NMDAR-dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity.

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC), underpinning a powerful mechanism for fine-tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt-dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR-134. Furthermore, NMDAR-dependent down-regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho-regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression to control dendritic spine morphology.

GSK-3ß regulates the synaptic expression of NMDA receptors via phosphorylation of phosphatidylinositol 4 kinase type IIα.

Deregulation of GSK-3ß is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK-3ß becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK-3ß plays a role in the induction of NMDA receptor-dependent long-term depression (LTD) and several substrates for GSK-3ß have been identified in this form of synaptic plasticity, including KLC-2, PSD-95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK-3ß, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK-3ß substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor-mediated synaptic transmission but substantially reduces NMDA receptor-mediated synaptic transmission. Furthermore, the ability of the selective GSK-3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor-mediated currents was occluded in shRNA-PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA-resistant wild-type construct, but not by a mutant construct that cannot be phosphorylated by GSK-3ß. These data suggest that GSK-3ß phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.

Calcium-Permeable AMPA Receptors Mediate the Induction of the Protein Kinase A-Dependent Component of Long-Term Potentiation in the Hippocampus.

Two forms of NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) at hippocampal CA1 synapses can be distinguished based on their sensitivity to inhibitors of protein kinase A (PKA). The PKA-dependent form requires multiple episodes of high-frequency stimulation (HFS) or theta burst stimuli (TBS) with a spacing between episodes in the order of minutes. To investigate the mechanism by which spaced episodes induce the PKA-dependent form of LTP, we have compared, in interleaved experiments, spaced (s) and compressed (c) TBS protocols in the rat CA1 synapses. We find that LTP induced by sTBS, but not that induced by cTBS, involves the insertion of calcium-permeable (CP) AMPARs, as assessed using pharmacological and electrophysiological criteria. Furthermore, a single TBS when paired with rolipram [4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one], to activate PKA, generates an LTP that also involves the insertion of CP-AMPARs. These data demonstrate that the involvement of CP-AMPARs in LTP is critically determined by the timing of the induction trigger and is associated specifically with the PKA-dependent form of LTP. SIGNIFICANCE STATEMENT: Long-term potentiation is a family of synaptic mechanisms that are believed to be important for learning and memory. Two of the most extensively studied forms are triggered by the synaptic activation of NMDA receptors and expressed by changes in AMPA receptor function. They can be distinguished on the basis of their requirement for activation of a protein kinase, PKA. We show that the PKA-dependent form also involves the transient insertion of calcium-permeable AMPA receptors. These results have implications for relating synaptic plasticity to learning and memory and suggest a specific linkage between PKA activation and the rapid synaptic insertion of calcium-permeable AMPA receptors during long-term potentiation.

APP fragment controls both ionotropic and non-ionotropic signaling of NMDA receptors.

NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.

Selective Recruitment of Presynaptic and Postsynaptic Forms of mGluR-LTD.

In area CA1 of the hippocampus, long-term depression (LTD) can be induced by activating group I metabotropic glutamate receptors (mGluRs), with the selective agonist DHPG. There is evidence that mGluR-LTD can be expressed by either a decrease in the probability of neurotransmitter release [P(r)] or by a change in postsynaptic AMPA receptor number. However, what determines the locus of expression is unknown. We investigated the expression mechanisms of mGluR-LTD using either a low (30 µM) or a high (100 µM) concentration of (RS)-DHPG. We found that 30 µM DHPG generated presynaptic LTD that required the co-activation of NMDA receptors, whereas 100 µM DHPG resulted in postsynaptic LTD that was independent of the activation of NMDA receptors. We found that both forms of LTD occur at the same synapses and that these may constitute the population with the lowest basal P(r). Our results reveal an unexpected complexity to mGluR-mediated synaptic plasticity in the hippocampus.

PDZD8 Disruption Causes Cognitive Impairment in Humans, Mice, and Fruit Flies.

BACKGROUND: The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability. METHODS: Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene's function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing. RESULTS: Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum-anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits. CONCLUSIONS: These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.

Neuronal calcium sensors and synaptic plasticity.

Calcium entry plays a major role in the induction of several forms of synaptic plasticity in different areas of the central nervous system. The spatiotemporal aspects of these calcium signals can determine the type of synaptic plasticity induced, e.g. LTP (long-term potentiation) or LTD (long-term depression). A vast amount of research has been conducted to identify the molecular and cellular signalling pathways underlying LTP and LTD, but many components remain to be identified. Calcium sensor proteins are thought to play an essential role in regulating the initial part of synaptic plasticity signalling pathways. However, there is still a significant gap in knowledge, and it is only recently that evidence for the importance of members of the NCS (neuronal calcium sensor) protein family has started to emerge. The present minireview aims to bring together evidence supporting a role for NCS proteins in plasticity, focusing on emerging roles of NCS-1 and hippocalcin.

