Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells.

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).

Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

Sustained rescue of prefrontal circuit dysfunction by antidepressant-induced spine formation.

The neurobiological mechanisms underlying the induction and remission of depressive episodes over time are not well understood. Through repeated longitudinal imaging of medial prefrontal microcircuits in the living brain, we found that prefrontal spinogenesis plays a critical role in sustaining specific antidepressant behavioral effects and maintaining long-term behavioral remission. Depression-related behavior was associated with targeted, branch-specific elimination of postsynaptic dendritic spines on prefrontal projection neurons. Antidepressant-dose ketamine reversed these effects by selectively rescuing eliminated spines and restoring coordinated activity in multicellular ensembles that predict motivated escape behavior. Prefrontal spinogenesis was required for the long-term maintenance of antidepressant effects on motivated escape behavior but not for their initial induction.

Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity.

Phosphorylation of the transcription factor CREB is thought to be important in processes underlying long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or providing synaptic input, or how synapse-to-nucleus communication is mediated. We found that Ca(2+)-dependent nuclear CREB phosphorylation was rapidly evoked by synaptic stimuli including, but not limited to, those that induced potentiation and depression of synaptic strength. In striking contrast, high frequency action potential firing alone failed to trigger CREB phosphorylation. Activation of a submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase.

Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments.

Dendritic arbors are critical for the information processing capability of central neurons, but quantitative analysis of their membrane properties has been hampered by their geometrical complexity. Here, we have focused on an important source of Ca2+ entry in dendrites, the voltage-gated Ca2+ channels, by applying the whole-cell voltage-clamp technique to isolated dendritic segments ("dendrosomes") from rat hippocampal neurons. We found that low voltage-activated T-type Ca2+ channels provide a significantly larger fraction of the Ca2+ influx in dendrites than their counterparts in cell bodies. Surprisingly, 60%-70% of the high voltage-activated Ca2+ current in dendrosomes was N and P/Q type, and these channels were susceptible to neurotransmitter inhibition, suggesting a novel physiological role for G protein-regulated Ca2+ channel modulation in controlling dendritic excitability and Ca2+ signaling.

Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.

Platelet-activating factor (PAF), an alkylether phospholipid, is produced in the brain when it is subjected to various stimuli. Using a Xenopus oocyte expression system, we obtained evidence for functional PAF receptor mRNA expression in rat brain. The presence of the PAF receptor was confirmed and shown to be quite ubiquitous in the CNS by RNA blot and radioligand binding studies. To investigate the neuronal functions of PAF, intracellular Ca2+ increase elicited by nanomolar PAF application was analyzed in cultured rat hippocampal cells. Fractions of NMDA-responsive cells and non-NMDA-responsive cells were shown to respond to PAF, suggesting a potential role for PAF in the Ca2+ signaling pathway in the hippocampus.

A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons.

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.

Histamine H3R receptor activation in the dorsal striatum triggers stereotypies in a mouse model of tic disorders.

Tic disorders affect ~5% of the population and are frequently comorbid with obsessive-compulsive disorder, autism, and attention deficit disorder. Histamine dysregulation has been identified as a rare genetic cause of tic disorders; mice with a knockout of the histidine decarboxylase (Hdc) gene represent a promising pathophysiologically grounded model. How alterations in the histamine system lead to tics and other neuropsychiatric pathology, however, remains unclear. We found elevated expression of the histamine H3 receptor in the striatum of Hdc knockout mice. The H3 receptor has significant basal activity even in the absence of ligand and thus may modulate striatal function in this knockout model. We probed H3R function using specific agonists. The H3 agonists R-aminomethylhistamine (RAMH) and immepip produced behavioral stereotypies in KO mice, but not in controls. H3 agonist treatment elevated intra-striatal dopamine in KO mice, but not in controls. This was associated with elevations in phosphorylation of rpS6, a sensitive marker of neural activity, in the dorsal striatum. We used a novel chemogenetic strategy to demonstrate that this dorsal striatal activity is necessary and sufficient for the development of stereotypy: when RAMH-activated cells in the dorsal striatum were chemogenetically activated (in the absence of RAMH), stereotypy was recapitulated in KO animals, and when they were silenced the ability of RAMH to produce stereotypy was blocked. These results identify the H3 receptor in the dorsal striatum as a contributor to repetitive behavioral pathology.

