The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures.

Finding new targets to control or reduce seizure activity is essential to improve the management of epileptic patients. We hypothesized that activation of the pre-synaptic and inhibitory metabotropic glutamate receptor type 7 (mGlu7) reduces spontaneous seizures. We tested LSP2-9166, a recently developed mGlu7/4 agonist with unprecedented potency on mGlu7 receptors, in two paradigms of epileptogenesis. In a model of chemically induced epileptogenesis (pentylenetetrazole systemic injection), LSP2-9166 induces an anti-epileptogenic effect rarely observed in preclinical studies. In particular, we found a bidirectional modulation of seizure progression by mGlu4 and mGlu7 receptors, the latter preventing kindling. In the intra-hippocampal injection of kainic acid mouse model that mimics the human mesial temporal lobe epilepsy, we found that LSP2-9166 reduces seizure frequency and hippocampal sclerosis. LSP2-9166 also acts as an anti-seizure drug on established seizures in both models tested. Specific modulation of the mGlu7 receptor could represent a novel approach to reduce pathological network remodeling.

Regulation of postsynaptic function by the dementia-related ESCRT-III subunit CHMP2B.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.

The stoichiometry of scaffold complexes in living neurons - DLC2 functions as a dimerization engine for GKAP.

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.

Type 1 metabotropic glutamate receptors (mGlu1) trigger the gating of GluD2 delta glutamate receptors.

The orphan GluD2 receptor belongs to the ionotropic glutamate receptor family but does not bind glutamate. Ligand-gated GluD2 currents have never been evidenced, and whether GluD2 operates as an ion channel has been a long-standing question. Here, we show that GluD2 gating is triggered by type 1 metabotropic glutamate receptors, both in a heterologous expression system and in Purkinje cells. Thus, GluD2 is not only an adhesion molecule at synapses but also works as a channel. This gating mechanism reveals new properties of glutamate receptors that emerge from their interaction and opens unexpected perspectives regarding synaptic transmission and plasticity.

Rho-GTPase-activating protein interacting with Cdc-42-interacting protein 4 homolog 2 (Rich2): a new Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase-activating protein that controls dendritic spine morphogenesis.

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.

Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABAergic synapses in the rodent cerebellar cortex.

Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.

Shank3-Rich2 interaction regulates AMPA receptor recycling and synaptic long-term potentiation.

Synaptic long-term potentiation (LTP) is a key mechanism involved in learning and memory, and its alteration is associated with mental disorders. Shank3 is a major postsynaptic scaffolding protein that orchestrates dendritic spine morphogenesis, and mutations of this protein lead to mental retardation and autism spectrum disorders. In the present study we investigated the role of a new Shank3-associated protein in LTP. We identified the Rho-GAP interacting CIP4 homolog 2 (Rich2) as a new Shank3 partner by proteomic screen. Using single-cell bioluminescence resonance energy transfer microscopy, we found that Rich2-Shank3 interaction is increased in dendritic spines of mouse cultured hippocampal neurons during LTP. We further characterized Rich2 as an endosomal recycling protein that controls AMPA receptor GluA1 subunit exocytosis and spine morphology. Knock-down of Rich2 with siRNA, or disruption of the Rich2-Shank3 complex using an interfering mimetic peptide, inhibited the dendritic spine enlargement and the increase in GluA1 subunit exocytosis typical of LTP. These results identify Rich2-Shank3 as a new postsynaptic protein complex involved in synaptic plasticity.

RNAi silencing of P/Q-type calcium channels in Purkinje neurons of adult mouse leads to episodic ataxia type 2.

Episodic ataxia type-2 (EA2) is a dominantly inherited human neurological disorder caused by loss of function mutations in the CACNA1A gene, which encodes the CaV2.1 subunit of P/Q-type voltage-gated calcium channels. It remains however unknown whether the deficit of cerebellar CaV2.1 in adult is in direct link with the disease. To address this issue, we have used lentiviral based-vector RNA interference (RNAi) to knock-down CaV2.1 expression in the cerebellum of adult mice. We show that suppression of the P/Q-type channels in Purkinje neurons induced motor abnormalities, such as imbalance and ataxic gait. Interestingly, moderate channel suppression caused no basal ataxia, while ß-adrenergic activation and exercise mimicked stress induced motor disorders. Moreover, stress-induced ataxia was stable, non-progressive and totally abolished by acetazolamide, a carbonic anhydrase inhibitor used to treat EA2. Altogether, these data reveal that P/Q-type channel suppression in adult mice supports the episodic status of EA2 disease.

