Autism-related deficits via dysregulated eIF4E-dependent translational control.

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Postnatal deamidation of 4E-BP2 in brain enhances its association with raptor and alters kinetics of excitatory synaptic transmission.

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Selective regulation of GluA subunit synthesis and AMPA receptor-mediated synaptic function and plasticity by the translation repressor 4E-BP2 in hippocampal pyramidal cells.

The eukaryotic initiation factor 4E-binding protein-2 (4E-BP2) is a repressor of cap-dependent mRNA translation and a major downstream effector of the mammalian target of rapamycin (mTOR) implicated in hippocampal long-term synaptic plasticity and memory. Yet, synaptic mechanisms regulated by 4E-BP2 translational repression remain unknown. Combining knock-out mice, whole-cell recordings, spine analysis, and translation profiling, we found that 4E-BP2 deletion selectively upregulated synthesis of glutamate receptor subunits GluA1 and GluA2, facilitating AMPA receptor (AMPAR)-mediated synaptic transmission and affecting translation-dependent chemically induced late long-term potentiation (cL-LTP). In 4E-BP2 knock-out (4E-BP2(-/-)) mice, evoked and miniature EPSCs were increased, an effect mimicked by short-hairpin RNA knockdown of 4E-BP2 in wild-type mice, indicating that 4E-BP2 level regulates basal transmission at mature hippocampal AMPAR-containing synapses. Remarkably, in 4E-BP2(-/-) mice, the AMPA to NMDA receptor (NMDAR) EPSC ratio was increased, without affecting NMDAR-mediated EPSCs. The enhanced AMPAR function concurred with increased spine density and decreased length resulting from greater proportion of regular spines and less filopodia in 4E-BP2(-/-) mice. Polysome profiling revealed that translation of GluA1 and GluA2 subunits, but not GluN1 or GluN2A/B, was selectively increased in 4E-BP2(-/-) hippocampi, consistent with unaltered I-V relation of EPSCs mediated by GluA1/GluA2 heteromers. Finally, translation-dependent cL-LTP of unitary EPSCs was also affected in 4E-BP2(-/-) mice, lowering induction threshold and removing mTOR signaling requirement while impairing induction by normal stimulation. Thus, translational control through 4E-BP2 represents a unique mechanism for selective regulation of AMPAR synthesis, synaptic function, and long-term plasticity, important for hippocampal-dependent memory processes.

CREB-dependent transcriptional control and quantal changes in persistent long-term potentiation in hippocampal interneurons.

Mounting evidence indicates an important role of long-term synaptic plasticity in hippocampal inhibitory interneurons in learning and memory. The cellular and molecular mechanisms that underlie such persistent changes in synaptic function in interneurons remain, however, largely undetermined. A transcription- and translation-dependent form of long-term potentiation was uncovered at excitatory synapses onto hippocampal interneurons in oriens-alveus (OA-INs) which is induced by activation of type 1 metabotropic glutamate receptors (cL-LTP(mGluR1)). Here, we use (1) a combination of pharmacological siRNA knock-down and overexpression approaches to reveal the molecular mechanisms of transcriptional control via cAMP response element-binding protein (CREB) during induction, and (2) quantal analysis to identify synaptic changes during maintenance of cL-LTP(mGluR1) in rat hippocampus. Induction stimulated CREB phosphorylation in OA-INs via extracellular signal-regulated protein kinase (ERK) signaling. Also, CREB knockdown impaired cL-LTP(mGluR1), whereas CREB overexpression facilitated the induction, demonstrating a necessary and permissive role of CREB via ERK signaling in transcriptional control in cL-LTP(mGluR1). Quantal analysis of synaptic responses during cL-LTP(mGluR1) maintenance revealed an increased number of quanta released, corresponding to enhanced transmitter release and a larger quantal size, indicating enhanced responsiveness to individual quanta. Fluctuation analysis of synaptic currents uncovered an increase in conductance and number of functional postsynaptic receptors contributing to single quanta. Our findings indicate that CREB-dependent transcription is a necessary permissive switch for eliciting persistent presynaptic and postsynaptic quantal changes at excitatory synapses in inhibitory local circuits, uncovering cell type-specific coupling of induction and expression mechanisms during persistent synaptic plasticity which may contribute to hippocampal long-term memory processes.

Activity-dependent compartmentalized regulation of dendritic Ca2+ signaling in hippocampal interneurons.

