Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain.

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states.

FGF8 acts as a classic diffusible morphogen to pattern the neocortex.

Gain- and loss-of-function experiments have demonstrated that a source of fibroblast growth factor (FGF) 8 regulates anterior to posterior (A/P) patterning in the neocortical area map. Whether FGF8 controls patterning as a classic diffusible morphogen has not been directly tested. We report evidence that FGF8 diffuses through the mouse neocortical primordium from a discrete source in the anterior telencephalon, forms a protein gradient across the entire A/P extent of the primordium, and acts directly at a distance from its source to determine area identity. FGF8 immunofluorescence revealed FGF8 protein distributed in an A/P gradient. Fate-mapping experiments showed that outside the most anterior telencephalon, neocortical progenitor cells did not express Fgf8, nor were they derived from Fgf8-expressing cells, suggesting that graded distribution of FGF8 results from protein diffusion from the anterior source. Supporting this conclusion, a dominant-negative high-affinity FGF8 receptor captured endogenous FGF8 at a distance from the FGF8 source. New FGF8 sources introduced by electroporation showed haloes of FGF8 immunofluorescence indicative of FGF8 diffusion, and surrounding cells reacted to a new source of FGF8 by upregulating different FGF8-responsive genes in concentric domains around the source. Reducing endogenous FGF8 with the dominant-negative receptor in the central neocortical primordium induced cells to adopt a more posterior area identity, demonstrating long-range area patterning by FGF8. These observations support FGF8 as a classic diffusible morphogen in neocortex, thereby guiding future studies of neocortical pattern formation.

The persistent release of HMGB1 contributes to tactile hyperalgesia in a rodent model of neuropathic pain.

BACKGROUND: High-mobility group box-1 protein (HMGB1) is a nuclear protein that regulates gene expression throughout the body. It can also become cytoplasmic and function as a neuromodulatory cytokine after tissue damage or injury. The manner in which HMGB1 influences the peripheral nervous system following nerve injury is unclear. The present study investigated the degree to which HMGB1 signaling contributes to the maintenance of neuropathic pain behavior in the rodent. RESULTS: Redistribution of HMGB1 from the nucleus to the cytoplasm occurred in both sensory neurons derived from a tibial nerve injured (TNI) rat and in a sensory neuron-like cell line following exposure to a depolarizing stimulus. We also observe that exogenous administration of HMGB1 to acutely dissociated sensory neurons derived from naïve or TNI rodents elicit increased excitability. Furthermore systemic injection of glycyrrhizin (50 mg/kg; i.p.), a known inhibitor of HMGB1, reversed TNI-induced mechanical hyperalgesia at fourteen days and three months following nerve injury. CONCLUSIONS: We have identified that a persistent endogenous release of HMGB1 by sensory neurons may be a potent, physiologically relevant modulator of neuronal excitability. More importantly, the use of the anti-inflammatory compound and known inhibitor of HMGB1, glycyrrhizin, has the ability to diminish persistent pain behavior in a model of peripheral neuropathy, presumably through its ability to neutralize the cyotkine. The identification of HMGB1 as a potential therapeutic target may contribute to a better understanding of mechanisms associated with chronic pain syndromes.

Neuroexcitatory effects of morphine-3-glucuronide are dependent on Toll-like receptor 4 signaling.

BACKGROUND: Multiple adverse events are associated with the use of morphine for the treatment of chronic non-cancer pain, including opioid-induced hyperalgesia (OIH). Mechanisms of OIH are independent of opioid tolerance and may involve the morphine metabolite morphine-3-glucuronide (M3G). M3G exhibits limited affinity for opioid receptors and no analgesic effect. Previous reports suggest that M3G can act via the Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) heterodimer in the central nervous system to elicit pain. METHODS: Immunoblot and immunocytochemistry methods were used to characterize the protein expression of TLR4 present in lumbar dorsal root ganglion (DRG). Using in vitro intracellular calcium and current clamp techniques, we determined whether TLR4 activation as elicited by the prototypical agonists of TLR4, lipopolysaccharide (LPS) and M3G, contributed to changes in intracellular calcium and increased excitation. Rodents were also injected with M3G to determine the degree to which M3G-induced tactile hyperalgesia could be diminished using either a small molecule inhibitor of the MD-2/TLR4 complex in rats or TLR4 knockout mice. Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 µm < DRG diameter <45 µm) for both control and M3G-treated neurons to determine the potential influence on voltage-gated sodium channels (NaVs). RESULTS: We observed that TLR4 immunoreactivity was present in peptidergic and non-peptidergic sensory neurons in the DRG. Non-neuronal cells in the DRG lacked evidence of TLR4 expression. Approximately 15% of assayed small- and medium-diameter sensory neurons exhibited a change in intracellular calcium following LPS administration. Both nociceptive and non-nociceptive neurons were observed to respond, and approximately 40% of these cells were capsaicin-insensitive. Increased excitability observed in sensory neurons following LPS or M3G could be eliminated using Compound 15, a small molecule inhibitor of the TLR4/MD-2 complex. Likewise, systemic injection of M3G induced rapid tactile, but not thermal, nociceptive behavioral changes in the rat, which were prevented by pre-treating animals with Compound 15. Unlike TLR4 wild-type mice, TLR4 knockout mice did not exhibit M3G-induced hyperalgesia. As abnormal pain sensitivity is often associated with NaVs, we predicted that M3G acting via the MD-2/TLR4 complex may affect the density and gating of NaVs in sensory neurons. We show that M3G increases tetrodotoxin-sensitive and tetrodotoxin-resistant (NaV1.9) current densities. CONCLUSIONS: These outcomes provide evidence that M3G may play a role in OIH via the TLR4/MD-2 heterodimer complex and biophysical properties of tetrodotoxin-sensitive and tetrodotoxin-resistant NaV currents.

