Eugene E. Fibuch

Modulation of D2R-NR2B Interactions in Response to Cocaine

Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.

Regulation of mitogen‐activated protein kinases by glutamate receptors

Glutamate receptors regulate gene expression in neurons by activating intracellular signaling cascades that phosphorylate transcription factors within the nucleus. The mitogen‐activated protein kinase (MAPK) cascade is one of the best characterized cascades in this regulatory process. The Ca2+‐permeable ionotropic glutamate receptor, mainly the NMDA receptor subtype, activates MAPKs through a biochemical route involving the Ca2+‐sensitive Ras‐guanine nucleotide releasing factor, Ca2+/calmodulin‐dependent protein kinase II, and phosphoinositide 3‐kinase. The metabotropic glutamate receptor (mGluR), however, activates MAPKs primarily through a Ca2+‐insensitve pathway involving the transactivation of receptor tyrosine kinases. The adaptor protein Homer also plays a role in this process. As an information superhighway between surface glutamate receptors and transcription factors in the nucleus, active MAPKs phosphorylate specific transcription factors (Elk‐1 and CREB), and thereby regulate distinct programs of gene expression. The regulated gene expression contributes to the development of multiple forms of synaptic plasticity related to long‐lasting changes in memory function and addictive properties of drugs of abuse. This review, by focusing on new data from recent years, discusses the signaling mechanisms by which different types of glutamate receptors activate MAPKs, features of each MAPK cascade in regulating gene expression, and the importance of glutamate/MAPK‐dependent synaptic plasticity in memory and addiction.

Stability of surface NMDA receptors controls synaptic and behavioral adaptations to amphetamine

Plastic changes in glutamatergic synapses that lead to endurance of drug craving and addiction are poorly understood. We examined the turnover and trafficking of NMDA receptors and found that chronic exposure to the psychostimulant amphetamine (AMPH) induced selective downregulation of NMDA receptor NR2B subunits in the confined surface membrane pool of rat striatal neurons at synaptic sites. This downregulation was a long-lived event and was a result of the destabilization of surface-expressed NR2B caused by accelerated ubiquitination and degradation of crucial NR2B-anchoring proteins by the ubiquitin-proteasome system. The biochemical loss of synaptic NR2B further translated to the modulation of synaptic plasticity in the form of long-term depression at cortico-accumbal glutamatergic synapses. Behaviorally, genetic disruption of NR2B induced and restoration of NR2B loss prevented behavioral sensitization to AMPH. Our data identify NR2B as an important regulator in the remodeling of excitatory synapses and persistent psychomotor plasticity in response to AMPH.

Activity-Dependent Modulation of Limbic Dopamine D3 Receptors by CaMKII

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is central to synaptic transmission. Here we show that synaptic CaMKIIalpha binds to the N-terminal region of the third intracellular loop of the limbic dopamine D3 receptor (D3R). This binding is Ca(2+) sensitive and is sustained by autophosphorylation of CaMKII, providing an unrecognized route for the Ca(2+)-mediated regulation of D3Rs. The interaction of CaMKIIalpha with D3Rs transforms D3Rs into a biochemical substrate of the kinase and promotes the kinase to phosphorylate D3Rs at a selective serine site (S229). In accumbal neurons in vivo, CaMKIIalpha is recruited to D3Rs by rising Ca(2+) to increase the CaMKIIalpha-mediated phosphorylation of D3Rs, thereby transiently inhibiting D3R efficacy. Notably, the D3R inhibition is critical for integrating dopamine signaling to control behavioral sensitivity to the psychostimulant cocaine. Our data identify CaMKIIalpha as a recruitable regulator of dopamine receptor function. By binding and phosphorylating limbic D3Rs, CaMKIIalpha modulates dopamine signaling and psychomotor function in an activity-dependent manner.

Role of Protein Phosphatase 2A in mGluR5-regulated MEK/ERK Phosphorylation in Neurons

The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Gαq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2.

Effects of Isoflurane Anesthesia and Muscle Paralysis on Respiratory Mechanics in Normal Man

In five healthy adult male volunteers in the supine position, respiratory mechanics and functional residual capacity (FRC) were studied in the awake state (control) and with muscle paralysis and mechanical ventilation during isoflurane anesthesia (inspired concentrations, 1 and 2 per cent). In eight of nine instances, FRC was less during isoflurane anesthesia compared with control. Static compliance of the total respiratory system (Crs) decreased consistently during anesthesia and that of the lung (Ct) decreased in eight of nine instances; static compliance of the chest wall (Cπ) did not change. Average pulmonary resistance (Rl) was significantly higher during anesthesia. The decrease in FRC and increase in Rl appear to be somewhat less than those reported for other anesthetics. Increasing the inspired isoflurane concentration to 2 per cent had no further significant effect on FRC, Crs C19 Cl and Rl Arterial blood pressure was decreased significantly and heart rate remained unchanged during anesthesia with I per cent isoflurane; with 2 per cent isoflurane, blood pressure was further significantly decreased and heart rate did not change significantly.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

Background: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-d-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. Methods: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Results: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist–induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Conclusions: Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

In vivo regulation of Homer1a expression in the striatum by cocaine.