Repeated stimulation of dopamine D1-like receptor and hyperactivation of mTOR signaling lead to generalized seizures, altered dentate gyrus plasticity, and memory deficits.

The acute activation of the dopamine D1-like receptors (D1R) is involved in a plethora of functions ranging from increased locomotor activity to the facilitation of consolidation, storage, and retrieval of memories. Although much less characterized, epileptiform activities, usually triggered by disruption of the glutamate and GABA balance, have also been reported to involve the dopaminergic transmission. Using a combination of biochemical, immunohistochemical, electrophysiological, and behavioral approaches we have investigated the consequences of repeated stimulation of D1R using the selective D1R-like agonist SKF81297. Here, we report that repeated systemic administration of SKF81297 induces kindled seizures in mice. These seizure episodes parallel the hyperactivation of the mTOR signaling in the hippocampus, leading to disrupted long-term potentiation (LTP) in the dentate gyrus (DG) and altered recognition memories. The mTOR inhibitor rapamycin delays the development of SKF81297-induced kindled seizures, and rescues LTP in the DG and object recognition. Our results show that repeated stimulation of D1R is sufficient to induce generalized seizures leading to the overactivation of mTOR signaling, disrupted hippocampal plasticity, and impaired long-term recognition memories. This work highlights the interest of mTOR inhibitors as therapeutic strategies to reverse plasticity and cognitive deficits.

GKAP-DLC2 interaction organizes the postsynaptic scaffold complex to enhance synaptic NMDA receptor activity.

At glutamatergic brain synapses, scaffolding proteins regulate receptor location and function. The targeting and organization of scaffolding proteins in the postsynaptic density (PSD) is poorly understood, but it is known that a core protein of the glutamatergic receptor postsynaptic scaffold complex, guanylate-kinase-associated protein (GKAP) interacts with dynein light chain 2 (DLC2, also known as DYNLL2), a protein associated with molecular motors. In the present study, we combined BRET imaging, immunostaining and electrophysiological recording to assess the role of the GKAP-DLC2 interaction in the functional organization of the glutamatergic synapse. We found that GKAP-DLC2 interaction in dendritic spine stabilizes scaffolding protein expression at the PSD and enhances synaptic NMDA receptor activity. Moreover, the GKAP-DLC2 functional interaction is favored by sustained synaptic activity. These data identify a regulatory pathway of synaptic transmission that depends on activity-induced remodelling of the postsynaptic scaffold protein complex.

GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies.

G protein-coupled receptors (GPCRs) are key transmembrane recognition molecules for regulatory signals such as light, odors, taste hormones, and neurotransmitters. In addition to activating guanine nucleotide binding proteins (G proteins), GPCRs associate with a variety of GPCR-interacting proteins (GIPs). GIPs contain structural interacting domains that allow the formation of large functional complexes involved in G protein-dependent and -independent signaling. At the cellular level, other functions of GIPs include targeting of GPCRs to subcellular compartments and their trafficking to and from the plasma membrane. Recently, roles of GPCR-GIP interactions in central nervous system physiology and pathologies have been revealed. Here, we highlight the role of GIPs in some important neurological and psychiatric disorders, as well as their potential for the future development of therapeutic drugs.

Epileptiform activity induces vascular remodeling and zonula occludens 1 downregulation in organotypic hippocampal cultures: role of VEGF signaling pathways.