Bone morphogenetic protein signaling in the developing telencephalon controls formation of the hippocampal dentate gyrus and modifies fear-related behavior.

The cortical hem is an embryonic signaling center that generates bone morphogenetic proteins (BMPs) and acts as an organizer for the hippocampus. The role of BMP signaling in hippocampal neurogenesis, however, has not been established. We therefore generated mice that were deficient in Bmpr1b constitutively, and deficient in Bmpr1a conditionally in the dorsal telencephalon. In double mutant male and female mice, the dentate gyrus (DG) was dramatically smaller than in control mice, reflecting decreased production of granule neurons at the peak period of DG neurogenesis. Additionally, the pool of cells that generates new DG neurons throughout life was reduced, commensurate with the smaller size of the DG. Effects of diminished BMP signaling on the cortical hem were at least partly responsible for these defects in DG development. Reduction of the DG and its major extrinsic output to CA3 raised the possibility that the DG was functionally compromised. We therefore looked for behavioral deficits in double mutants and found that the mice were less responsive to fear- or anxiety-provoking stimuli, whether the association of the stimulus with fear or anxiety was learned or innate. Given that no anatomical defects appeared in the double mutant telencephalon outside the DG, our observations support a growing literature that implicates the hippocampus in circuitry mediating fear and anxiety. Our results additionally indicate a requirement for BMP signaling in generating the dorsalmost neuronal lineage of the telencephalon, DG granule neurons, and in the development of the stem cell niche that makes neurons in the adult hippocampus.

Challenging the catechism of therapeutics for chronic neuropathic pain: Targeting CaV2.2 interactions with CRMP2 peptides.

Chronic neuropathic pain management is a worldwide concern. Pharmaceutical companies globally have historically targeted ion channels as the therapeutic catechism with many blockbuster successes. Remarkably, no new pain therapeutic has been approved by European or American regulatory agencies over the last decade. This article will provide an overview of an alternative approach to ion channel drug discovery: targeting regulators of ion channels, specifically focusing on voltage-gated calcium channels. We will highlight the discovery of an anti-nociceptive peptide derived from a novel calcium channel interacting partner - the collapsin response mediator protein 2 (CRMP2). In vivo administration of this peptide reduces pain behavior in a number of models of neuropathic pain without affecting sympathetic-associated cardiovascular activity, memory retrieval, sensorimotor function, or depression. A CRMP2-derived peptide analgesic, with restricted access to the CNS, represents a completely novel approach to the treatment of severe pain with an improved safety profile. As peptides now represent one of the fastest growing classes of new drugs, it is expected that peptide targeting of protein interactions within the calcium channel complex may be a paradigm shift in ion channel drug discovery.

Chemokine signaling and the management of neuropathic pain.

Chemokines and chemokine receptors are widely expressed in the nervous system, where they play roles in the regulation of stem cell migration, axonal path finding, and neurotransmission. Chemokine signaling is also of key importance in the regulation of neuroinflammatory responses. The expression of the chemokine monocyte chemoattractant protein 1 (MCP1) and its receptor (CCR2) is upregulated by dorsal root ganglia neurons in rodent models of neuropathic pain. MCP1 increases the excitability of nociceptive neurons after a peripheral nerve injury, and disruption of MCP1 signaling blocks the development of neuropathic pain. In the spinal cord, microglial cells expressing CCR2 are thought to play an active role in the initiation and maintenance of pain hypersensitivity, and MCP1 may also alter the excitability of spinal neurons in some cases. Other predominant sites of CCR2 activation are found in the peripheral nervous system, thereby explaining, at least in some circumstances, the rapid anti-nociceptive effects of peripherally administered CCR2 antagonists. In this article we discuss the relative roles of CCR2 activation in the peripheral and central nervous systems in relation to the phenomenon of neuropathic pain.