Ca2+-dependent regulation in neuronal gene expression.

Ca2+ is an important signal-transduction molecule that plays a role in many intracellular signaling pathways. Recent advances have indicated that in neurons, Ca2+-controlled signaling mechanisms cooperate in order to discriminate amongst incoming cellular inputs. Ca2+-dependent transcriptional events can thereby be made selectively responsive to bursts of synaptic activity of specific intensity or duration.

Critical dependence of cAMP response element-binding protein phosphorylation on L-type calcium channels supports a selective response to EPSPs in preference to action potentials.

Activity-dependent gene expression in neurons shows a remarkable ability to differentiate between different types of stimulation: orthodromic inputs that engage synaptic transmission are much more effective than antidromic stimuli that do not. We have studied the basis of such selectivity in cultured hippocampal neurons in which nuclear cAMP response element-binding protein (CREB) phosphorylation is induced by synaptic activity but not by action potential (AP) stimulation in the absence of EPSPs, although spikes by themselves generate large elevations in intracellular Ca(2+). Previous work has shown that Ca(2+) entry through L-type Ca(2+) channels plays a dominant role in triggering calmodulin mobilization and activation of calmodulin-dependent kinases that phosphorylate CREB, raising the possibility that L-type channels contribute to the selective response to EPSPs rather than APs. Accordingly, we performed voltage-clamp experiments to compare the currents carried by L-type channels during depolarizing waveforms that approximated APs or dendritic EPSPs. The integrated current generated by L-type channels was significantly less after mock APs than with EPSP-like depolarizations. The difference was traced to two distinct factors. Compared with other channels, L-type channels activated at relatively negative potentials, favoring their opening with EPSP stimulation; they also exhibited relatively slow activation kinetics, weighing against their contribution during an AP. The relative ineffectiveness of APs as a stimulus for CREB phosphorylation could be overcome by exposure to the agonist Bay K8644, which potentiated the AP-induced influx through L-type channels by approximately 10-fold. Under normal conditions, the unique biophysical properties of L-type channels allow them to act as a kinetic filter to support spike-EPSP discrimination.

Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.

Remifentanil-based anaesthesia increases the incidence of postoperative surgical site infection.

BACKGROUND: Surgical site infection (SSI) after colorectal surgery is the leading cause of postoperative morbidity. Opioids induce immunosuppression through activation of µ-opioid receptors expressed on leucocytes, and through opioid withdrawal. A high dose of opioid administered as remifentanil during surgery may induce immunosuppression, leading to the development of SSI. AIM: The purpose of this study was to investigate the influence of remifentanil on the development of SSI. METHODS: Adult patients who underwent elective colorectal surgery from January 2009 to December 2012 (N = 286) were prospectively investigated according to the guidelines of the US Centers for Disease Control and Prevention. After exclusion of 51 patients, propensity matching was performed in 235 patients. To reduce the influence of selection on SSIs, propensity score pairwise matching was performed for patients maintained with remifentanil and for patients maintained with fentanyl. FINDINGS: The number of patients who developed SSI was higher after remifentanil-based anaesthesia compared with fentanyl-based anaesthesia [11.6% (17/146) vs 3.4% (3/89), remifentanil vs fentanyl, P = 0.03] before propensity matching. Propensity matching yielded 61 pairs of patients anaesthetized with remifentanil or fentanyl, and corrected several biases in the preoperative patient characteristics. After propensity matching, the number of patients who developed SSI was still higher after remifentanil-based anaesthesia than after fentanyl-based anaesthesia [16.4% (10/61) vs 3.3% (2/61), remifentanil vs fentanyl, P = 0.029]. CONCLUSION: Remifentanil-based anaesthesia increased the incidence of SSI. A possible reason may be opioid-induced immunosuppression or opioid withdrawal-induced immunosuppression.

The role of calcium in activity-dependent neuronal gene regulation.