Recent studies suggest that blood-brain barrier (BBB) permeability contributes to epileptogenesis in symptomatic epilepsies. We have previously described angiogenesis, aberrant vascularization, and BBB alteration in drug-refractory temporal lobe epilepsy. Here, we investigated the role of vascular endothelial growth factor (VEGF) in an in vitro integrative model of vascular remodeling induced by epileptiform activity in rat organotypic hippocampal cultures. After kainate-induced seizure-like events (SLEs), we observed an overexpression of VEGF and VEGF receptor-2 (VEGFR-2) as well as receptor activation. Vascular density and branching were significantly increased, whereas zonula occludens 1 (ZO-1), a key protein of tight junctions (TJs), was downregulated. These effects were fully prevented by VEGF neutralization. Using selective inhibitors of VEGFR-2 signaling pathways, we found that phosphatidylinositol 3-kinase is involved in cell survival, protein kinase C (PKC) in vascularization, and Src in ZO-1 regulation. Recombinant VEGF reproduced the kainate-induced vascular changes. As in the kainate model, VEGFR-2 and Src were involved in ZO-1 downregulation. These results showed that VEGF/VEGFR-2 initiates the vascular remodeling induced by SLEs and pointed out the roles of PKC in vascularization and Src in TJ dysfunction, respectively. This suggests that Src pathway could be a therapeutic target for BBB protection in epilepsies.

Diversity of metabotropic glutamate receptor-interacting proteins and pathophysiological functions.

In the mammalian brain, the large majority of excitatory synapses express pre- and postsynaptic glutamate receptors. These are ion channels and G protein-coupled membrane proteins that are organized into functional signaling complexes. Here, we will review the nature and pathophysiological functions of the scaffolding proteins associated to these receptors, focusing on the G protein-coupled subtypes.

Extracellular interactions between GluR2 and N-cadherin in spine regulation.

Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. Here we show that the first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. Our data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation.

Gelatinase Biosensor Reports Cellular Remodeling During Epileptogenesis.

Epileptogenesis is the gradual process responsible for converting a healthy brain into an epileptic brain. This process can be triggered by a wide range of factors, including brain injury or tumors, infections, and status epilepticus. Epileptogenesis results in aberrant synaptic plasticity, neuroinflammation and seizure-induced cell death. As Matrix Metalloproteinases (MMPs) play a crucial role in cellular plasticity by remodeling the extracellular matrix (ECM), gelatinases (MMP-2 and MMP-9) were recently highlighted as key players in epileptogenesis. In this work, we engineered a biosensor to report in situ gelatinase activity in a model of epileptogenesis. This biosensor encompasses a gelatinase-sensitive activatable cell penetrating peptide (ACPP) coupled to a TAMRA fluorophore, allowing fluorescence uptake in cells displaying endogenous gelatinase activities. In a preclinical mouse model of temporal lobe epilepsy (TLE), the intrahippocampal kainate injection, ACPPs revealed a localized distribution of gelatinase activities, refining temporal cellular changes during epileptogenesis. The activity was found particularly but not only in the ipsilateral hippocampus, starting from the CA1 area and spreading to dentate gyrus from the early stages throughout chronic epilepsy, notably in neurons and microglial cells. Thus, our work shows that ACPPs are suitable molecular imaging probes for detecting the spatiotemporal pattern of gelatinase activity during epileptogenesis, suggesting their possible use as vectors to target cellular reactive changes with treatment for epileptogenesis.

D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson's disease.

Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.

Knock-in mice lacking the PDZ-ligand motif of mGluR7a show impaired PKC-dependent autoinhibition of glutamate release, spatial working memory deficits, and increased susceptibility to pentylenetetrazol.

