mGluR-dependent LTD Research Articles

Role for Astrocytes in mGluR-Dependent LTD in the Neocortex and Hippocampus.

Astroglia are an active element of brain plasticity, capable to release small molecule gliotransmitters by various mechanisms and regulate synaptic strength. While importance of glia-neuron communications for long-term potentiation has been rather widely reported, research into role for astrocytes in long-depression (LTD) is just gaining momentum. Here, we explored the role for astrocytes in the prominent form of synaptic plasticity-mGluR-dependent LTD. We found out the substantial contribution of the Group I receptors, especially mGluR1 subtype, into Ca2+-signaling in hippocampal and neocortical astrocytes, which can be activated during synaptic stimulation used for LTD induction. Our data demonstrate that mGluR receptors can activate SNARE-dependent release of ATP from astrocytes which in turn can directly activate postsynaptic P2X receptors in the hippocampal and neocortical neurons. The latter mechanism has recently been shown to cause the synaptic depression via triggering the internalisation of AMPA receptors. Using mouse model of impaired glial exocytosis (dnSNARE mice), we demonstrated that mGluR-activated release of ATP from astrocytes is essential for regulation of mGluR-dependent LTD in CA3-CA1 and layer 2/3 synapses. Our data also suggest that astrocyte-related pathway relies mainly on mGluR1 receptors and act synergistically with neuronal mechanisms dependent mainly on mGluR5.

Autism-associated Shank3 mutations alter mGluR expression and mGluR-dependent but not NMDA receptor-dependent long-term depression.

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor-mediated synaptic responses in neurons, which is thought to underlie ASD-related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD-associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform-specific deficits induced by ASD-associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR-dependent synaptic plasticity (R,S-3,5-dihydroxyphenylglycine [DHPG]-induced long-term depression [LTD]) as well as N-methyl-D-aspartate receptor (NMDAR)-dependent LTD. ASD-associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR-dependent LTD without altering NMDAR-dependent LTD. Our data show that the specific perturbation in mGluR-dependent synaptic plasticity occurs in neurons expressing ASD-associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.

Association of mGluR-Dependent LTD of Excitatory Synapses with Endocannabinoid-Dependent LTD of Inhibitory Synapses Leads to EPSP to Spike Potentiation in CA1 Pyramidal Neurons.

The input-output relationships in neural circuits are determined not only by synaptic efficacy but also by neuronal excitability. Activity-dependent alterations of synaptic efficacy have been extensively investigated, but relatively less is known about how the neuronal output is modulated when synaptic efficacy changes are associated with neuronal excitability changes. In this study, we demonstrate that paired pulses of low-frequency stimulation (PP-LFS) induced metabotropic glutamate receptor (mGluR)-dependent LTD at Schaffer collateral (SC)-CA1 synapses in Sprague Dawley rats (both sexes), and this LTD was associated with EPSP to spike (E-S) potentiation, leading to the increase in action potential (AP) outputs. Threshold voltage (Vth) for APs evoked by synaptic stimulation and that by somatic current injection were hyperpolarized significantly after PP-LFS. Blockers of GABA receptors mimicked and occluded PP-LFS effects on E-S potentiation and Vth hyperpolarization, suggesting that suppression of GABAergic mechanisms is involved in E-S potentiation after PP-LFS. Indeed, IPSCs and tonic inhibitory currents were reduced after PP-LFS. The IPSC reduction was accompanied by increased paired-pulse ratio, and abolished by AM251, a blocker for Type 1 cannabinoid receptors, suggesting that PP-LFS suppresses presynaptic GABA release by mGluR-dependent endocannabinoids signaling. By contrast, a Group 1 mGluR agonist, 3, 5-dihydroxyphenylglycine, induced LTD at SC-CA1 synapses but failed to induce significant IPSC reduction and AP output increase. We propose that mGluR signaling that induces LTD coexpression at excitatory and inhibitory synapses regulates an excitation-inhibition balance to increase neuronal output in CA1 neurons.SIGNIFICANCE STATEMENT Long-lasting forms of synaptic plasticity are usually associated with excitability changes, the ability to fire action potentials. However, excitability changes have been regarded to play subsidiary roles to synaptic plasticity in modifying neuronal output. We demonstrate that, when metabotropic glutamate receptor-dependent LTD is induced by paired pulses of low-frequency stimulation, the action potential output in response to a given input paradoxically increases, indicating that increased excitability is more powerful than synaptic depression. This increase is mediated by the suppression of a presynaptic GABA release via metabotropic glutamate receptor-dependent endocannabinoid signaling. Our study shows that neuronal output changes do not always follow the direction of synaptic plasticity at excitatory synapses, highlighting the importance of regulating inhibitory tone via endocannabinoid signaling.

