The α isoform of cGMP-dependent protein kinase 1 (PKG1α) is expressed and functionally important in intrinsic primary afferent neurons of the guinea pig enteric nervous system.

BACKGROUND: Intrinsic primary afferent neurons (IPANs) enable the gut to manifest reflexes in the absence of CNS input. PKG1α is selectively expressed in a subset of neurons in dorsal root ganglia (DRG) and has been linked to nociception and long-term hyperexcitability. METHODS: We used immunoblotting, immunocytochemistry, and in vitro assays of IPAN-dependent enteric functions to test hypotheses that subsets of primary neurons of the ENS and DRG share a reliance on PKG1α expression. KEY RESULTS: PKG1α immunoreactivity was demonstrated in immunoblots from isolated myenteric ganglia. PKG1α, but not PKG1ß, immunoreactivity, was coincident with that of neuronal markers (HuC/D; ß3-tubulin) in both enteric plexuses. PKG1α immunoreactivity also co-localized with the immunoreactivities of the IPAN markers, calbindin (100%; myenteric plexus) and cytoplasmic NeuN (98 ± 1% submucosal plexus). CGRP-immunoreactive DRG neurons, identified as visceral afferents by retrograde transport, were PKG1α-immunoreactive. We used intraluminal cholera toxin to determine whether PKG1α was necessary to enable stimulation of the mucosa to activate Fos in enteric neurons. Tetrodotoxin (1.0 µM), low Ca2+ /high Mg2+ media, and the PKG inhibitor, N46 (100 µM), all inhibited Fos activation in myenteric neurons. N46 also concentration dependently inhibited peristaltic reflexes in isolated preparations of distal colon (IC50  = 83.3 ± 1.3 µM). CONCLUSIONS & INFERENCES: These data suggest that PKG1α is present and functionally important in IPANs and visceral afferent nociceptive neurons.

A novel inhibitor of active protein kinase G attenuates chronic inflammatory and osteoarthritic pain.

Activating PKG-1α induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1α would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1α with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1α and LTH. Injecting complete Freund's adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1α in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1α appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1α antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.

Extremely low frequency electromagnetic fields activate the ERK cascade, increase hsp70 protein levels and promote regeneration in Planaria.

PURPOSE: To use regenerating Planaria Dugesia dorotocethala as a model to determine whether an intermittent modulated extremely low frequency electro-magnetic field (ELF-EMF) produces elevated levels of the heat shock protein hsp70 and stimulates intracellular pathways known to be involved in injury and repair. We focused on serum response element (SRE) binding through the extra-cellular signal-regulated kinase (ERK) cascade. MATERIALS AND METHODS: Planaria were transected equidistant between the tip of the head and the tip of the tail. Individual head and tail portions from the same worm were exposed to a 60 Hertz 80 milliGauss ELF-EMF for 1 h twice daily for 15 days post-transection under carefully controlled exposure conditions. The regenerating heads and tails were photographed and the lengths measured at three-day intervals. In other experiments, the timing of the appearance of pigmented eyes was monitored in the tail portion at 12-h intervals following transection in both ELF-EMF exposed and sham control. In some experiments protein lysates were analysed for hsp70 levels, doubly phosphorylated (pp)-ERK, Elk-1 kinase activity and serum response factor (SRF)-SRE binding. RESULTS: ELF-EMF exposure during the initial 3-days post-surgery caused a significant increase in regeneration for both heads and tails, but especially tails. The first appearance of eyes occurred at day seven post-transection in tail portions exposed to ELF-EMF. In the sham control tail samples the initial appearance of eyes occurred 48 h later. Concurrently, ELF-EMF-exposed heads and tails exhibited an elevation in the level of hsp70 protein, an activation of an ERK cascade, and an increase in SRF-SRE binding. CONCLUSION: Exposures to a modulated sinusoidal ELF-EMF were delivered by a Helmholtz configuration at a frequency of 60 Hz and 80 mG twice a day for one hour. This is accompanied by an increase in hsp70 protein levels, activation of specific kinases and upregulation of transcription factors that are generally associated with repair processes.

Synaptogenesis regulates axotomy-induced activation of c-Jun-activator protein-1 transcription.

