Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis.

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

Postsynaptic D2 dopamine receptor supersensitivity in the striatum of mice lacking TAAR1.

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor (GPCR) known to modulate dopaminergic system through several mechanisms. Mice lacking this receptor show a higher sensitivity to dopaminergic stimuli, such as amphetamine; however, it is not clear whether D1 or D2 dopamine receptors and which associated intracellular signaling events are involved in this modulation. In the striatum of TAAR1 knock out (TAAR1-KO mice) we found that D2, but not D1, dopamine receptors were over-expressed, both in terms of mRNA and protein levels. Moreover, the D2 dopamine receptor-related G protein-independent AKT/GSK3 signaling pathway was selectively activated, as indicated by the decrease of phosphorylation of AKT and GSK3ß. The decrease in phospho-AKT levels, suggesting an increase in D2 dopamine receptor activity in basal conditions, was associated with an increase of AKT/PP2A complex, as revealed by co-immunoprecipitation experiments. Finally, we found that the locomotor activation induced by the D2 dopamine receptor agonist quinpirole, but not by the full D1 dopamine receptor agonist SKF-82958, was increased in TAAR1-KO mice. These data demonstrate pronounced supersensitivity of postsynaptic D2 dopamine receptors in the striatum of TAAR1-KO mice and indicate that a close interaction of TAAR1 and D2 dopamine receptors at the level of postsynaptic structures has important functional consequences.

Photoinactivation of glutamate receptors by genetically encoded unnatural amino acids.

Ionotropic glutamate receptors (iGluRs) are ubiquitous in the mammalian brain, and the AMPA-subtype is essential for fast, glutamate-activated postsynaptic currents. We incorporated photoactive crosslinkers into AMPA receptors using genetically encoded unnatural amino acid mutagenesis in a mammalian cell line. Receptors rescued by incorporation of unnatural amino acids, including p-benzoyl-l-phenylalanine (BzF, also known as Bpa), had largely similar properties to wild-type channels and were expressed at similar levels. BzF incorporation at subunit interfaces afforded photocrosslinking of subunits, as assessed by biochemical experiments. In electrophysiological recordings, BzF incorporation allowed selective and potent UV-driven photoinactivation of both homomeric (GluA2) and heteromeric (GluA2:GluA1) AMPA receptors. State dependence of trapping at two sites in the lower lobe of the ligand binding domain is consistent with deformation of these domains as well as intersubunit rearrangements during AMPA receptor desensitization.

A conformational intermediate in glutamate receptor activation.

Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ∼30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

5-HT1A receptor agonists enhance pyramidal cell firing in prefrontal cortex through a preferential action on GABA interneurons.

5-HT(1A) receptors (5-HT1AR) are expressed by pyramidal and γ-aminobutyric acidergic (GABAergic) neurons in medial prefrontal cortex (mPFC). Endogenous serotonin inhibits mPFC pyramidal neurons via 5-HT1AR while 5-HT1AR agonists, given systemically, paradoxically excite ventral tegmental area-projecting pyramidal neurons. This enhances mesocortical dopamine function, a process involved in the superior efficacy of atypical antipsychotic drugs on negative and cognitive symptoms of schizophrenia. Moreover, the 5-HT1AR-induced increase of pyramidal discharge may also contribute to the maintenance of activity patterns required for working memory, impaired in schizophrenia. Given the importance of these processes, we examined the neurobiological basis of pyramidal activation through 5-HT1AR using the prototypical agent 8-OH-DPAT. (±)8-OH-DPAT (7.5 µg/kg i.v.) increased discharge rate and c-fos expression in rat mPFC pyramidal neurons. Local blockade of GABA(A) inputs with gabazine (SR-95531) avoided (±)8-OH-DPAT-induced excitations of pyramidal neurons. Moreover, (±)8-OH-DPAT administration reduced the discharge rate of mPFC fast-spiking GABAergic interneurons at doses exciting pyramidal neurons. Activation of other 5-HT1AR subpopulations (raphe nuclei or hippocampus) does not appear to contribute to pyramidal excitations. Overall, the present data suggest a preferential action of (±)8-OH-DPAT on 5-HT1AR in GABAergic interneurons. This results in pyramidal disinhibition and subsequent downstream excitations of subcortical structures reciprocally connected with PFC, such as midbrain dopaminergic neurons.

Reduced D2-mediated signaling activity and trans-synaptic upregulation of D1 and D2 dopamine receptors in mice overexpressing the dopamine transporter.