S66. Therapeutic effects of an AMPA receptor antagonist on fragile X syndrome and cerebellar coordination disorders/involuntary movements

Introduction To examine the effectiveness of an AMPA receptor antagonist (perampanel: PER) for treating cerebellar coordination disorders/involuntary movements with the genetic loss of FMRP protein, which regulates AMPA receptor expression in Purkinje cells. Fragile X syndrome causes mental retardation and cerebellar motor disorders through CGG repeat expansion in the 5′-untranslated region of the FMR1 gene (chX 27.3). The FMRP protein encoded by the FMR1 gene is responsible for translational repression of the signaling of a metabotropic glutamate receptor, mGluR. In FMRP-protein knockout animals, abnormal phenotypes are caused by mGluR-dependent LTD, resulting in morphological abnormalities of the hippocampal neuronal dendrites, which were improved by mGluR antagonist administration ( Bear et al., 2004 ). In Japan, PER is covered by insurance as a combination therapy for epilepsy. Methods The present study was approved by the ethics committee of our hospital, with consent obtained from patients and their families. Two patients with fragile X syndrome (16-year-old male with FMR1-full mutation and 68-year-old female with FMRI-premutation) were included. One case of Usher syndrome and two cases of LCCA were included as disease controls. The administration of PER was started at 2 mg/day, and maintained at 4 mg/day in week 2 and later to examine effectiveness for 12 weeks. Motor symptoms and involuntary movements were evaluated using an international standard score, ICARS, and MDS-UPDRS part III-3, respectively 15–18. AMPA-R protein (130 m/kDa) expressed on the peripheral blood lymphocytes before drug administration was examined by Western blotting (MyBioSource, AMPA receptor (GluR4) rabbit polyclonal antibody). Results Motor and gait functions were markedly improved in the cases of fragile X syndrome (ICARS-motor; −9, gait; −3). Involuntary movement scores were improved in all cases (−12). AMPA-R protein on lymphocytes was detected in the cases of fragile X syndrome, but was not in the disease controls. Conclusion PER is effective for treating cerebellar involuntary movements in fragile X syndrome, and may have LTP/disinhibiting effects through AMPA-R blocking in Purkinje cells. Changes in lymphocyte AMPA-R expression level suggested that AMPA-R expression pattern might be changed in fragile X syndrome.

MicroRNA-137 Controls AMPA-Receptor-Mediated Transmission and mGluR-Dependent LTD.

Mutations affecting the levels of microRNA miR-137 are associated with intellectual disability and schizophrenia. However, the pathophysiological role of miR-137 remains poorly understood. Here, we describe a highly conserved miR-137-binding site within the mRNA encoding the GluA1 subunit of AMPA-type glutamate receptors (AMPARs) and confirm that GluA1 is a direct target of miR-137. Postsynaptic downregulation of miR-137 at the CA3-CA1 hippocampal synapse selectively enhances AMPAR-mediated synaptic transmission and converts silent synapses to active synapses. Conversely, miR-137 overexpression selectively reduces AMPAR-mediated synaptic transmission and silences active synapses. In addition, we find that miR-137 is transiently upregulated in response to metabotropic glutamate receptor 5 (mGluR5), but not mGluR1 activation. Consequently, acute interference with miR-137 function impedes mGluR-LTD expression. Our findings suggest that miR-137 is a key factor in the control of synaptic efficacy and mGluR-dependent synaptic plasticity, supporting the notion that glutamatergic dysfunction contributes to the pathogenesis of miR-137-linked cognitive impairments.

A Cdh1-APC/FMRP Ubiquitin Signaling Link Drives mGluR-Dependent Synaptic Plasticity in the Mammalian Brain

Deregulation of synaptic plasticity may contribute to the pathogenesis of developmental cognitive disorders. In particular, exaggerated mGluR-dependent LTD is featured in fragile X syndrome, but the mechanisms that regulate mGluR-LTD remain incompletely understood. We report that conditional knockout of Cdh1, the key regulatory subunit of the ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC), profoundly impairs mGluR-LTD in the hippocampus. Mechanistically, we find that Cdh1-APC operates in the cytoplasm to drive mGluR-LTD. We also identify the fragile X syndrome protein FMRP as a substrate of Cdh1-APC. Endogenous Cdh1-APC forms a complex with endogenous FMRP, and knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. Knockout of FMRP suppresses, and expression of an FMRP mutant protein that fails to interact with Cdh1 phenocopies, the Cdh1 knockout phenotype of impaired mGluR-LTD. These findings define Cdh1-APC and FMRP as components of a novel ubiquitin signaling pathway that regulates mGluR-LTD in the brain.