The activator protein-1 (AP1) transcription complex remains active for long periods after axotomy, but its activity diminishes during target contact. This raises the possibility that the function of this complex is regulated by the synaptic connections. Using Aplysia californica, we found that crushing peripheral nerves in vivo enhanced AP1 binding in the sensory neurons that lasted for weeks and then declined as regeneration was completed. The AP1 complex in Aplysia is a c-Jun homodimer. Its activation, after axotomy, is mediated by Aplysia c-Jun-N-terminal kinase (apJNK), which enters the nucleus of sensory neurons and phosphorylates c-Jun at Ser-73 (p73-c-Jun). Active AP1 in the sensory neurons did not mediate apoptosis and was not involved in the appearance of the long-term hyperexcitability that develops in these cells after axotomy, and blocking the activation of apJNK in vitro did not influence neurite outgrowth. In contrast, the levels of activated apJNK and p73-c-Jun declined markedly when sensory neurons formed synapses with motor neuron L7 in vitro. Furthermore, inhibiting the pathway accelerated synaptogenesis between sensory neurons and L7. These data suggest that positive and negative modulation of the JNK-c-Jun-AP1 pathway functions in alerting the nucleus to the loss and gain of synapses, respectively.

mRNAs encoding the Aplysia homologues of fasciclin-I and beta-thymosin are expressed only in the second phase of nerve injury and are differentially segregated in axons regenerating in vitro and in vivo.

Studies using Aplysia californica have demonstrated that transcription after nerve injury occurs during a rapid, transient first phase and a delayed, prolonged second phase. Although the second phase is especially important for regeneration, the mRNAs produced during this phase have not been identified. We characterized two such mRNAs following axotomy. One encodes a novel fasciclin-I homologue, Aplysia fasciclin-like protein (apFasP), and the other encodes Aplysia beta-thymosin (apbetaT). In addition to mRNA synthesis, proteins required for regeneration must be available at the site of growth, and the transport and local translation of certain extrasomatic mRNAs aids in this process. We found apbetaT and apFasP proteins and mRNA at growth cones in vitro. However, only the mRNA for apbetaT was present in regenerating axons in vivo. This implies that the membrane protein apFasP is supplied by rapid transport from the soma, whereas the soluble apbetaT is synthesized locally.

Activation of c-Jun and ATF-2 in primate motor cranial nerve nuclei is not associated with apoptosis following axotomy.

Nerve transection induces complex changes in gene regulation and expression that can have profound phenotypic effects on the fate of axotomized neurons. The transcription factors c-Jun and ATF-2 (activating transcription factor-2) are components of a regulatory network that mediates survival, regeneration, and apoptosis following axotomy in rodents. The activation and function of c-Jun and ATF-2 after nerve injury have not been examined in primates. Using a novel model of cranial nerve injury in baboons, we have examined the temporality of c-Jun activation (phosphorylation) in cranial nerve (CN) III and CN VI neurons and ATF-2 activation in CN VI neurons at 2, 4, and 9 days post-injury by immunohistochemistry. Furthermore, we have addressed whether the activation of these factors is associated with apoptosis by the TUNEL assay. We report that activated c-Jun is present in CN III and CN VI neurons ipsilateral to axotomy at 2, 4, and 9 days post-injury, but not in neurons contralateral to injury. Additionally, CN VI neurons ipsilateral to injury at 4 and 9 days contain activated ATF-2. Furthermore, no evidence of TUNEL reactivity was observed in either nucleus, regardless of laterality, at any of the examined time points. These findings suggest that activation of both c-Jun and ATF-2 does not mediate apoptosis in axotomized primate CN III and CN VI neurons at time points up to 9 days. This report serves as a basic inquiry into the neuronal response to cranial nerve injury in primates.

Bridge over troubled waters.

Spinal cord injury interrupts connections between the brain and spinal cord, rather than producing large-scale damage. Reconnecting severed axons with their prior targets is a primary objective of spinal cord repair. Despite progress, this goal will probably not be attained soon because many problems remain to be solved. We discuss an alternative for promoting motor function after spinal damage by bridging the injury. We highlight a novel spinal injury bridge that we have developed to reconnect spinal motor circuits below the injury with the brain. A spinal nerve that exits above the injury is disconnected and inserted into the cord caudal to injury. Motor axons in the inserted nerve regenerate into the cord and synapse on neurons producing a novel circuit to bypass the injury.

