP2 contribute to regulation of pathological pain states. In the present study, we demonstrate a novel role for CRMP2 in spinal nociceptive transmission. We found that, of six possible post-translational modifications, three phosphorylation sites on CRMP2 were critical for regulating calcium influx in dorsal root ganglion sensory neurons. Of these, only CRMP2 phosphorylated at serine 522 by cyclin-dependent kinase 5 (Cdk5) contributed to spinal neurotransmission in a bidirectional manner. Accordingly, expression of a non-phosphorylatable CRMP2 (S522A) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), whereas expression of a constitutively phosphorylated CRMP2 (S522D) increased the frequency of sEPSCs. The presynaptic nature of CRMP2's actions was further confirmed by pharmacological antagonism of Cdk5-mediated CRMP2 phosphorylation with S-N-benzy-2-acetamido-3-methoxypropionamide ((S)-lacosamide; (S)-LCM) which (i) decreased sEPSC frequency, (ii) increased paired-pulse ratio, and (iii) reduced the presynaptic distribution of CaV2.2 and NaV1.7, two voltage-gated ion channels implicated in nociceptive signaling. (S)-LCM also inhibited depolarization-evoked release of the pro-nociceptive neurotransmitter calcitonin gene-related peptide (CGRP) in the spinal cord. Increased CRMP2 phosphorylation in rats with spared nerve injury (SNI) was decreased by intrathecal administration of (S)-LCM resulting in a loss of presynaptic localization of CaV2.2 and NaV1.7. Together, these findings indicate that CRMP2 regulates presynaptic excitatory neurotransmission in spinal cord and may play an important role in regulating pathological pain. Novel targeting strategies to inhibit CRMP2 phosphorylation by Cdk5 may have great potential for the treatment of chronic pain.

N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca(2+) channel complex. Nat. Med. 17, 822-829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388-402), "L1") and the distal C terminus (CaV1.2(2014-2028) "Ct-dis") that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2'-3'-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.

Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.

Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.

Neuropathic pain results from nerve injuries that cause ectopic firing and increased nociceptive signal transmission due to activation of key membrane receptors and channels. The dysregulation of trafficking of voltage-gated ion channels is an emerging mechanism in the etiology of neuropathic pain. We identify increased phosphorylation of collapsin response mediator protein 2 (CRMP2), a protein reported to regulate presynaptic voltage-gated calcium and sodium channels. A spared nerve injury (SNI) increased expression of a cyclin dependent kinase 5 (Cdk5)-phosphorylated form of CRMP2 in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) in the ipsilateral (injured) versus the contralateral (non-injured) sites. Biochemical fractionation of spinal cord from SNI rats revealed the increase in Cdk5-mediated CRMP2 phosphorylation to be enriched to pre-synaptic sites. CRMP2 has emerged as a central node in assembling nociceptive signaling complexes. Knockdown of CRMP2 using a small interfering RNA (siRNA) reversed SNI-induced mechanical allodynia implicating CRMP2 expression as necessary for neuropathic pain. Intrathecal expression of a CRMP2 resistant to phosphorylation by Cdk5 normalized SNI-induced mechanical allodynia, whereas mimicking constitutive phosphorylation of CRMP2 resulted in induction of mechanical allodynia in naïve rats. Collectively, these results demonstrate that Cdk5-mediated CRMP2 phosphorylation is both necessary and sufficient for peripheral neuropathic pain.

Migraine is one of the world's most common neurological disorders. Current acute migraine treatments have suboptimal efficacy, and new therapeutic options are needed. Approaches targeting calcitonin gene related peptide (CGRP) signaling are clinically effective, but small molecule antagonists have not been advanced because of toxicity. In this study, we explored the axonal growth/specification collapsin response mediator protein 2 (CRMP2) as a novel “druggable” target for inhibiting CGRP release and for potential relevance for treatment of migraine pain. Collapsin response mediator protein 2 has been demonstrated to regulate N-type voltage-gated Ca2+ channel activity and Ca2+-dependent CGRP release in sensory neurons. The coexpression of CRMP2 with N-type voltage-gated Ca2+ channel and CGRP in trigeminal ganglia (TGs) sensory neurons suggested the possibility of a novel approach to regulate CGRP release in the trigeminal system. Screening protocols surprisingly revealed that (S)-lacosamide ((S)-LCM), an inactive analog of the clinically approved small molecule antiepileptic drug (R)-lacosamide (Vimpat), inhibited CRMP2 phosphorylation by cyclin-dependent kinase 5 in rat TG slices and decreased depolarization-evoked Ca2+ influx in TG cells in culture. (S)-LCM significantly blocked capsaicin-evoked CGRP release from dural nerve terminals in the rat in ex vivo cranial cup preparation. Additionally, cephalic and extracephalic cutaneous allodynia induced in rats by activation of dural nociceptors with a cocktail of inflammatory mediators, was inhibited by oral administration of (S)-LCM. The confirmation of CRMP2 as an upstream mediator of CGRP release, together with the brain penetrance of this molecule suggests (S)-LCM as a potential therapy for acute migraine.