Coordination between Translation and Degradation Regulates Inducibility of mGluR-LTD.

SUMMARYDendritic protein homeostasis is crucial for most forms of long-term synaptic plasticity, and its dysregulation is linked to a wide range of brain disorders. Current models of metabotropic glutamate receptor mediated long-term depression (mGluR-LTD) suggest that rapid, local synthesis of key proteins is necessary for the induction and expression of LTD. Here, we find that mGluR-LTD can be induced in the absence of translation if the proteasome is concurrently inhibited. We report that enhanced pro-teasomal degradation during the expression of mGluR-LTD depletes dendritic proteins and inhibits subsequent inductions of LTD. Moreover, proteasome inhibition can rescue mGluR-LTD in mice null for the RNA binding protein Sam68, which we show here lack mGluR-dependent translation and LTD. Our study provides mechanistic insights for how changes in dendritic protein abundance regulate mGluR-LTD induction. We propose that Sam68-mediated translation helps to counterbalance degradation, ensuring that protein levels briefly remain above a permissive threshold during LTD induction.

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.

F-box Only Protein 2 (Fbxo2) Regulates Amyloid Precursor Protein Levels and Processing

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

A form of synaptically induced metabotropic glutamate receptor-dependent long-term depression that does not require postsynaptic calcium

The calcium control hypothesis posits that postsynaptic calcium increases are required to trigger synaptic plasticity, with large increases inducing LTP and small increases inducing LTD. In CA1 of the hippocampus, however, LTD induced by chemical activation of metabotropic glutamate receptors (agonist-LTD) is independent of increases in postsynaptic calcium. Here we tested whether LTD induced by pairing of presynaptic stimulation with postsynaptic depolarization (synaptic-LTD) is similarly calcium-independent. This protocol induced an NMDA-dependent LTP when paired at 0mV, which was converted to mGluR-dependent LTD when paired at −20mV. The LTD was not blocked by calcium chelation, blockers of L- or T-type voltage-dependent calcium channels, or hyperpolarization to −70mV. We conclude that synaptically induced mGluR-dependent LTD, like agonist induced mGluR LTD, does not require calcium influx for its induction.

Rapid Synthesis of the X-Linked Mental Retardation Protein OPHN1 Mediates mGluR-Dependent LTD through Interaction with the Endocytic Machinery

Activation of group I metabotropic glutamate receptors leads to long-term depression (mGluR-LTD). Alterations in this form of plasticity have been linked to drug addiction and cognitive disorders. A key characteristic of mGluR-LTD is its dependence on rapid protein synthesis; however, the identities of the proteins mediating LTD remain elusive. Here, we identify the X-linked mental retardation protein OPHN1 as a molecule essential for mGluR-LTD in the hippocampus. mGluR-LTD induction elicits rapid dendritic OPHN1 synthesis, which is dependent on mGluR1 activation and independent of fragile X mental retardation protein (FMRP). This response is essential for mGluR-LTD, as acute blockade of OPHN1 synthesis impedes LTD. mGluR-induced OPHN1 mediates LTD and associated persistent decreases in surface AMPARs via interactions with endophilin A2/3. Importantly, this role of OPHN1 is separable from its effects on basal synaptic strength, which require OPHN1's Rho-GAP activity and interaction with Homer1b/c. Thus, our data establish a role for rapid OPHN1 synthesis in mGluR-LTD.

Reverse engineering of metabotropic glutamate receptor-dependent long-term depression in the hippocampus

This study focused on metabotropic glutamate receptor-dependent long-term depression (mGluR–LTD) in the hippocampus. This form of LTD is suggested to play a key role in learning, memory and the plasticity of behaviour. Recent advances have started to uncover the underlying mechanisms of mGluR-LTD [1]. However, it is not completely clear how these mechanisms are linked and it is believed that several crucial mechanisms still remain to be revealed. The two main objectives of this study were (i) to quantify the dynamics of mGluR-LTD responses by dynamic data-based models and (ii) to identify underlying dominant processes of mGluR-LTD by applying mathematical system identification methods. In recent years, more and more researchers advocate the use of a top-down modelling approach (reverse engineering) for improving the knowledge of biological systems [2,3]. The drug dihydroxyphenylglycine (DHPG) was used to induce mGluR-LTD in rat brain slices (table ​(table1).1). The drug was applied for different durations (5min, 15min, 2 hours) and in different concentrations (15mM, 30mM). In addition, also different sampling intervals (5min, 30s, 90s) were used. Table 1 Overview of the experiments For the modelling, discrete-time Transfer Functions (TF) models were used. The models described the relation between the DHPG application (input) and the long-term depression responses (output). All models were very accurate (all RT2-values higher than 0,94) and reliably estimated. For a 2 hours application of 30 µM DHPG sampled with a frequency of 1/30s, the time-constant of the mGluR-LTD response was 92s. Thus, the models for high sampling rate indicated that a sampling interval of 30s would be ideal to minimize information loss of the dynamics of mGluR-LTD responses. Interestingly, it was suggested that there are three dominant sub-processes underlying mGluR-LTD: one fast sub-process, one slow sub-process and an immediate sub-process. This study suggests that the dynamic data-based modelling approach can be a valuable tool for reverse engineering of mGluR-dependent LTD responses.