PolyADP-ribose polymerase-1 (PARP-1) and the evolution of learning and memory.

PARP-1 is a multifunctional enzyme that can modulate gene expression. Cohen-Armon et al.(1) found that a homologue of PARP-1 is activated in the Aplysia nervous system as the animal responds to an aversive stimulus, which leads to sensitization, and during a more complex form of learning that involves feeding behavior. Significantly, inhibiting PARP-1 activation blocked the learning. Several key pathways in Aplysia neurons are activated both during learning and after injury, suggesting that mechanisms of learning evolved from primitive responses to injury. Since PARP-1 is evolutionarily conserved as a responder to various forms of stress, the finding that PARP-1 is activated during learning supports this idea.

Activity-dependent transcription regulation of PSD-95 by neuregulin-1 and Eos.

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. Here we report that, in the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD-Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of gamma-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD-Eos complex provides a molecular basis for activity-dependent synaptic plasticity.

A neuronal isoform of protein kinase G couples mitogen-activated protein kinase nuclear import to axotomy-induced long-term hyperexcitability in Aplysia sensory neurons.

The induction of a long-term hyperexcitability (LTH) in vertebrate nociceptive sensory neurons (SNs) after nerve injury is an important contributor to neuropathic pain in humans, but the signaling cascades that induce this LTH have not been identified. In particular, it is not known how injuring an axon far from the cell soma elicits changes in gene expression in the nucleus that underlie LTH. The nociceptive SNs of Aplysia (ap) develop an LTH with electrophysiological properties after axotomy similar to those of mammalian neurons and are an experimentally useful model to examine these issues. We cloned an Aplysia PKG (cGMP-dependent protein kinase; protein kinase G) that is homologous to vertebrate type-I PKGs and found that apPKG is activated at the site of injury in the axon after peripheral nerve crush. The active apPKG is subsequently retrogradely transported to the somata of the SNs, but apPKG activity does not appear in other neurons whose axons are injured. In the soma, apPKG phosphorylates apMAPK (Aplysia mitogen-activated protein kinase), resulting in its entry into the nucleus. Surprisingly, studies using recombinant proteins in vivo and in vitro indicate that apPKG directly phosphorylates the threonine moiety in the T-E-Y activation site of apMAPK when the -Y- site contains a phosphate. We used inhibitors of nitric oxide synthase, soluble guanyl cyclase, or PKG after nerve injury, and found that each prevented the appearance of the LTH. Moreover, blocking apPKG activation prevented the nuclear import of apMAPK. Consequently, the nitric oxide-PKG-MAPK pathway is a potential target for treatment of neuropathic pain.

Pathways that elicit long-term changes in gene expression in nociceptive neurons following nerve injury: contributions to neuropathic pain.

Chronic neuropathic pain following nerve injury or inflammation is mediated by transcription-dependent changes in neurons that comprise the nociceptive pathway. Among these changes is often a long-term hyperexcitability (LTH) in primary nociceptors that persists long after the lesion has healed. LTH is manifest by a reduction in threshold and an increased tendency to fire action potentials. This increased excitability activates higher order neurons in the pathway, leading to the perception of pain. Efforts to ameliorate chronic pain would therefore benefit if we understood how LTH is induced, but studies toward this goal are impeded by the complexity and heterogeneity of vertebrate nervous systems. Fortunately, LTH is an evolutionarily conserved mechanism that underlies defensive behaviors across phyla, including invertebrates. Thus, the same electrophysiological changes that underlie LTH in vertebrate nociceptive neurons are seen in their counterparts in the experimentally favorable mollusk Aplysia californica. Nociceptive neurons of Aplysia are readily accessible and large enough to approach using a variety of cell and molecular approaches not possible in higher organisms. Studies of the molecular cascades activated by injury to Aplysia peripheral nerves has focused on a group of positive injury signals that are retrogradely transported from the injury site in the axon to the cell nucleus where they regulate gene transcription. One of these, protein kinase G, is activated by nitric oxide synthetase and its activation in axons is required for the induction of LTH after injury. This pathway, and the transcriptional events that it activates, are targets for therapeutic intervention for chronic pain.