Effect of emodin on neuropathic pain transmission mediated by P2X 2/3 receptor of primary sensory neurons

Deletion of Crmp4 attenuates CSPG-induced inhibition of axonal growth and induces nociceptive recovery after spinal cord injury.

Neuropathic pain (NP) is a chronic and intractable disease that is widely present in the general population. It causes painful behavior and even mood changes such as anxiety and depression by altering the metabolism of substances. However, there have been limited metabolomics studies conducted in relation to neuropathic pain. Therefore, in this study, the effects of NP on metabolites in serum and the dorsal root ganglion (DRG) were investigated using a non-targeted metabolomics approach detected by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) to uncover differential metabolites and affected metabolic pathways associated with NP. Sixty mice were divided into the following two groups: a chronic constriction injury (CCI) of the sciatic nerve group and a sham group (n = 30, each). After 7 days of CCI modeling, the metabolite profiles of serum and the DRG were analyzed using GC/LC-MS for both the CCI and sham groups of mice. Multivariate analysis revealed differential metabolites and altered metabolic pathways between the CCI and sham groups. In the CCI group, our findings provided insights into the complex phospholipid, amino acid and acylcarnitine metabolic perturbations of DRG metabolism. In addition, phospholipid metabolic disorders and impaired glucose metabolism were observed in the serum. Moreover, the metabolic differences in the DRG and serum were correlated with each other. The results from this untargeted metabolomics study provide a perspective on the metabolic impact of NP on serum and the DRG.

Dual leucine zipper kinase (DLK) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family mainly involved in neuronal degeneration. However, the role of DLK signaling in the neuropathic pain has not yet been fully determined. Chronic constrictive injury (CCI) was conducted by four 3-0 chromic gut ligatures loosely ligated around the sciatic nerve. Escalated DLK expression over the dorsal root ganglion was observed from one to four rings of CCI. Remarkable expression of DLK was observed in primary dorsal root ganglion cells culture subjected to electrical stimulation and attenuated by DLK short hairpin RNA (shRNA) treatment. Intrathecal injection of DLK shRNA attenuates the expression of DLK over the dorsal root ganglion and hippocampus neurons and increased the threshold of mechanical allodynia and decreased thermal hyperalgesia. In CatWalk gait analysis, significant decreases of print area, maximum contact maximum intensity, stand phase, single stance, and regular index by CCI were alleviated by the DLK shRNA administration. In conclusion, the expression of DLK was up-regulated in chronic constrictive injury and attenuated by the administration of DLK shRNA, which paralleled the improvement of neurobehavior of neuropathic pain. The modulation of DLK expression is a potential clinic treatment option for neuropathic pain.

PurposeNeuropathic pain is a chronic intractable disease characterized by allodynia and hyperalgesia. Effective treatments are unavailable because of the complicated mechanisms of neuropathic pain. Transient receptor potential canonical 6 (TRPC6) is a nonselective calcium (Ca2+)-channel protein related to hyperalgesia. Dexmedetomidine (Dex) is an alpha-2 (α2) adrenoreceptor agonist that mediates intracellular Ca2+ levels to alleviate pain. However, the relationship between TRPC6 and Dex is currently unclear. We speculated that the α2 receptor agonist would be closely linked to the TRPC6 channel. We aimed to investigate whether Dex relieves neuropathic pain by the TRPC6 pathway in the dorsal root ganglia (DRG).MethodsThe chronic constriction injury (CCI) model was established in male rats, and we evaluated the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL). The expression of TRPC6 and Iba-1 in the DRG were analyzed using quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence assay. The levels of inflammatory cytokines were measured using an enzyme-linked immunosorbent assay.ResultsCompared with the CCI normal saline group, both the MWT and TWL were significantly improved after 7 days of Dex administration. Results demonstrated that TRPC6 expression was increased in the DRG following CCI but was suppressed by Dex. In addition, multiple administrations of Dex inhibited the phosphorylation level of p38 mitogen-activated protein kinase and the upregulation of neuroinflammatory factors.ConclusionThe results of this study demonstrated that Dex exhibits anti-nociceptive and anti-inflammatory properties in a neuropathic pain model. Moreover, our findings of the CCI model suggested that Dex has an inhibitory effect on TRPC6 expression in the DRG by decreasing the phosphorylation level of p38 in the DRG.

