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author:("skuse, Kenji")
1.  Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain* 
The Journal of Biological Chemistry  2013;288(48):34638-34646.
Background: VGF is a neuropeptide involved in chronic pain.
Results: VGF-derived peptide TLQP-21 activates macrophages. We identified gC1qR as a receptor for TLQP-21.
Conclusion: TLQP-21 and gC1qR are involved in chronic pain pathways.
Significance: TLQP-21 and gC1qR may be drug targets for chronic pain treatment.
VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca2+ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca2+ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.
PMCID: PMC3843076  PMID: 24106277
Calcium Intracellular Release; Macrophages; Neurons; Neuropeptide; Pain; VGF; gC1qR; Neuropathic Pain; Sensory Neurons
2.  Correction: Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability 
PLoS ONE  2013;8(5):10.1371/annotation/55db09f9-cce9-44cc-8501-5f97d1d1e6a1.
PMCID: PMC3651323
3.  Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability 
PLoS ONE  2012;7(8):e40079.
Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. NaV1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. NaV1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for NaV1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated NaV1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that NaV1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that NaV1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-β-cyclodextrin (MβCD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and NaV1.8. By calcium imaging we demonstrated that the lack of association between rafts and NaV1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of NaV1.8 in nociceptors and highlight the importance of the association between NaV1.8 and lipid rafts in the control of nociceptor excitability.
PMCID: PMC3411591  PMID: 22870192
4.  In vitro and intrathecal siRNA mediated KV1.1 knock-down in primary sensory neurons 
Molecular and Cellular Neurosciences  2011;48(3-3):258-265.
KV1.1 is a Shaker homologue K+ channel that contributes to the juxta-paranodal membrane conductance in myelinated axons, and is blocked by fampridine (4-aminopyridine), used to treat the symptoms of multiple sclerosis. The present experiments investigate KV1.1 function in primary sensory neurons and A-fibres, and help define its characteristics as a drug-target using sequence specific small-interfering RNAs (siRNAs). siRNA (71 nM) was used to knock-down functional expression of KV1.1 in sensory neurons (> 25 μm in apparent diameter) in culture, and was also delivered intrathecally in vivo (9.3 μg). K+ channel knock-down in sensory neurons was found to make the voltage-threshold for action potential generation significantly more negative than in control (p = 0.02), led to the breakdown of accommodation and promoted spontaneous action potential firing. Exposure to dendrotoxin-K (DTX-K, 10–100 nM) also selectively abolished K+ currents at negative potentials and made voltage-threshold more negative, consistent with KV1.1 controlling excitability close to the nominal resting potential of the neuron cell body, near − 60 mV. Introduction of one working siRNA sequence into the intrathecal space in vivo was associated with a small increase in the amplitude of the depolarising after-potential in sacral spinal roots (p < 0.02), suggesting a reduction in the number of working K+ channels in internodal axon membrane. Our study provides evidence that KV1.1 contributes to the control of peripheral sensory nerve excitability, and suggests that its characteristics as a putative drug target can be assessed by siRNA transfection in primary sensory neurons in vitro and in vivo.
PMCID: PMC3240745  PMID: 21903165
Sensory neuron; Axon; Potassium channel; Dendrotoxin-K; RNA interference; QPCR
5.  The trafficking of NaV1.8 
Neuroscience Letters  2010;486(2-13):78-83.
Research highlights
▶ The β3 subunit masks the ER retention signal of NaV1.8 and release the channel from the ER. ▶ p11 directly binds to NaV1.8 and help its translocation to the plasma membrane. ▶ PDZD2 is responsible for the functional expression of NaV1.8 on the plasma membrane. ▶ Contactin KO mice exhibit a reduction of NaV1.8 along unmyelinated axons in the sciatic nerve. ▶ PKA activation increases the NaV1.8 density on the membrane through direct phosphorylation.
The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel NaV1.8 is selectively expressed in sensory neurons. It has been reported that NaV1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive [1] and neuropathic [24] pain conditions. Thus NaV1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of NaV1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.
PMCID: PMC2977848  PMID: 20816723
Sodium Channel; Sensory Neuron; Pain; Trafficking
6.  A multi PDZ-domain protein Pdzd2 contributes to functional expression of sensory neuron-specific sodium channel NaV1.8 
The voltage-gated sodium channel NaV1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. NaV1.8 cannot be functionally expressed in non-neuronal cells even in the presence of β-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for NaV1.8. Here we report that Pdzd2 binds directly to the intracellular loops of NaV1.8 and NaV1.7. The endogenous NaV1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for NaV1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of NaV1.8 expression in sensory neurons.
PMCID: PMC2764382  PMID: 19607921
7.  Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain 
To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120 + ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.
PMCID: PMC2706986  PMID: 18606552
Neuropathic pain; HIV; Mechanical hypersensitivity; Microarray
8.  Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves 
Molecular Pain  2008;4:33.
Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.
PMCID: PMC2525634  PMID: 18700027

Results 1-8 (8)