The Probability of Neurotransmitter Release Governs AMPA Receptor Trafficking via Activity-Dependent Regulation of mGluR1 Surface Expression.

A major mechanism contributing to synaptic plasticity involves alterations in the number of AMPA receptors (AMPARs) expressed at synapses. Hippocampal CA1 synapses, where this process has been most extensively studied, are highly heterogeneous with respect to their probability of neurotransmitter release, P(r). It is unknown whether there is any relationship between the extent of plasticity-related AMPAR trafficking and the initial P(r) of a synapse. To address this question, we induced metabotropic glutamate receptor (mGluR) dependent long-term depression (mGluR-LTD) and assessed AMPAR trafficking and P(r) at individual synapses, using SEP-GluA2 and FM4-64, respectively. We found that either pharmacological or synaptic activation of mGluR1 reduced synaptic SEP-GluA2 in a manner that depends upon P(r); this process involved an activity-dependent reduction in surface mGluR1 that selectively protects high-P(r) synapses from synaptic weakening. Consequently, the extent of postsynaptic plasticity can be pre-tuned by presynaptic activity.

Effects of the antibiotic gentamicin on nicotinic acetylcholine receptors.

Medical treatment with the aminoglycosidic antibiotic gentamicin may produce side effects that include neuromuscular blockage and ototoxicity; which are believed to result from a dysfunction of nicotinic acetylcholine receptors (AChRs). Gentamicin is known to reversibly block ACh-currents generated by the activation of muscle-type alphabetagammadelta-AChR and neuronal alpha9-AChR. We studied the effects of gentamicin on heteromeric alphabetagammadelta-AChR and homomeric alpha7-AChR expressed in Xenopus oocytes. Prolonged treatment with gentamicin, and other antibiotics, differentially altered alphabetagammadelta- and alpha7-AChR responses. Specifically, gentamicin accelerated desensitization and did not reduce ACh-currents in oocytes expressing alphabetagammadelta-AChRs, whereas ACh-currents were reduced and desensitization was unaltered in oocytes expressing alpha7-AChRs. Moreover, acutely applied gentamicin acted as a competitive antagonist on both types of receptors and increased the rate of desensitization in alphabetagammadelta-AChR while reducing the rate of desensitization in alpha7-AChR. This data helps to better understand the action of gentamicin on muscle and nervous tissues, providing mechanistic insights that could eventually lead to improving the medical use of aminoglycosides.

The role of JAK-STAT signaling within the CNS.

JAK-STAT is an efficient and highly regulated system mainly dedicated to the regulation of gene expression. Primarily identified as functioning in hematopoietic cells, its role has been found critical in all cell types, including neurons. This review will focus on JAK-STAT functions in the mature central nervous system. Our recent research suggests the intriguing possibility of a non-nuclear role of STAT3 during synaptic plasticity. Dysregulation of the JAK-STAT pathway in inflammation, cancer and neurodegenerative diseases positions it at the heart of most brain disorders, highlighting the importance to understand how it can influence the fate and functions of brain cells.

The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity.

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation.

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.

Piperazine-2,3-dicarboxylic acid derivatives as dual antagonists of NMDA and GluK1-containing kainate receptors.

Competitive N-methyl-d-aspartate receptor (NMDAR) antagonists bind to the GluN2 subunit, of which there are four types (GluN2A-D). We report that some N(1)-substituted derivatives of cis-piperazine-2,3-dicarboxylic acid display improved relative affinity for GluN2C and GluN2D versus GluN2A and GluN2B. These derivatives also display subtype selectivity among the more distantly related kainate receptor family. Compounds 18i and (-)-4 were the most potent kainate receptor antagonists, and 18i was selective for GluK1 versus GluK2, GluK3 and AMPA receptors. Modeling studies revealed structural features required for activity at GluK1 subunits and suggested that S674 was vital for antagonist activity. Consistent with this hypothesis, replacing the equivalent residue in GluK3 (alanine) with a serine imparts 18i antagonist activity. Antagonists with dual GluN2D and GluK1 antagonist activity may have beneficial effects in various neurological disorders. Consistent with this idea, antagonist 18i (30 mg/kg ip) showed antinociceptive effects in an animal model of mild nerve injury.