The metabotropic glutamate receptor 7 (mGluR7) is widely expressed throughout the brain and primarily localized at presynaptic active zones, where it is thought to regulate neurotransmitter release. Protein interacting with C kinase 1 (PICK1), a postsynaptic density protein-95/disc-large tumor suppressor protein/zonula occludens-1 (PDZ)-domain protein, binds to the three C-terminal amino acids (-LVI) of the predominant mGluR7 splice variant, mGluR7a, and has been implicated in the synaptic clustering of this receptor. Here, we generated knock-in mice in which the C-terminal LVI coding sequence of exon 10 of the mGluR7 gene was replaced by three alanine codons (-AAA). Immunoprecipitation showed that the PICK1-mGluR7a interaction is disrupted in mGluR7a(AAA/AAA) mice. However, the synaptic localization of mGluR7a was not altered in cultured hippocampal neurons and brain sections prepared from the knock-in animals. In cerebellar granule cell cultures, the group III mGluR agonist l-AP-4 decreased the frequency of spontaneous excitatory currents in neurons derived from wild-type but not mGluR7a(AAA/AAA) mice, consistent with the interaction between mGluR7a and PICK1 being required for protein kinase C-mediated inhibition of glutamate release. At the behavioral level, the mGluR7a(AAA/AAA) mice showed no deficits in motor coordination, pain sensitivity, and anxiety but exhibited significant defects in hippocampus-dependent spatial working memory. In addition, they displayed a high susceptibility to the convulsant drug pentylenetetrazole. Together, these results indicate that PICK1 binding to the C-terminal region of mGluR7a is crucial for agonist-triggered presynaptic signaling in vivo.

Epac mediates PACAP-dependent long-term depression in the hippocampus.

Extensive work has shown that activation of the cAMP-dependent protein kinase A (PKA) is crucial for long-term depression (LTD) of synaptic transmission in the hippocampus, a phenomenon that is thought to be involved in memory formation. Here we studied the role of an alternative target of cAMP, the exchange protein factor directly activated by cyclic AMP (Epac). We show that pharmacological activation of Epac by the selective agonist 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT) induces LTD in the CA1 region. Paired-pulse facilitation of synaptic responses remained unchanged after induction of this LTD, suggesting that it depended on postsynaptic mechanisms. The 8-pCPT-induced LTD was blocked by the Epac signalling inhibitor brefeldin-A (BFA), Rap-1 antagonist geranylgeranyltransferase inhibitor (GGTI) and p38 mitogen activated protein kinase (P38-MAPK) inhibitor SB203580. This indicated a direct involvement of Epac in this form of LTD. As for other forms of LTD, a mimetic peptide of the PSD-95/Disc-large/ZO-1 homology (PDZ) ligand motif of the AMPA receptor subunit GluR2 blocked the Epac-LTD, suggesting involvement of PDZ protein interaction. The Epac-LTD also depended on mobilization of intracellular Ca(2+), proteasome activity and mRNA translation, but not transcription, as it was inhibited by thapsigargin, lactacystin and anisomycin, but not actinomycin-D, respectively. Finally, we found that the pituitary adenylate cyclase activating polypeptide (PACAP) can induce an LTD that was mutually occluded by the Epac-LTD and blocked by BFA or SB203580, suggesting that the Epac-LTD could be mobilized by stimulation of PACAP receptors. Altogether these results provided evidence for a new form of hippocampal LTD.

Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer.

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and beta-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.

Loss of X-linked mental retardation gene oligophrenin1 in mice impairs spatial memory and leads to ventricular enlargement and dendritic spine immaturity.

Loss of oligophrenin1 (OPHN1) function in human causes X-linked mental retardation associated with cerebellar hypoplasia and, in some cases, with lateral ventricle enlargement. In vitro studies showed that ophn1 regulates dendritic spine through the control of Rho GTPases, but its in vivo function remains unknown. We generated a mouse model of ophn1 deficiency and showed that it mimics the ventricles enlargement without affecting the cerebellum morphoanatomy. The ophn1 knock-out mice exhibit behavioral defects in spatial memory together with impairment in social behavior, lateralization, and hyperactivity. Long-term potentiation and mGluR-dependent long-term depression are normal in the CA1 hippocampal area of ophn1 mutant, whereas paired-pulse facilitation is reduced. This altered short-term plasticity that reflects changes in the release of neurotransmitters from the presynaptic processes is associated with normal synaptic density together with a reduction in mature dendritic spines. In culture, inactivation of ophn1 function increases the density and proportion of immature spines. Using a conditional model of loss of ophn1 function, we confirmed this immaturity defect and showed that ophn1 is required at all the stages of the development. These studies show that, depending of the context, ophn1 controls the maturation of dendritic spines either by maintaining the density of mature spines or by limiting the extension of new filopodia. Altogether, these observations indicate that cognitive impairment related to OPHN1 loss of function is associated with both presynaptic and postsynaptic alterations.