Bjarke Abrahamsen

SCN9A Mutations in Paroxysmal Extreme Pain Disorder: Allelic Variants Underlie Distinct Channel Defects and Phenotypes

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, or OMIM 167400), is an inherited condition characterized by paroxysms of rectal, ocular, or submandibular pain with flushing. A genome-wide linkage search followed by mutational analysis of the candidate gene SCN9A, which encodes hNa(v)1.7, identified eight missense mutations in 11 families and 2 sporadic cases. Functional analysis in vitro of three of these mutant Na(v)1.7 channels revealed a reduction in fast inactivation, leading to persistent sodium current. Other mutations in SCN9A associated with more negative activation thresholds are known to cause primary erythermalgia (PE). Carbamazepine, a drug that is effective in PEPD, but not PE, showed selective block of persistent current associated with PEPD mutants, but did not affect the negative activation threshold of a PE mutant. PEPD and PE are allelic variants with distinct underlying biophysical mechanisms and represent a separate class of peripheral neuronal sodium channelopathy.

The cell and molecular basis of mechanical, cold, and inflammatory pain.

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Nav1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.

VGLUT2-Dependent Sensory Neurons in the TRPV1 Population Regulate Pain and Itch

The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.

μO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that μO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Nav1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Nav1.8 but not Nav1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03–3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Nav1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

To examine the role of small RNAs in peripheral pain pathways, we deleted the enzyme Dicer in mouse postmitotic damage-sensing neurons. We used a Nav1.8-Cre mouse to target those nociceptors important for inflammatory pain. The conditional null mice were healthy with a normal number of sensory neurons and normal acute pain thresholds. Behavioral studies showed that inflammatory pain was attenuated or abolished. Inflammatory mediators failed to enhance excitability of Nav1.8+ sensory neurons from null mutant mice. Acute noxious input into the dorsal horn of the spinal cord was apparently normal, but the increased input associated with inflammatory pain measured using c-Fos staining was diminished. Microarray and quantitative real-time reverse-transcription PCR (qRT-PCR) analysis showed that Dicer deletion lead to the upregulation of many broadly expressed mRNA transcripts in dorsal root ganglia. By contrast, nociceptor-associated mRNA transcripts (e.g., Nav1.8, P2xr3, and Runx-1) were downregulated, resulting in lower levels of protein and functional expression. qRT-PCR analysis also showed lowered levels of expression of nociceptor-specific pre-mRNA transcripts. MicroRNA microarray and deep sequencing identified known and novel nociceptor microRNAs in mouse Nav1.8+ sensory neurons that may regulate nociceptor gene expression.

High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain

Little is known about the molecular basis of somatosensory mechanotransduction in mammals. We screened a library of peptide toxins for effects on mechanically activated currents in cultured dorsal root ganglion neurons. One conopeptide analogue, termed NMB-1 for noxious mechanosensation blocker 1, selectively inhibits (IC50 1 µM) sustained mechanically activated currents in a subset of sensory neurons. Biotinylated NMB-1 retains activity and binds selectively to peripherin-positive nociceptive sensory neurons. The selectivity of NMB-1 was confirmed by the fact that it has no inhibitory effects on voltage-gated sodium and calcium channels, or ligand-gated channels such as acid-sensing ion channels or TRPA1 channels. Conversely, the tarantula toxin, GsMTx-4, which inhibits stretch-activated ion channels, had no effects on mechanically activated currents in sensory neurons. In behavioral assays, NMB-1 inhibits responses only to high intensity, painful mechanical stimulation and has no effects on low intensity mechanical stimulation or thermosensation. Unexpectedly, NMB-1 was found to also be an inhibitor of rapid FM1-43 loading (a measure of mechanotransduction) in cochlear hair cells. These data demonstrate that pharmacologically distinct channels respond to distinct types of mechanical stimuli and suggest that mechanically activated sustained currents underlie noxious mechanosensation. NMB-1 thus provides a novel diagnostic tool for the molecular definition of channels involved in hearing and pressure-evoked pain.

Sensory Axon-Derived Neuregulin-1 Is Required for Axoglial Signaling and Normal Sensory Function But Not for Long-Term Axon Maintenance

Neuregulin-1 has a key role in mediating signaling between axons and Schwann cells during development. A limitation to studying its role in adulthood is the embryonic lethality of global Nrg1 gene deletion. We used the Cre-loxP system to generate transgenic mice in which neuregulin-1 is conditionally ablated in the majority of small-diameter and a proportion of large-diameter sensory neurons that have axons conducting in the C- and Aδ-fiber range, respectively. Sensory neuron-specific neuregulin-1 ablation resulted in abnormally large Remak bundles with axons clustered in “polyaxonal” pockets. The total number of axons in the sural nerve was unchanged, but a greater proportion was unmyelinated. In addition, we observed large-diameter axons that were in a 1:1 relationship with Schwann cells, surrounded by a basal lamina but not myelinated. There was no evidence of DRG or Schwann cell death; the markers of different DRG cell populations and cutaneous innervation were unchanged. These anatomical changes were reflected in a slowing of conduction velocity at the lower end of the A-fiber conduction velocity range and a new population of more rapidly conducting C-fibers that are likely to represent large-diameter axons that have failed to myelinate. Conditional neuregulin-1 ablation resulted in a reduced sensitivity to noxious mechanical stimuli. These findings emphasize the importance of neuregulin-1 in mediating the signaling between axons and both myelinating and nonmyelinating Schwann cells required for normal sensory function. Sensory neuronal survival and axonal maintenance, however, are not dependent on axon-derived neuregulin-1 signaling in adulthood.

A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain

Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Nav1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Nav1.8Cre-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2f/f;Nav1.8Cre mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Nav1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.

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.