The Jak/STAT pathway is involved in synaptic plasticity.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain.

Synergistic interactions between kainate and mGlu receptors regulate bouton Ca signalling and mossy fibre LTP.

It is currently unknown why glutamatergic presynaptic terminals express multiple types of glutamate receptors. We have addressed this question by studying both acute and long-term regulation of mossy fibre function in the hippocampus. We find that inhibition of both mGlu1 and mGlu5 receptors together can block the induction of mossy fibre LTP. Furthermore, mossy fibre LTP can be induced by the pharmacological activation of either mGlu1 or mGlu5 receptors, provided that kainate receptors are also stimulated. Like conventional mossy fibre LTP, chemically-induced mossy fibre LTP (chem-LTPm) depends on Ca²âº release from intracellular stores and the activation of PKA. Similar synergistic interactions between mGlu receptors and kainate receptors were observed at the level of Ca²âº signalling in individual giant mossy fibre boutons. Thus three distinct glutamate receptors interact, in both an AND and OR gate fashion, to regulate both immediate and long-term presynaptic function in the brain.

Synaptic plasticity in the hippocampal slice preparation.

Synaptic plasticity is the process by which the brain alters the strength of its synaptic connections, a fundamental function of the brain that enables individuals to learn from experience. The study of synaptic plasticity often involves the application of standard in vitro electrophysiological techniques to hippocampal slice preparations. This unit discusses many of the special considerations that are applicable for the optimal study of synaptic plasticity in this system. Most of these principles also apply to the study of synaptic plasticity in other brain slice preparations.

Muscarinic receptors induce LTD of NMDAR EPSCs via a mechanism involving hippocalcin, AP2 and PSD-95.

Although muscarinic acetylcholine receptors (mAChRs) and NMDA receptors (NMDARs) are important for synaptic plasticity, learning and memory, the manner in which they interact is poorly understood. We found that stimulation of muscarinic receptors, either by an agonist or by the synaptic release of acetylcholine, led to long-term depression (LTD) of NMDAR-mediated synaptic transmission. This form of LTD involved the release of Ca2+ from IP3-sensitive intracellular stores and was expressed via the internalization of NMDARs. Our results suggest that the molecular mechanism involves a dynamic interaction between the neuronal calcium sensor protein hippocalcin, the clathrin adaptor molecule AP2, the postsynaptic density enriched protein PSD-95 and NMDARs. We propose that hippocalcin binds to the SH3 region of PSD-95 under basal conditions, but it translocates to the plasma membrane on sensing Ca2+; in doing so, it causes PSD-95 to dissociate from NMDARs, permitting AP2 to bind and initiate their dynamin-dependent endocytosis.

Amyloid beta(1-42) peptide alters the gating of human and mouse alpha-bungarotoxin-sensitive nicotinic receptors.

The beta-amyloid(1-42) peptide (Abeta(1-42)), a major constituent of the Alzheimer's disease amyloid plaque, specifically binds to the neuronal alpha-bungarotoxin (alpha-BuTx)-sensitive alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). Accordingly, Abeta1-42 interferes with the function of alpha7 nAChRs in chick and rodent neurons. To gain insights into the human disease, we studied the action of Abeta(1-42) on human alpha7 nAChRs expressed in Xenopus oocytes. In voltage-clamped oocytes expressing the wild-type receptor, Abeta(1-42) blocked ACh-evoked currents. The block was non-competitive, required over 100 s to develop and was partially reversible. In oocytes expressing the mutant L248T receptor, Abeta(1-42) activated methyllycaconitine-sensitive currents in a dose-dependent manner. Peptide-evoked unitary events, recorded in outside-out patches, showed single-channel conductances and open duration comparable to ACh-evoked events. Abeta(1-42) had no effect on the currents evoked by glutamate, GABA or glycine in oocytes expressing human or mouse receptors for these transmitters. Muscle nAChRs are also alpha-BuTx-sensitive and we therefore investigated whether they respond to Abeta(1-42). In human kidney BOSC 23 cells expressing the fetal or adult mouse muscle nAChRs, Abeta(1-42) blocked ACh-evoked whole-cell currents, accelerating their decay. Outside-out single-channel recordings showed that the block was due to a reduced channel open probability and enhanced block upon ACh application. We also report that the inverse peptide Abeta(42-1), but not Abeta(40-1), partially mimicked the effects of the physiological Abeta(1-42) peptide. Possible implications for degenerative neuronal and muscular diseases are discussed.