Patrick L. Stemkowski

HIV-1 viral protein R causes peripheral nervous system injury associated with in vivo neuropathic pain

Painful peripheral neuropathy has become the principal neurological disorder in HIV/AIDS patients. Herein, we investigated the effects of a cytotoxic HIV‐1 accessory protein, viral protein R (Vpr), on the peripheral nervous system (PNS). Host and viral gene expression was investigated in peripheral nerves from HIV‐infected individuals and in HIV‐infected human dorsal root ganglion (DRG) cultures by RT‐PCR and immunocytochemistry. Cytosolic calcium ([Ca2+]) fluxes and neuronal membrane responses were analyzed in cultured DRGs. Neurobehavioral responses and cytokine levels were assessed in a transgenic mouse model in which the vpr transgene was expressed in an immunodeficient background (vpr/RAG1−/−). Vpr transcripts and proteins were detected in peripheral nerves and DRGs from HIV‐infected patients. Exposure of rat or human cultured DRG neurons to Vpr rapidly increased [Ca2+] and action potential frequency while increasing input resistance. HIV infection of human DRG cultures caused neurite retraction (P<0.05), accompanied by induction of interferon‐α (IFN‐α) transcripts (P<0.05). vpr/RAG1−/− mice expressed Vpr together with increased IFN‐α (P<0.05) in the PNS and also exhibited mechanical allodynia, unlike their vpr/RAG1−/− littermates (P<0.05). Herein, Vpr caused DRG neuronal damage, likely through cytosolic calcium activation and cytokine perturbation, highlighting Vprs contribution to HIV‐associated peripheral neuropathy and ensuing neuropathic pain.—Acharjee, S., Noorbakhsh, S., Stemkowski, P. L., Olechowski, C., Cohen, E. A., Ballanyi, K., Kerr, B., Pardo, C., Smith, P. A., Power, C. HIV‐1 viral protein R causes peripheral nervous system injury associated with in vivo neuropathic pain. FASEBJ. 24, 4343–4353 (2010). www.fasebj.org

Intrathecal administration of proteinase-activated receptor-2 agonists produces hyperalgesia by exciting the cell bodies of primary sensory neurons.

Proteinase-activated receptors (PARs) are a family of G-protein-coupled receptors that are activated by endogenous serine proteinases that cleave the N-terminal domain of the receptor unmasking a “tethered ligand” sequence. Trypsin and other agonists at PAR2 act on peripheral nerves to augment the transfer of nociceptive information. We tested whether PAR2 agonists also exert a spinal pronociceptive effect by i.t. administering the selective ligand, Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLI-GRL). This produced thermal and mechanical hyperalgesia in rats and mice and augmented mechanical and thermal hyperalgesia seen in the formalin inflammatory pain test. Effects of SLIGRL were abrogated in PAR2-deficient mice and were not seen with the inactive control peptide, Leu-Arg-Gly-Ile-Leu-Ser-NH2. Surprisingly, electrophysiological studies, using whole-cell recording from rat substantia gelatinosa neurons, failed to demonstrate an increase in excitatory transmission or neuronal excitability following treatment with SLIGRL or trypsin. In fact, the actions of trypsin were consistent with a decrease in dorsal horn excitability. SLIGRL and trypsin did, however, depolarize and increase the excitability of large, medium and small primary afferent, dorsal root ganglion neurons. The effects were associated with an increase in conductance at hyperpolarized potentials and a decrease in conductance at depolarized potentials. PAR2-like immunoreactivity was found in DRG but not in spinal dorsal horn. These results suggest that activation of DRG neuron cell bodies may account for the pronociceptive actions of i.t. applied PAR2 agonists. They also imply that pathophysiological release of PAR2-activating proteases in the vicinity of DRG neurons may produce profound effects on nociceptive processing in vivo.

Long-term IL-1β exposure causes subpopulation-dependent alterations in rat dorsal root ganglion neuron excitability.

The effect of interleukin-1β (IL-1β) on the electrical properties of sensory neurons was assessed at levels and exposure times comparable to those found in animal models of neuropathic pain. Experiments involved whole cell current-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined-medium, neuron-enriched cultures. Five- to six-day exposure to 100 pM IL-1β produced subpopulation-dependent effects on DRG neurons. These included an increase in the excitability of medium-diameter and small-diameter isolectin B(4) (IB(4))-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large-diameter neurons, while no effect was found in small-diameter IB(4)-negative neurons. Further characterization of changes in medium and small IB(4)-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Although the acute actions of IL-1β on sensory neurons have been well studied and related to acute and/or inflammatory pain, the present study shows how sensory neurons respond to long-term cytokine exposure. Such effects are relevant to understanding processes that contribute to the onset of neuropathic pain.

Possible role of phosphatidylinositol 4,5 bisphosphate in luteinizing hormone releasing hormone-mediated M-current inhibition in bullfrog sympathetic neurons.

