An Itch To Be Scratched

VGLUT2-Dependent Glutamate Release from Nociceptors Is Required to Sense Pain and Suppress Itch

γ-Aminobutyric acid type A receptors (GABA(A)Rs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X(2) receptors (P2X(2)Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA(A)Rs. Here, we investigated a possible "dynamic" interaction between GABA(A)Rs and P2X(2)Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X(2)Rs forms a transient complex with GABA(A)Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X(2)Rs and GABA(A)Rs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X(2)Rs results in a Ca(2+)-dependent as well as an apparently Ca(2+)-independent increase in the mobility and an enhanced degradation of GABA(A)Rs, whereas P2X(2)Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X(2)Rs and GABA(A)Rs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA(A)Rs.

Acid-sensing Ion Channels in Sensory Perception

Chronically CNS-Injured Adult Sensory Neurons Gain Regenerative Competence upon a Lesion of Their Peripheral Axon

The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75(exonIII-/-) mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase(-/-) mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

“Runx”ing towards Sensory Differentiation

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

Epidermal Nerve Fibers Modulate Keratinocyte Growth via Neuropeptide Signaling in an Innervated Skin Model

Activation of Extracellular Signal-Regulated Protein Kinase in Sensory Neurons After Noxious Gastric Distention and Its Involvement in Acute Visceral Pain in Rats

AAV Provides an Alternative for Gene Therapy of the Peripheral Sensory Nervous System

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Variants in Autophagy Genes Affect Susceptibility to Both Crohn's Disease and Helicobacter pylori Infection

In mammalian brain, tau, glycogen synthase kinase 3beta (GSK3beta), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3beta (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722-12728). The molecular mechanism by which 14-3-3 connects GSK3beta to tau within the complex is not clear. In this study, we find that GSK3beta within the tau phosphorylation complex is phosphorylated on Ser(9). From extracts of rat brain and rat primary cultured neurons, Ser(9)-phosphorylated GSK3beta precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser(9)-phosphorylated GSK3beta co-immunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3beta only when the kinase is phosphorylated on Ser(9). In transfected HEK-293 cells, 14-3-3 binds to Ser(9)-phosphorylated GSK3beta and does not bind to GSK3beta (S9A). Tau, on the other hand, binds to both GSK3beta (WT) and GSK3beta (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser(9)-phosphorylated GSK3beta by approximately 3-fold but not with GSK3beta (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser(9)-phosphorylated GSK3beta but not by GSK3beta (S9A). In transfected HEK-293 cells, Ser(9) phosphorylation suppresses GSK3beta-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser(9)-phosphorylated GSK3beta remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser(9)-phosphorylated GSK3beta to tau and Ser(9)-phosphorylated GSK3beta phosphorylates tau.