The dopamine transporter (DAT) regulates the temporal and spatial actions of dopamine by reuptaking this neurotransmitter into the presynaptic neurons. We recently generated transgenic mice overexpressing DAT (DAT-tg) that have a 3-fold increase in DAT protein levels which results in a 40% reduction of the extracellular DA concentration in the striatum. The aim of this study was to examine the effect of this reduction in dopaminergic tone on postsynaptic responses mediated by dopamine receptors. We report here that DAT-tg mice have increased levels of striatal D1 (30%) and D2 (approximately 60%) dopamine receptors with D1 receptor signaling components not significantly altered, as evidenced by unaffected basal or stimulated levels of phospho-GluR1 (Ser845) and phospho-ERK2. However, the novel D2 mediated Akt signaling is markedly altered in DAT-tg animals. In particular, there is a 300% increase in the basal levels of phospho-Akt in the striatum of DAT-tg, reflecting the reduced extracellular dopamine tone in these animals. This increase in basal pAkt levels can be pharmacologically recapitulated by partial dopamine depletion in WT mice treated with the selective tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (alpha-MPT). Behaviorally, DAT-tg animals demonstrate an augmented synergistic interaction between up-regulated D1 and D2 receptors, which results in increased climbing behavior in transgenic mice after stimulation with either apomorphine or a co-administration of selective D1 and D2 receptor agonists. In sum, our study reveals that hypodopaminegia caused by up-regulation of DAT results in significant alterations at postsynaptic receptor function with most notable dysregulation at the level of D2 receptor signaling.

Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics.

Since the unexpected discovery of the antipsychotic activity of chlorpromazine, a variety of therapeutic agents have been developed for the treatment of schizophrenia. Despite differences in their activities at various neurotransmitter systems, all clinically effective antipsychotics share the ability to interact with D2 class dopamine receptors (D2R). D2R mediate their physiological effects via both G protein-dependent and independent (beta-arrestin 2-dependent) signaling, but the role of these D2R-mediated signaling events in the actions of antipsychotics remains unclear. We demonstrate here that while different classes of antipsychotics have complex pharmacological profiles at G protein-dependent D2R long isoform (D2(L)R) signaling, they share the common property of antagonizing dopamine-mediated interaction of D2(L)R with beta-arrestin 2. Using two cellular assays based on a bioluminescence resonance energy transfer (BRET) approach, we demonstrate that a series of antipsychotics including haloperidol, clozapine, aripiprazole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone all potently antagonize the beta-arrestin 2 recruitment to D2(L)R induced by quinpirole. However, these antipsychotics have various effects on D2(L)R mediated G(i/o) protein activation ranging from inverse to partial agonists and antagonists with highly variable efficacies and potencies at quinpirole-induced cAMP inhibition. These results suggest that the different classes of clinically effective antipsychotics share a common molecular mechanism involving inhibition of D2(L)R/beta-arrestin 2 mediated signaling. Thus, selective targeting of D2(L)R/beta-arrestin 2 interaction and related signaling pathways may provide new opportunities for antipsychotic development.

Trace amine associated receptor 1 and movement control.

The recently discovered trace amine associated receptors (TAARs) represent attractive potential mediators of certain aspects of movement control. The TAAR that is best characterized, TAAR1, is particularly interesting because it can be activated by a variety of monoaminergic compounds including trace amines, amphetamines and dopamine metabolites. By using an experimental paradigm developed in our laboratory that involves a novel model of acute dopamine deficiency (DDD mice), and mice lacking TAAR1 (TAAR1 knockout mice), we explored the role of TAAR1 in movement control and actions of antiparkinsonian drugs. These investigations may eventually bring novel approaches to the pharmacology of Parkinson's disease.

Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.

The dopamine transporter (DAT) plays a key role in the regulation of dopaminergic signaling wherein it controls both the spatial and temporal actions of dopamine. Here we evaluated the behavioral and neurochemical consequences of increased DAT function by generating DAT transgenic mice (DAT-tg) that overexpress the transporter. These mice were generated by pronuclear injection of a bacterial artificial chromosome containing the mouse DAT locus, yielding an anatomical expression pattern of DAT-tg identical to WT. In DAT-tg mice there is a 3-fold increase in the levels of total and membrane-expressed DAT, but synaptic plasma membrane fractions of DAT-tg mice show only a 30% increase in transporter levels. Functional studies reveal that in the DAT-tg animals there is a 50% increase in the rate of dopamine (DA) uptake resulting in extracellular levels of DA that are decreased by approximately 40%. Behaviorally, DAT-tg animals display similar locomotor stimulation when treated with DAT blockers such as GBR12909, methylphenidate, and cocaine. However, these mice demonstrate markedly increased locomotor responses to amphetamine compared with WT animals. Furthermore, compared with controls, there is a 3-fold greater increase in the amount of DA released by amphetamine in DAT-tg mice that correlates with the 3-fold increase in protein expression. Finally, DAT-tg animals show reduced operant responding for natural reward while displaying preference for amphetamine at much lower doses (0.2 and 0.5 mg/kg) than WT mice (2 mg/kg). These results suggest that overexpression of DAT leads to a marked increase in sensitivity to psychomotor and rewarding properties of amphetamine.