Activity-dependent regulation of synaptic inputs in neurons is controlled by highly compartmentalized and dynamic dendritic calcium signaling. Among multiple Ca(2+) mechanisms operating in neuronal dendrites, voltage-sensitive Ca(2+) channels (VSCCs) represent a major source of Ca(2+) influx; however, their use-dependent implication, regulation, and function in different types of central neurons remain widely unknown. Using two-photon microscopy to probe Ca(2+) signaling in dendrites of hippocampal oriens/alveus interneurons, we found that intense synaptic activity or local activation of mGluR5 induced long-lasting potentiation of action potential evoked Ca(2+) transients. This potentiation of dendritic Ca(2+) signaling required mGluR5-induced intracellular Ca(2+) release and PKC activation and was expressed as a selective compartmentalized potentiation of L-type VSCCs. Thus, in addition to mGluR1a-dependent synaptic plasticity, hippocampal interneurons in the feedback inhibitory circuit demonstrate a novel form of mGluR5-induced dendritic plasticity. Given an implication of L-type VSCCs in the induction of Hebbian LTP at interneuron excitatory synapses, their activity-dependent regulation may represent a powerful mechanism for regulating synaptic plasticity.

Persistent transcription- and translation-dependent long-term potentiation induced by mGluR1 in hippocampal interneurons.

Hippocampal interneurons synchronize the activity of large neuronal ensembles during memory consolidation. Although the latter process is manifested as increases in synaptic efficacy which require new protein synthesis in pyramidal neurons, it is unknown whether such enduring plasticity occurs in interneurons. Here, we uncover a long-term potentiation (LTP) of transmission at individual interneuron excitatory synapses which persists for at least 24 h, after repetitive activation of type-1 metabotropic glutamate receptors [mGluR1-mediated chemical late LTP (cL-LTP(mGluR1))]. cL-LTP(mGluR1) involves presynaptic and postsynaptic expression mechanisms and requires both transcription and translation via phosphoinositide 3-kinase/mammalian target of rapamycin and MAP kinase kinase-extracellular signal-regulated protein kinase signaling pathways. Moreover, cL-LTP(mGluR1) involves translational control at the level of initiation as it is prevented by hippuristanol, an inhibitor of eIF4A, and facilitated in mice lacking the cap-dependent translational repressor, 4E-BP. Our results reveal novel mechanisms of long-term synaptic plasticity that are transcription and translation-dependent in inhibitory interneurons, indicating that persistent synaptic modifications in interneuron circuits may contribute to hippocampal-dependent cognitive processes.

Fluctuation analysis of tetanic rundown (short-term depression) at a corticothalamic synapse.

Hypothetical scenarios for "tetanic rundown" ("short-term depression") of synaptic signals evoked by stimulus trains differ in evolution of quantal amplitude (Q) and covariances between signals. With corticothalamic excitatory postsynaptic currents (EPSCs) evoked by 2.5- to 20-Hz trains, we found Q (estimated using various corrections of variance/mean ratios) to be unchanged during rundown and close to the size of stimulus-evoked "miniatures". Except for covariances, results were compatible with a depletion model, according to which incomplete "refill" after probabilistic quantal release entails release-site "emptying". For five neurons with 20 train repetitions at each frequency, there was little between-neuron variation of rundown; pool-refill rate increased with stimulus frequency and evolved during rundown. Covariances did not fit the depletion model or theoretical alternatives, being excessively negative for adjacent EPSCs early in trains, absent at equilibrium, and anomalously positive for some nonadjacent EPSCs. The anomalous covariances were unaltered during pharmacological blockade of receptor desensitization and saturation. These findings suggest that pool-refill rate and release probability at each release site are continually modulated by antecedent outputs in its neighborhood, possibly via feedback mechanisms. In all data sets, sampling errors for between-train variances were much less than theoretical, warranting reconsideration of the probabilistic nature of quantal transmitter release.

Spermine modulates neuronal excitability and NMDA receptors in juvenile gerbil auditory thalamus.

Medial geniculate body (MGB) neurons process synaptic inputs from auditory cortex. Corticothalamic stimulation evokes glutamatergic excitatory postsynaptic potentials (EPSPs) that vary markedly in amplitude and duration during development. The EPSP decay phase is prolonged during second postnatal week but then shortens, significantly, until adulthood. The EPSP prolongation depends on spermine interactions with a polyamine-sensitive site on receptors for N-methyl-D-aspartate (NMDA). We examined effects of spermine application on EPSPs, firing modes, and membrane properties in gerbil MGB neurons during the P14 period of highest polyamine sensitivity. Spermine slowed EPSP decay and promoted firing on EPSPs, without changing passive membrane properties. Spermine increased membrane rectification on depolarization, which is mediated by tetrodotoxin (TTX)-sensitive, persistent Na(+) conductance. As a result, spermine lowered threshold and increased tonic firing evoked with current injection by up to approximately 150%. These effects were concentration-dependent (ED(50)=100 microM), reversible, and eliminated by NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV). In contrast, spermine increased dV/dt of the low threshold Ca(2+) spike (LTS) and burst firing, evoked from hyperpolarized potentials. LTS enhancement was greater at -55 mV than at hyperpolarized potentials and did not result from persistent Na(+) conductance or glutamate receptor mechanisms. In summary, spermine increased excitability by modulating NMDA receptors in juvenile gerbil neurons.