Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes c-fiber nociceptors with broad action potentials and high nav1.9 expression

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4+) and small dark neurons had similar size distributions. We examined IB4-binding levels in >120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4+ neurons were classified as strongly or weakly IB4+. All strongly IB4+ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, ~50% were strongly IB4+, ~20% were weakly IB4+ and ~30% were IB4–. A{delta} low-threshold mechanoreceptive (LTM) neurons were weakly IB4+ or IB4–. All 33 A-fiber nociceptors and all 44 A{alpha}/beta-LTM neurons examined were IB4–. IB4+ compared with IB4– C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, ~35% were trkA+/IB4+ but with negatively correlated immunointensities; 26% were IB4+/trkA–, and 35% were IB4–/trkA+. We conclude that strongly IB4+ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties

Investigating the mechanisms of action of VGF-derived peptides in the nervous system

The VGF neurosecretory protein, first identified as a nerve growth factor (NGF) inducible gene product, is selectively synthesised predominantly in neuronal and neuroendocrine cells. The ~68 kDa VGF protein sequence is rich in paired basic amino acids, and thus the protein undergoes endoproteolytic cleavage to produce smaller peptides, which are stored in dense core vesicles and released upon stimulation via the regulated secretory pathway both in vitro and in vivo. Several of these VGF-derived peptides have been characterised and are involved in energy homeostasis, reproductive processes, synaptic plasticity as well as pain modulation. A number of studies have observed an increase in VGF gene expression in various pain models and more recently the VGF-derived peptides, TLQP-21, LQEQ-19 and TLQP-62 showed direct modulation of inflammatory and neuropathic pain when applied in vivo. The molecular mechanisms of action of VGF-derived peptides are not well understood and were investigated in this study. The TLQP-21 peptide, but not LQEQ-19, was shown to dose-dependently induce an increase in intracellular Ca2+ levels from cellular internal stores in brain- and spinal cord-derived primary microglia, in >65 % of the cell population in vitro. Three hour treatment of primary microglia with TLQP-21 (100 nM) induced a 2.78 fold increase in Ccl11 and a 2.28 fold decrease in Cxcl9 gene expression levels relative to the vehicle control (Student's t-test; p ≤ 0.05). Biochemical analysis using affinity chromatography and LC-MS/MS techniques identified the gC1q-R protein as a potential binding partner / receptor for TLQP-21. The gC1q-R protein is a ubiquitously expressed, multi-compartmental protein involved in complement activation, inflammatory processes and the plasma bradykinin formation pathway. These results tentatively suggest that TLQP-21 may contribute to the modulation of pain through activation of primary microglia and potentially involve interactions with components of the complement system. The findings highlight the importance of VGF-derived peptides in pain research and could lead to new perspectives and targets for pain therapeutics.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigations into the mechanisms underlying HIV-1 gp120-associated neurotoxicity

HIV-associated distal sensory polyneuropathy is a frequent (~40% prevalence) complication of HIV infection and treatment. It is characterised by a dying back pattern of axonal degeneration, predominantly of nociceptors usually accompanied by neuropathic pain. The HIV envelope glycoprotein, gp120, has recently been identified as a key mediator of axonal degeneration both in vitro and in vivo. We hypothesised that gp120 interacts, in a chemokine receptor-dependant manner, with primary sensory neurons either directly or indirectly via macrophages and/or Schwann cells. Neurite outgrowth of cultured adult rat dorsal root ganglia (DRG) cells was used to assess direct or indirect neurotoxicity. Gp120 induced concentration-dependent neurite degeneration 24h after exposure, which was not restricted to phenotypic subsets of DRG cells. However, gp120 localisation studies indicated that only a minority (approx. 10%) of neurons internalised gp120 before the onset of neurite degeneration suggesting that the direct toxicity was not a predominant mechanism. Therefore, indirect mechanisms of neurotoxicity were investigated. Application of gp120-conditioned macrophage or Schwann cell media to DRG neuronal cultures revealed that gp120-conditioned media also had the capacity to induce neurite degeneration. Using qPCR it was shown that 4hr exposure to gp120 increased transcription of cytokine–related genes, in cultured Schwann cells and macrophages. These gp120-mediated effects were then explored in vivo. The cytokine expression profiles 4hrs following intradermal gp120 injection in rats were similar to the gene upregulation observed in vitro. These findings highlight the complexity of gp120-mediated mechanisms and indicate that macrophages and Schwann cells may play a key indirect role in the pathogenesis of HIV-associated peripheral neuropathy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Na_V1.8 associates with lipid rafts in the sciatic nerve.