BackgroundNeuroinflammation is an important driver of acute and chronic pain states. Therefore, targeting molecular mediators of neuroinflammation may present an opportunity for developing novel pain therapies. In preclinical models of neuroinflammatory pain, calcitonin gene-related peptide (CGRP), substance P and high mobility group box 1 protein (HMGB1) are molecules synthesized and released by sensory neurons which activate inflammation and pain. High-frequency electrical nerve stimulation (HFES) has achieved clinical success as an analgesic modality, but the underlying mechanism is unknown. Here, we reasoned that HFES inhibits neuroinflammatory mediator release by sensory neurons to reduce pain.MethodsUtilizing in vitro and in vivo assays, we assessed the modulating effects of HFES on neuroinflammatory mediator release by activated sensory neurons. Dorsal root ganglia (DRG) neurons harvested from wildtype or transgenic mice expressing channelrhodopsin-2 (ChR2) were cultured on micro-electrode arrays, and effect of HFES on optogenetic- or capsaicin-induced neuroinflammatory mediator release was determined. Additionally, the effects of HFES on local neuroinflammatory mediator release and hyperalgesia was assessed in vivo using optogenetic paw stimulation and the neuropathic pain model of chronic constriction injury (CCI) of the sciatic nerve.ResultsLight- or capsaicin-evoked neuroinflammatory mediator release from cultured transgenic DRG sensory neurons was significantly reduced by concurrent HFES (10 kHz). In agreement with these findings, elevated levels of neuroinflammatory mediators were detected in the affected paw following optogenetic stimulation or CCI and were significantly attenuated using HFES (20.6 kHz for 10 min) delivered once daily for 3 days.ConclusionThese studies reveal a previously unidentified mechanism for the pain-modulating effect of HFES in the setting of acute and chronic nerve injury. The results support the mechanistic insight that HFES may reset sensory neurons into a less pro-inflammatory state via inhibiting the release of neuroinflammatory mediators resulting in reduced inflammation and pain.

BACKGROUND:Spinal cord stimulation (SCS) is an emerging, minimally invasive procedure used to treat patients with intractable chronic pain conditions. Although several signaling pathways have been proposed to account for SCS-mediated pain relief, the precise mechanisms remain poorly understood. Recent evidence reveals that injured sensory neuron-derived colony-stimulating factor 1 (CSF1) induces microglial activation in the spinal cord, contributing to the development of neuropathic pain (NP). Here, we tested the hypothesis that SCS relieves pain in a rat model of chronic constriction injury (CCI) by attenuating microglial activation via blocking CSF1 to the spinal cord.METHODS:Sprague-Dawley rats underwent sciatic nerve ligation to induce CCI and were implanted with an epidural SCS lead. SCS was delivered 6 hours per day for 5 days. Some rats received a once-daily intrathecal injection of CSF1 for 3 days during SCS.RESULTS:Compared with naive rats, CCI rats had a marked decrease in the mechanical withdrawal threshold of the paw, along with increased microglial activation and augmented CSF1 levels in the spinal dorsal horn and dorsal root ganglion, as measured by immunofluorescence or Western blotting. SCS significantly increased the mechanical withdrawal threshold and attenuated microglial activation in the spinal dorsal horn in CCI rats, which were associated with reductions in CSF1 levels in the spinal dorsal horn and dorsal roots but not dorsal root ganglion. Moreover, intrathecal injection of CSF1 completely abolished SCS-induced changes in the mechanical withdrawal threshold and activation of microglia in the spinal dorsal horn in CCI rats.CONCLUSIONS:SCS reduces microglial activation in the spinal cord and alleviates chronic NP, at least in part by inhibiting the release of CSF1 from the dorsal root ganglion ipsilateral to nerve injury.

Effects of adrenergic stimulus on the activities of Ca2+ and K+ channels of dorsal root ganglion neurons in a neuropathic pain model