The glutamate receptor adaptor protein Homer is concentrated in the postsynaptic density of excitatory synapses and is critical for normal operation of synaptic transmission. In this study, we investigated the responsiveness of Homer family proteins to dopamine stimulation with the psychostimulant cocaine in rat striatal neurons both in vivo and in vitro. We found that a single dose of cocaine specifically induced a rapid and transient increase in protein levels of the Homer1a, but not Homer1b/c and Homer2a/b, isoforms in the striatum. This selective Homer1a induction was mediated primarily through activation of dopamine D1, but not D2, receptors. Both protein kinase A and Ca2+/calmodulin-dependent protein kinases are important for mediating the cocaine stimulation of Homer1a expression. At the transcriptional level, cAMP response element-binding protein serves as a prime transcription factor transmitting the signals derived from D1 receptors and associative pathways to the CaCRE sites within the Homer1a promoter. From a functional perspective, non–cross-linking Homer1a, once induced, competed with the cross-linking isoforms of Homer proteins (Homer1b/c and Homer2a/b) to uncouple the connection of group I metabotropic glutamate receptors (mGluRs) with inositol-1,4,5-triphosphate receptors. These results indicate that cocaine possesses the ability to stimulate Homer1a expression in striatal neurons through a specific synapse-to-nucleus pathway. Moreover, inducible Homer1a expression may represent a transcription-dependent mechanism underlying the dynamic regulation of submembranous macromolecular complex formation between group I mGluRs and their anchoring proteins.

Phosphorylation of glutamate receptors: A potential mechanism for the regulation of receptor function and psychostimulant action

Ionotropic glutamate receptors, N‐methyl‐d‐aspartate receptors (NMDARs) and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptors (AMPARs), are densely distributed in the mammalian brain and actively regulate a variety of cellular activities. Expression and function of these receptors are also under a tight regulation by many molecular mechanisms. Protein phosphorylation represents one of the important mechanisms for the posttranslational modulation of these receptors. Constitutive and regulatory phosphorylation occurs at distinct sites (serine, threonine, or tyrosine) on the intracellular C‐terminal domain of almost all subunits capable of assembling a functional channel. Several key protein kinases, such as protein kinase A, protein kinase C, Ca2+/calmodulin‐dependent protein kinases, and tyrosine kinases are involved in the site‐specific catalyzation and regulation of NMDAR and AMPAR phosphorylation. Through the phosphorylation mechanism, these protein kinases as well as protein phosphatases control biochemi cal properties (biosynthesis, delivery, and subunit assembling), subcellular distribution, and interactions of these receptors with various synaptic proteins, which ultimately modify the efficacy and strength of excitatory synapses containing NMDARs and AMPARs and many forms of synaptic plasticity. Emerging evidence shows that psychostimulants (cocaine and amphetamine) are among effective agents that profoundly alter the phosphorylation status of both receptors in striatal neurons in vivo. Thus, psychostimulants may modulate NMDAR and AMPAR function through the phosphorylation mechanism to shape the excitatory synaptic plasticity related to additive properties of drugs of abuse.

Inhibition of glutamatergic activation of extracellular signal-regulated protein kinases in hippocampal neurons by the intravenous anesthetic propofol.

Background:Intravenous anesthetics cause amnesia, but the underlying molecular mechanisms are poorly understood. Recent studies reveal a significant role of extracellular signal–regulated protein kinases (ERKs) in controlling synaptic plasticity and memory formation. As a major synapse-to-nucleus superhighway, ERK transmits N-methyl-d-aspartate (NMDA) receptor signals to inducible transcriptional events essential for NMDA receptor–dependent forms of synaptic plasticity and memory. This study investigated the role of the widely used intravenous anesthetic propofol in regulating NMDA receptor–dependent ERK phosphorylation. Methods:The possible effect of propofol on NMDA receptor–mediated ERK phosphorylation was detected in cultured rat hippocampal neurons with Western blot analysis. Results:The authors found that propofol at clinical relevant concentrations (1–10 &mgr;m) reduced NMDA receptor–mediated ERK phosphorylation. This reduction was independent of &ggr;-aminobutyric acid transmission. The inhibition of the NMDA receptor seems to contribute to the effect of propofol on NMDA-stimulated ERK phosphorylation, because propofol reduced constitutive NMDA receptor NR1 subunit phosphorylation and impaired NMDA receptor–mediated Ca2+ influx. Furthermore, by inhibiting the ERK pathway, propofol blocked NMDA receptor–dependent activation of two key transcription factors, Elk-1 and cyclic adenosine monophosphate response element–binding protein (CREB), and, as a result, attenuated Elk-1/CREB–dependent reporter gene (c-Fos) expression. Conclusions:These results suggest that propofol possesses the ability to inhibit NMDA receptor activation of the ERK pathway and subsequent transcriptional activities in hippocampal neurons. These findings indicate a new avenue to explore a transcription-dependent mechanism that may underlie anesthetic interference with synaptic plasticity related to amnesic properties of intravenous anesthetics.