Engineering novel spinal circuits to promote recovery after spinal injury.

We have developed an innovative way to establish a functional bridge around a spinal lesion. We disconnected the T13 nerve from its muscle targets, leaving the proximal end intact. The cut end was inserted either into an intact spinal cord, to assess regeneration of T13 axons into the cord and synapse formation with spinal neurons, or caudal to a hemisection at L2/3, to assess restoration of function below the injury. Four to 28 weeks later, anterograde tracers indicated that axons from the inserted T13 nerve regenerated into the ventral horn, the intermediate zone, and dorsal horn base, both in intact and hemisected animals. Antibodies to cholinergic markers showed that many regenerating axons were from T13 motoneurons. Electrical stimulation of the T13 nerve proximal to the insertion site 4 weeks or more after insertion into the intact cord evoked local field potentials in the intermediate zone and ventral horn, which is where T13 axons terminated. Stimulation of T13 in 71% of the animals (8 hemisected, 7 intact) evoked contraction of the back or leg muscles, depending on the level of insertion. Animals in which T13 was inserted caudal to hemisection had significantly less spasticity and muscle wasting and greater mobility at the hip, knee, ankle, and digits in the ipsilateral hindlimb than did animals with a hemisection only. Thus, T13 motor axons form novel synapses with lumbosacral motor circuits. Because the T13 motor neurons retain their connections to the brain, these novel circuits might restore voluntary control to muscles paralyzed below a spinal lesion.

Rapid electrical and delayed molecular signals regulate the serum response element after nerve injury: convergence of injury and learning signals.

Axotomy elicits changes in gene expression, but little is known about how information from the site of injury is communicated to the cell nucleus. We crushed nerves in Aplysia californica and the sciatic nerve in the mouse and found short- and long-term activation of an Elk1-SRF transcription complex that binds to the serum response element (SRE). The enhanced short-term binding appeared rapidly and was attributed to the injury-induced activation of an Elk1 kinase that phosphorylates Elk1 at ser383. This kinase is the previously described Aplysia (ap) ERK2 homologue, apMAPK. Nerve crush evoked action potentials that propagated along the axon to the cell soma. Exposing axons to medium containing high K(+), which evoked a similar burst of spikes, or bathing the ganglia in 20 microM serotonin (5HT) for 20 min, activated the apMAPK and enhanced SRE binding. Since 5HT is released in response to electrical activity, our data indicate that the short-term process is initiated by an injury-induced electrical discharge that causes the release of 5HT which activates apMAPK. 5HT is also released in response to noxious stimuli for aversive learning. Hence, apMAPK is a point of convergence for injury signals and learning signals. The delay before the onset of the long-term SRE binding was reduced when the crush was closer to the ganglion and was attributed to an Elk1 kinase that is activated by injury in the axon and retrogradely transported to the cell body. Although this Elk1 kinase phosphorylates mammalian rElk1 at ser383, it is distinct from apMAPK.

Histologic analysis of Schwann cell migration and peripheral nerve regeneration in the autogenous venous nerve conduit (AVNC).

Over the last two decades, the autogenous venous nerve conduit (AVNC) has been established as an effective treatment modality for the repair of nerve gaps less than 3 cm. In this study, the spatial-temporal progression of Schwann-cell migration and peripheral-nerve regeneration across a 10-mm gap bridged by a venous conduit was examined, using immunoctyochemical techniques. Histologic analysis revealed that the process of nerve regeneration through an AVNC occurs in four phases: the hematoma phase, cellular migration phase, axonal advancement phase, and myelination and maturation phase. The authors found that: 1) the lumen of the vein conduit remains patent throughout the process of nerve regeneration; 2) Schwann cells migrate into the vital space of the vessel lumen from the proximal and distal nerve stumps; 3) axonal growth into the conduit lags behind Schwann-cell migration; 4) Schwann cells migrate to the regenerating axons to form mature nodes of Ranvier when the distal stump is present; and 5) mechanical injury alone is sufficient to induce axonal outgrowth from the proximal nerve stump.