Pharmacological inactivation of the small GTPase Rac1 impairs long-term plasticity in the mouse hippocampus

Neuronal development involves several discrete morphological steps requiring migration of newborn neurons to characteristic locations, extension of axons and dendrites into proper target regions, and formation of synapses with appropriate partners. Small GTPases such as Rac1, are believed to be critical regulators of these processes. We have previously reported that Rac1 is highly expressed in mouse hippocampus, where NMDA receptor activation causes Rac1 to translocate to the membrane in a manner similar to that observed in other non-neuronal cells. Additionally Rac1 has been seen to play a role in activation of signal transduction pathways associated with hippocampal learning and memory. Because of the established role of LTP and LTD in learning and memory processes, in this study we investigate whether Rac1 plays also an active and critical role in these types of long-term synaptic plasticity. We found that activation of Rac1 is associated with long-term plasticity, both LTP and LTD. Rac1 appears to have a transient role during the induction of NMDA receptor-dependent LTP, but does not have an effect on LTP maintenance and expression. Similar results were found for NMDA receptor-dependent induction of LTD, while mGluR-dependent LTD was shown to be significantly altered but not abolished. The results of these experiments provide essential knowledge regarding the signaling mechanisms that underlie synaptic plasticity, as well as learning and memory processes, which in turn offers insights into the basis of diseases involving memory impairment, such as Fragile X syndrome, Alzheimer’s disease, William’s syndrome, Angelman syndrome (AS), and schizophrenia.

Enhanced Polyubiquitination of Shank3 and NMDA Receptor in a Mouse Model of Autism

We have created a mouse genetic model that mimicsa human mutation of Shank3 that deletes the C terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the wild-type (WT) gene product and results in >90% reduction of Shank3 at synapses. This "gain-of-function" phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of postsynaptic density proteins, spine morphology, and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder.

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Long-term potentiation persists in an occult state following mGluR-dependent depotentiation

Depotentiation, the reversal of long-term potentiation (LTP), can be induced by activation of metabotropic glutamate receptors (mGluRs) or NMDA receptors (NMDARs). Although NMDAR-dependent depotentiation is due to a protein phosphatase-dependent erasure of LTP, the notion that mGluR-dependent depotentiation also involves LTP erasure is controversial. To address this issue we used electrophysiological and biochemical approaches to investigate mGluR-dependent depotentiation in hippocampal slices. Activating group I mGluRs with ( R, S)-3,5-dihydroxyphenylglycine (DHPG) induced robust depotentiation in both the CA1 and CA3 regions of hippocampal slices. Western immunoblotting of samples prepared from DHPG-treated slices revealed, however, that activation of group I mGluRs causes a transient increase in phosphorylation of AMPA receptor GluR1 subunits at sites crucial for LTP and under some conditions causes persistent activation of αCamKII. The paradoxical ability of DHPG to induce depotentiation while at the same time activating signaling pathways involved in LTP suggests that LTP might not be erased by mGluR-dependent depotentiation. Consistent with this, DHPG-induced depotentiation did not restore the ability of high-frequency stimulation to induce LTP at synapses that had previously undergone saturating levels of LTP. In addition, blocking the expression of DHPG-induced LTD revealed hidden LTP at depotentiated synapses. Our results indicate that LTP and mGluR-dependent LTD can co-exist at excitatory synapses.

Altered Presynaptic Vesicle Release and Cycling during mGluR-Dependent LTD

The site of modification of synaptic transmission during long-term plasticity in the mammalian hippocampus remains controversial. Here we used a fluorescent marker of presynaptic activity, FM 1-43, to directly image presynaptic function during metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at CA3-CA1 excitatory synapses in acute hippocampal slices. We found a significant decrease in the rate of FM 1-43 release in response to synaptic stimulation following induction of mGluR-LTD, providing direct evidence for altered presynaptic function. Moreover, we found that mGluR-LTD causes several changes in FM dye release properties that are consistent with a change in the mode of vesicle cycling, possibly involving a switch from a full fusion mode of release to a “kiss-and-run” mode of release through the transient opening of a fusion pore.