Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2.

Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. Here we report a unique feature of hippocampal slices from GCN2(-/-) mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2(-/-) slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2(-/-) mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2(-/-) mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway.

Kv4.3-mediated A-type K+ currents underlie rhythmic activity in hippocampal interneurons.

Hippocampal-dependent learning and memory processes are associated with theta frequency rhythmic activity. Interneuron and pyramidal cell network interactions underlie this activity, but contributions of interneuron voltage-gated membrane conductances remain unclear. We show that interneurons at the CA1 lacunosum-moleculare (LM) and radiatum (RAD) junction (LM/RAD) display voltage-dependent subthreshold membrane potential oscillations (MPOs) generated by voltage-gated tetrodotoxin-sensitive Na+ and 4-aminopyridine (4-AP)-sensitive K+ currents. They also exhibit prominent 4-AP-sensitive A-type K+ currents, with gating properties showing activation at subthreshold membrane potentials. We found that LM/RAD cells are part of specific interneuron subpopulations expressing the K+ channel subunit Kv4.3 and their transfection with Kv4.3 small interfering RNA selectively impaired A-type K+ currents and MPOs. Thus, our findings reveal a novel function of Kv4.3-mediated A-type K+ currents in the generation of intrinsic MPOs in specific subpopulations of interneurons that may participate in hippocampal theta-related rhythmic activity.

Forskolin induction of late-LTP and up-regulation of 5' TOP mRNAs translation via mTOR, ERK, and PI3K in hippocampal pyramidal cells.

The late phase of long-term potentiation (LTP) requires activation of the mammalian target of rapamycin (mTOR) pathway and synthesis of new proteins. mTOR regulates protein synthesis via phosphorylation of 4E-binding proteins (4E-BPs) and S6K, and via selective up-regulation of 5' terminal oligopyrimidine (5' TOP) mRNAs that encode components of the translational machinery. In this study, we explored the regulation of 5' TOP mRNAs during late-LTP (L-LTP). Synaptic plasticity was studied at Schaffer collateral--CA1 pyramidal cell synapses in rat organotypic hippocampal slices. Forskolin, an adenylate cyclase activator, induced L-LTP in organotypic slices that was mTOR-dependent. To determine if 5' TOP mRNAs are specifically up-regulated during L-LTP, we generated a 5' TOP-myr-dYFP reporter to selectively monitor 5' TOP translation. Confocal imaging experiments in cultured slices revealed an increase in somatic and dendritic fluorescence after forskolin treatment. This up-regulation was dependent on an intact TOP sequence and was mTOR, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-dependent. Our findings indicate that forskolin induces L-LTP in hippocampal neurons and up-regulates 5' TOP mRNAs translation via mTOR, suggesting that up-regulation of the translational machinery is a candidate mechanism for the stabilization of LTP.

The V2 vasopressin receptor stimulates ERK1/2 activity independently of heterotrimeric G protein signalling.

The V2 vasopressin receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein beta arrestin. Here we report that this activating pathway is independent of G alpha s, G alpha i, G alpha q or G betagamma and that the V2R-mediated activation of G alpha s inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the beta arrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and beta arrestin prevents signal propagation to the nucleus. Beta arrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the beta arrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that beta arrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.

Targeting soluble Abeta peptide with Tramiprosate for the treatment of brain amyloidosis.

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in Alzheimer's disease (AD). Neurotoxicity results from the conformational transition of Abeta from random-coil to beta-sheet and its oligomerization. Among a series of ionic compounds able to interact with soluble Abeta, Tramiprosate (3-amino-1-propanesulfonic acid; 3APS; Alzhemedtrade mark) was found to maintain Abeta in a non-fibrillar form, to decrease Abeta(42)-induced cell death in neuronal cell cultures, and to inhibit amyloid deposition. Tramiprosate crosses the murine blood-brain barrier (BBB) to exert its activity. Treatment of TgCRND8 mice with Tramiprosate resulted in significant reduction (approximately 30%) in the brain amyloid plaque load and a significant decrease in the cerebral levels of soluble and insoluble Abeta(40) and Abeta(42) (approximately 20-30%). A dose-dependent reduction (up to 60%) of plasma Abeta levels was also observed, suggesting that Tramiprosate influences the central pool of Abeta, changing either its efflux or its metabolism in the brain. We propose that Tramiprosate, which targets soluble Abeta, represents a new and promising therapeutic class of drugs for the treatment of AD.

mGluR1/5 subtype-specific calcium signalling and induction of long-term potentiation in rat hippocampal oriens/alveus interneurones.