<p>Lipid rafts were extracted from the sciatic nerve. After centrifugation on an Iodixanol density gradient, fractions were analysed by western blotting and dot blot analysis to assess lipid raft isolation and Na_V1.8 partitioning between lipid rafts and the non-raft portions of the membrane. Flotillin1 and GM1 were used as a protein and lipid marker of lipid rafts, respectively. Transferrin receptor was used as a marker of non-raft portions. In the sciatic nerve the totality of Na_V1.8 is associated with lipid rafts. M represents protein ladder, the recovered fractions are numbered from 1 (top fraction) to 9 (bottom fraction).</p

Na_V1.8 is distributed in clusters along the axons of small, unmyelinated DRG neurons <i>in vitro</i>.

<p>Endogenous Na_V1.8 was immuno-localised in cultured DRG neurons after 2 DIV. Small-diameter neurons were identified by morphology (A, right panel) and by the immuno-reactivity for Peripherin (B, right panel). The region framed by the dotted square in A is magnified in the inset below. Na_V1.8 is distributed in distinct puncta along the neurites, of small-diameter neurons (A, B; arrows pinpoint example of clusters, which are distributed throughout the neurites). Na_V1.8 was also found to be enriched at the level of the cell bodies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040079#pone-0040079-g001" target="_blank">Figure 1A</a>, asterisk). The fluorescent construct Na_V1.8-DsRed2 was visualised in DRG neurons. The image shows Na_V1.8-DsRed2 distributed in clusters along the axon of DRG neuron (C, left panel). The discontinuous distribution of the fluorescent construct has been mapped by quantifying pixel intensity along the neurite (C, graph; right panel). Also, the fluorescent construct Na_V1.8-DsRed2 colocalises with GM1 puncta along the neurite of DRG neurons, as shown by the superimposed images of Na_V1.8-DsRed2 and GM1 (merge). Scale bars are 20 μm.</p

Effect of lipid raft depletion on the speed of propagation of Fluo-4 signals upon mechano-stimulation of the neurites.

<p>Box plot show that upon 7KC and MβCD treatments the speed of propagation (expressed in μm/sec) of the mechanically-evoked depolarisation is lower, compared to Control (CTR)- and Cholesterol (CHOL)-treated cells. * = p<0.05 vs. CTR. Mann-Whitney U Test.</p

Na_V1.8 associates with lipid rafts in DRG neurons <i>in vitro</i>.

<p>Lipid rafts were extracted from DRG neurons after 2 DIV. After centrifugation on an Iodixanol density gradient fractions were analysed by western blotting and dot blot analysis to assess lipid raft isolation and Na_V1.8 partitioning between lipid rafts and the non-raft portions of the membrane. Flotillin1 and GM1 were used as a protein and lipid marker of lipid rafts, respectively. Transferrin receptor was used a marker of non-raft portions. Na_V1.8 is associated with both lipid rafts and non-raft portions of the membrane (A). Incorporation of 7KC into the neuronal plasma-membranes impairs lipid raft stability. In this condition total Na_V1.8 is associated with the non-raft portion of the membrane (B). Depletion of cholesterol from the neuronal membrane, by using MβCD, leads to lipid rafts disruption. Na_V1.8 is only associated with the soluble, non-raft, portion of the membrane upon this treatment (C). M represents protein ladder, the recovered fractions are numbered from 1 (top fraction) to 9 (bottom fraction).</p