A beta-arrestin 2 signaling complex mediates lithium action on behavior.

Besides their role in desensitization, beta-arrestin 1 and 2 promote the formation of signaling complexes allowing G protein-coupled receptors (GPCR) to signal independently from G proteins. Here we show that lithium, a pharmacological agent used for the management of psychiatric disorders such as bipolar disorder, schizophrenia, and depression, regulates Akt/glycogen synthase kinase 3 (GSK3) signaling and related behaviors in mice by disrupting a signaling complex composed of Akt, beta-arrestin 2, and protein phosphatase 2A. When administered to beta-arrestin 2 knockout mice, lithium fails to affect Akt/GSK3 signaling and induce behavioral changes associated with GSK3 inhibition as it does in normal animals. These results point toward a pharmacological approach to modulating GPCR function that affects the formation of beta-arrestin-mediated signaling complexes.

NF-kappaB pathway: a target for preventing beta-amyloid (Abeta)-induced neuronal damage and Abeta42 production.

Beta-amyloid (Abeta) peptides are key proteins in the pathophysiology of Alzheimer's disease (AD). While Abeta42 aggregates very rapidly to form early diffuse plaques, supplemental Abeta40 deposition is required to form mature neuritic plaques. We here investigated the role of nuclear factor-kappaB (NF-kappaB) pathway in Abeta40-mediated neuronal damage and amyloid pathology. In rat primary neurons and human postmitotic neuronal cells, the Abeta peptide induced a dose-dependent neuronal death, reduced the levels of the anti-apoptotic protein Bcl-XL, enhanced the cytosolic release of cytochrome c, and elicited the intracellular accumulation and secretion of Abeta42 oligomers. Moreover, Abeta40 activated the NF-kappaB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression. As inhibitors of the NF-kappaB pathway, we tested the capability of a double-stranded kappaB decoy oligonucleotide, the anti-inflammatory drug aspirin and the selective IkappaB kinase 2 inhibitor, AS602868, to modify the Abeta40-mediated effects. These treatments, transiently applied before Abeta exposure, completely inhibited p50/p65 nuclear translocation and neuronal damage. The kappaB decoy also inhibited the Abeta-induced release of cytochrome c, restored the levels of Bcl-XL, and prevented intraneuronal accumulation and secretion of Abeta42. These results open up interesting perspectives on the development of novel strategies targeting out NF-kappaB p50/p65 dimers for pharmacological intervention in AD.

Leptin increases axonal growth cone size in developing mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3beta.

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3beta (GSK3beta), an event inactivating this kinase. Leptin-mediated GSK3beta phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr(db/db) mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3beta phosphorylation and mimicked by the GSK3beta inhibitor SB216763. At concentrations preventing GSK3beta phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3beta activity.

Gene expression profile activated by the chemokine CCL5/RANTES in human neuronal cells.

Differentiated human NT2-N neurons were shown to express CCR5 and CXCR4 chemokine receptor mRNA and protein, and to be responsive to the chemokines CCL5 and CXCL12. Using cDNA microarray technology, CCL5 was found to induce a distinct transcriptional program, with reproducible induction of 46 and 9 genes after 2 and 8 hr of treatment, respectively. Conversely, downregulation of 20 and 7 genes was observed after 2 and 8 hr of treatment, respectively. Modulation of a selected panel of CCL5-responsive genes was also confirmed by quantitative RT-PCR and Western blot and compared to gene expression changes induced by CXCL12 treatment. Gene clustering identified distinct functional subsets of CCL5-responsive molecules, and a significant number of expressed sequence tags encoding unknown genes. CCL5-responsive genes comprise a significant number of enzymes, transcription factors, and miscellaneous molecules involved in neuronal survival and differentiation, including neurite outgrowth and synaptogenesis. Our results suggest that CCL5 biological functions might go beyond its recognized chemotactic activity in the central nervous system, in particular with regard to the control of neural plasticity events both during development and in postnatal life.