Synaptic transmission is a key signaling event, whereby an action potential-induced release of chemical neurotransmitters again generates a positive or negative electrical activity via opening of postsynaptic channels. Thereafter, information spreads through space, from the postsynaptic membranes to the dendrites, to the soma, to the nucleus, to the presynaptic terminals and, in some cases, back to the originally stimulated synapses. Furthermore, information is also often converted in time, either by shifting the phase of electrical activity during the integration of EPSPs and IPSPs into the generation of an action potential, or by triggering a long-lasting cascade of enzymatic or protein-protein interaction-mediated events in the cytoplasm and in the nucleus. Recent studies of the signaling from the synapse to the nucleus now allow us to consider how various patterns of synaptic activity could couple with activation of specific nuclear transcription factors and thus regulate neuronal gene expression. The critical importance of Ca(2+)-dependent signaling processes in such regulatory events will be discussed below.

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor.

The human platelet-activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5'-noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF-kappa B and Sp-1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP-1, AP-2, and Sp-1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL-1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Leukotriene A4 hydrolase from guinea pig lung: the presence of two catalytically active forms.

Leukotriene A4 hydrolase was purified to apparent homogeneity from the guinea pig lung. The molecular weight was determined to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited two active forms with different pI values (5.7 and 5.4) depending on the presence or absence of SH-reducing reagents during purification procedures. No significant differences were observed between both forms of the enzyme as regards the catalytic properties. The N-terminal 20 amino acid sequence (PEVVDTXSLASPATVXRTKH) showed a 90% identity to the human enzyme with a constitutive substitution of Ile-3 and Ser-14 (human) by Val-3 and Thr-14 (guinea pig), respectively.

Activation of mitogen-activated protein kinase and arachidonate release via two G protein-coupled receptors expressed in the rat hippocampus.

Platelet-activating factor and somatostatin receptors, two G protein-coupled receptors expressed in the rat hippocampus, were analyzed for the downstream signaling pathways in Chinese hamster ovary cells stably expressing each receptor. Ligand stimulation to each CHO cell line induced (1) inhibition of forskolin-induced accumulation of cAMP, (2) arachidonate release, and (3) activation of mitogen-activated protein kinase and MAP kinase kinase. In contrast, inositol phosphate breakdown was seen only in the PAF-stimulated CHO cells. The induction of these signals accompanied no detectable Ras activation. Suppression of the signals by pertussis toxin was almost complete for the somatostatin receptor but partial for the PAF receptor, suggesting that the somatostatin receptor couples only with PTX-sensitive G protein, while the PAF receptor couples with both PTX-sensitive and -insensitive G proteins. A model of G protein-mediated signaling pathways was proposed in which the signals from Gi and those from Gq converge at MAP kinase kinase and lead to arachidonate release. The present system using CHO cells is useful for analyzing signaling pathways from G proteins to MAP kinase kinase and will thereby provide clues for understanding the mechanisms underlying the physiological and pathological events mediated by PAF, somatostatin, and other G protein-coupled receptors in the central nervous system and other tissues.

Citron, a Rho target that affects contractility during cytokinesis.

The small GTPase Rho, which regulates cell shape, is thought to contribute to cytokinesis. Recently, Citron was characterized as a Rho target. This large protein contains a Ser/Thr kinase domain related to that of ROCK, another Rho effector. Both endogenous Citron and recombinant Citron localize to the cleavage furrow in dividing cells and to the midbody in post-mitotic cells. Moreover, overexpression of Citron deleted from its C-terminal sequence caused abnormal contractions specifically during cytokinesis, resulting in the formation of multinucleated cells. Cell shape, F-actin, intermediate filaments, and microtubules appeared essentially normal in these cells during interphase. Thus, Citron is a Rho effector that appears to function during cytokinesis, modulating its contractile process. In brain, however, Citron is highly expressed in a subset of neurons as a brain-specific isoform that lacks a kinase domain, Citron-N. This protein accumulates in synapses and associates to the NMDA receptor via interaction with the adaptor protein PSD95, suggesting that the function of Citron is specialized in the neurons.