Luteinizing hormone releasing hormone (LHRH) is a physiological modulator of neuronal excitability in bullfrog sympathetic ganglia (BFSG). Actions of LHRH involve suppression of the noninactivating, voltage‐dependent M‐type K+ channel conductance (gM). We found, using whole‐cell recordings from these neurons, that LHRH‐induced suppression of gM was attenuated by the phospholipase C (PLC) inhibitor U73122 (10 µm) but not by the inactive isomer U73343 (10 µm). Buffering internal Ca2+ to 117 nm with intracellular 20 mm BAPTA + 8 mm Ca2+ or to < 10 nm with intracellular 20 mm BAPTA + 0.4 mm Ca2+ did not attenuate LHRH‐induced gM suppression. Suppression of gM by LHRH was not antagonized by the inositol 1,4,5 trisphosphate (InsP3) receptor antagonist heparin (∼ 300 µm). Preventing phosphatidylinositol‐4,5‐bisphosphate (PIP2) synthesis by blocking phosphatidylinositol‐4‐kinase with wortmannin (10 µm) or with the nonhydrolysable ATP analogue AMP‐PNP (3 mm) prolonged recovery of LHRH‐induced gM suppression. This effect was not produced by blocking phosphatidyl inositol‐3‐kinase with LY294002 (10 µm). Rundown of gM was attenuated when cells were dialysed with 240 µm di‐octanoyl PIP2 or 240 µm di‐octanoyl phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) but not with 240 µm di‐octanoyl phosphatidylcholine. LHRH‐induced gM suppression was competitively antagonized by dialysis with 240 µm di‐octanoyl PIP2, but not with di‐octanoyl phosphatidylcholine. These results would be expected if LHRH‐induced gM suppression reflects a PLC‐mediated decrease in plasma membrane PIP2 levels.

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 μM) or PGB (10 μM) were less effective than 20 μM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 μM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.

Suppression of network activity in dorsal horn by gabapentin permeation of TRPV1 channels: implications for drug access to cytoplasmic targets.

The effectiveness of gabapentin (GBP) in the treatment of neuropathic pain depends on access to the α2δ-1 accessory subunit of voltage-gated Ca(2+) channels. Access may be limited by its rate of entry via the neuronal system L-neutral amino acid transporter. The open pore of capsaicin-activated TRPV1 channel admits organic molecules such as local anesthetics and we calculated that GBP entry via this route would be 500× more rapid than via the transporter. Capsaicin should therefore increase GBP effectiveness. We used a quaternary GBP derivative (Q-GBP) as sole charge carrier in whole-cell recording experiments on rat dorsal root ganglion (DRG) neurons. Under these conditions, capsaicin produced a capsazepine-sensitive inward current thereby confirming Q-GBP permeation of TRPV1 channels. We have previously established that 5-6 days exposure to 100 μM GBP decreases excitability of dorsal horn neurons whereas 10 μM is ineffective. Excitability was monitored using confocal Ca(2+) imaging of rat spinal cord slices in organotypic culture. GBP effectiveness was augmented by transient exposures of cultures to capsaicin and robust suppression of excitability was seen with 10 μM GBP. Experiments with an inhibitor of the neutral amino acid transporter, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH 300 μM), showed the actions of GBP seen in the presence of capsaicin were independent of its entry by this route. Capsaicin potentiation of GBP depression of dorsal horn activity may therefore reflect drug permeation of TRPV1 channels. Agonist activation of TRP channels may provide a means for improving drug access to cytoplasmic targets in selective neuronal populations defined on the basis of type of TRP channel expressed.

Understanding and Treating Neuropathic Pain

Maladaptive neuropathic pain results from injury or disease of the nervous system. It is typically chronic and frequently intractable. Standard analgesics, such as opioids, are of little use, while gabapentinoids (pregabalin and gabapentin) are not universally effective. In peripherally generated neuropathic pain, an initial inflammatory response releases a variety of mediators, including cytokines and prostaglandins that alter ion channel expression in primary afferent neurons. This initiates ectopic activity in sensory nerves and results in the release of ATP and a second group of mediators from primary afferent terminals. The level of spinal microglial activation is altered such that microglia releases a third set of mediators, notably brain-derived neurotrophic factor (BDNF), in the spinal dorsal horn. Through various mechanisms, BDNF increases excitatory synaptic transmission whilst decreasing inhibitory transmission. The resulting “central sensitization” contributes to hyperalgesia, causalgia, and allodynia that are associated with neuropathic pain. It is suggested that targeting ion channels in the sensory nerves and excitatory transmission in the dorsal horn may lead to urgently needed new treatments for neuropathic pain. It is also suggested that the effectiveness of gabapentinoids may be increased by combining these agents with the TRPV1 agonist capsaicin.