A Neuronal Isoform of Protein Kinase G Couples Mitogen-Activated Protein Kinase Nuclear Import to Axotomy-Induced Long-Term Hyperexcitability inAplysiaSensory Neurons

Abstract

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.

Memory-Like Alterations inAplysiaAxons after Nerve Injury or Localized Depolarization

“…The observation of depolarization-induced LTH in intact axons shows that axonal LTH can be produced by mechanisms other than damming of Na ϩ channels at the proximal stump of a severed axon (Devor and Govrin-Lippmann, 1983;Devor et al, 1993). The distance of the treated nerve segment from the neuronal soma (2-4 cm) and the blockade of synaptic transmission in the ganglion during induction and testing of LTH, coupled with reported rates of axonal transport in Aplysia (ϳ1.5 mm/hr) (Ambron et al, 1992;Gunstream et al, 1995;Sung et al, 2004), suggests that the axonal LTH seen 24 hr after treatment does not depend on transport of retrograde signals that produce transcriptional or translational effects in the soma Sung et al, 2004) and subsequent anterograde transport of plasticity-mediating molecules to the axonal test site.…”

Role of Nitric Oxide in Classical Conditioning of Siphon Withdrawal inAplysia

“…N -nitro-L-arginine (500 M) blocked conditioning (F ϭ 7.80; p Ͻ 0.01 for the interaction of drug and pairing overall and on the posttest), suggesting that NO is involved. Because NO is thought to act by stimulating cGMP and cGMPdependent protein kinase (PKG) in many systems, including Aplysia neurons (Mothet et al, 1996a;Jacklet, 1999, 2001;Sung et al, 2004;Bodnarova et al, 2005), we tested an inhibitor of PKG, KT5823 (2 M), which also blocked conditioning (F ϭ 8.32; p Ͻ 0.01 overall and on the posttest). Neither drug had significant effects on the amplitude of the initial response to the CS or US.…”

“…However, it is not clear whether NO might act through cGMP in LE sensory neurons. Although physiological evidence suggests that cGMP is present in VC sensory neurons in the pleural ganglia (Lewin and Walters, 1999;Sung et al, 2004), there is no detectable staining for guanylyl cyclase (Martasek et al, 2004) (L. Moroz and M. Bodnarova, Figure 8. Injecting botulinum toxin into an LFS motor neuron reduces facilitation.…”

Enhanced Transmission at a Spinal Synapse TriggeredIn Vivoby an Injury Signal Independent of Altered Synaptic Activity

“…The question can be addressed by distinguishing signals generated when axon damage interrupts central (retrograde) transport of constitutive molecules (negative injury signals) versus ones that are newly introduced, activated, or modified by axon injury (positive injury signals) (Ambron and Walters, 1996;Perlson et al, 2004;McMahon et al, 2005). Both negative and positive injury signals have been shown to initiate synaptic change (Lessmann, 1998;Mendell et al, 1999;Woolf and Costigan, 1999;Mendell and Arvanian, 2002;Kohno et al, 2003;Du and Poo, 2004;Sung et al, 2004;McMahon et al, 2005). For IA-MN synapses, deprivation of muscle-derived neurotrophin NT3 may contribute to the decline in EPSP amplitude that occurs Ͼ1 week after nerve section (Mendell et al, 2001).…”

“…This Ca 2ϩ signal drives protein kinase pathways (Ji and Strichartz, 2004) with the capacity to exert retrograde effects on synaptic transmission . For example, protein kinase G is a positive retrograde injury signal that increases neuronal excitability (Sung et al, 2004(Sung et al, , 2006 and has the potential to meet our criteria.…”