Hippocampal inhibitory interneurones demonstrate pathway- and synapse-specific rules of transmission and plasticity, which are key determinants of their role in controlling pyramidal cell excitability. Mechanisms underlying long-term changes at interneurone excitatory synapses, despite their importance, remain largely unknown. We use two-photon calcium imaging and whole-cell recordings to determine the Ca2+ signalling mechanisms linked specifically to group I metabotropic glutamate receptors (mGluR1alpha and mGluR5) and their role in hebbian long-term potentiation (LTP) in oriens/alveus (O/A) interneurones. We demonstrate that mGluR1alpha activation elicits dendritic Ca2+ signals resulting from Ca2+ influx via transient receptor potential (TRP) channels and Ca2+ release from intracellular stores. By contrast, mGluR5 activation produces dendritic Ca2+ transients mediated exclusively by intracellular Ca2+ release. Using Western blot analysis and immunocytochemistry, we show mGluR1alpha-specific extracellular signal-regulated kinase (ERK1/2) activation via Src in CA1 hippocampus and, in particular, in O/A interneurones. Moreover, we find that mGluR1alpha/TRP Ca2+ signals in interneurone dendrites are dependent on activation of the Src/ERK cascade. Finally, this mGluR1alpha-specific Ca2+ signalling controls LTP at interneurone synapses since blocking either TRP channels or Src/ERK and intracellular Ca2+ release prevents LTP induction. Thus, our findings uncover a novel molecular mechanism of interneurone-specific Ca2+ signalling, critical in regulating synaptic excitability in hippocampal networks.

Reciprocal regulation of agonist and inverse agonist signaling efficacy upon short-term treatment of the human delta-opioid receptor with an inverse agonist.

Rapid regulation of receptor signaling by agonist ligands is widely accepted, whereas short-term adaptation to inverse agonists has been little documented. In the present study, guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding and cAMP accumulation assays were used to assess the consequences of 30-min exposure to the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) (1 microM) on delta-opioid receptor signaling efficacy. ICI174864 pretreatment increased maximal effect (E(max)) for the partial agonist Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP) at the two levels of the signaling cascade, whereas E(max) values for more efficacious agonists like (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) and bremazocine were increased in [(35)S]GTPgammaS binding but not in cAMP accumulation assays. Pre-exposure to ICI174864 also induced a shift to the left in dose-response curves for bremazocine and TIPP. On the other hand, E(max) for the inverse agonist H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH was reduced in both assays, but no changes in potency were observed. For the weaker inverse agonist naloxone, E(max) in [(35)S]GTPgammaS binding was drastically modified because the drug turned from inverse agonist to agonist after ICI174864 pretreatment. Likewise, ICI174864 turned from inverse agonist to agonist when tested in cAMP accumulation assays. In both cases, inversion of efficacy was concomitant with marked increase in potency for agonist effects. Together with functional changes, short-term treatment with ICI174864 reduced basal receptor phosphorylation and increased immunoreactivity for Galpha(i3) in membrane preparations. Functional consequences of ICI174864 pretreatment were simulated in the cubic ternary complex model by increasing receptor/G protein coupling or G protein amount available for interaction with the receptor. Taken together, these data show that inverse agonists may induce rapid regulation in receptor signaling efficacy.

Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.

It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that beta2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 thus behaving as dual efficacy ligands. ERK1/2 activation by dual efficacy ligands was not affected by ADP-ribosylation of Galphai and could be observed in S49-cyc- cells lacking Galphas indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1/2 activation in a Gs/i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of beta-arrestin and was abolished in mouse embryonic fibroblasts lacking beta-arrestin 1 and 2. The role of beta-arrestin was further confirmed by showing that transfection of beta-arrestin 2 in these knockout cells restored ICI118551 promoted ERK1/2 activation. ICI118551 and propranolol also promoted beta-arrestin recruitment to the receptor. Taken together, these observations suggest that beta-arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on beta-arrestin for their positive signaling activity. This phenomenon is not unique to beta2-adrenergic ligands because SR121463B, an inverse agonist on the V2 vasopressin receptor-stimulated adenylyl cyclase, recruited beta-arrestin and stimulated ERK1/2. These results point to a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners.