Federica Bertaso

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 Ca2+, 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.

PICK1 uncoupling from mGluR7a causes absence-like seizures

Absence epilepsy is a neurological disorder that causes a recurrent loss of consciousness and generalized spike-and-wave discharges on an electroencephalogram (EEG). The role of metabotropic glutamate receptors (mGluRs) and associated scaffolding proteins in absence epilepsy has been unclear to date. We investigated a possible role for these proteins in absence epilepsy, focusing on the mGluR7a receptor and its PDZ-interacting protein, protein interacting with C kinase 1 (PICK1), in rats and mice. Injection of a cell-permeant dominant-negative peptide or targeted mutation of the mGluR7a C terminus, both of which disrupt the interaction between the receptor and PDZ proteins, caused behavioral symptoms and EEG discharges that are characteristic of absence epilepsy. Inactivation of the Pick1 gene also facilitated pharmacological induction of the absence epilepsy phenotype. The cortex and thalamus, which are known to participate in absence epilepsy, were involved, but the hippocampus was not. Our results indicate that disruption of the mGluR7a-PICK1 complex is sufficient to induce absence epilepsy–like seizures in rats and mice, thus providing, to the best of our knowledge, the first animal model of metabotropic glutamate receptor–PDZ protein interaction in absence epilepsy.

Homer1a-dependent crosstalk between NMDA and metabotropic glutamate receptors in mouse neurons.

Background A large number of evidences suggest that group-I metabotropic glutamate receptors (mGluR1a, 1b, 1c, 5a, 5b) can modulate NMDA receptor activity. Interestingly, a physical link exists between these receptors through a Homer-Shank multi-protein scaffold that can be disrupted by the immediate early gene, Homer1a. Whether such a versatile link supports functional crosstalk between the receptors is unknown. Methodology/Principal Findings Here we used biochemical, electrophysiological and molecular biological approaches in cultured mouse cerebellar neurons to investigate this issue. We found that Homer1a or dominant negative Shank3 mutants that disrupt the physical link between the receptors allow inhibition of NMDA current by group-I mGluR agonist. This effect is antagonized by pertussis toxin, but not thapsigargin, suggesting the involvement of a G protein, but not intracellular calcium stores. Also, this effect is voltage-sensitive, being present at negative, but not positive membrane potentials. In the presence of DHPG, an apparent NMDA “tail current” was evoked by large pulse depolarization, only in neurons transfected with Homer1a. Co-immunoprecipitation experiments showed interaction between G-protein βγ subunits and NMDA receptor in the presence of Homer1a and group-I mGluR agonist. Conclusions/Significance Altogether these results suggest a direct inhibition of NMDA receptor-channel by Gbetagamma subunits, following disruption of the Homer-Shank3 complex by the immediate early gene Homer1a. This study provides a new molecular mechanism by which group-I mGluRs could dynamically regulate NMDA receptor function.

Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons.

gamma-aminobutyric acid type B (GABA(B)) receptor is an allosteric complex made of two subunits, GABA(B1) and GABA(B2). GABA(B2) plays a major role in the coupling to G protein whereas GABA(B1) binds GABA. It has been shown that GABA(B) receptor activates ERK(1/2) in neurons of the central nervous system, but the molecular mechanisms underlying this event are poorly characterized. Here, we demonstrate that activation of GABA(B) receptor by either GABA or the selective agonist baclofen induces ERK(1/2) phosphorylation in cultured cerebellar granule neurons. We also show that CGP7930, a positive allosteric regulator specific of GABA(B2), alone can induce the phosphorylation of ERK(1/2). PTX, a G(i/o) inhibitor, abolishes both baclofen and CGP7930-mediated-ERK(1/2) phosphorylation. Moreover, both baclofen and CGP7930 induce ERK-dependent CREB phosphorylation. Furthermore, by using LY294002, a PI-3 kinase inhibitor, and a C-term of GRK-2 that has been reported to sequester Gbetagamma subunits, we demonstrate the role of Gbetagamma in GABA(B) receptor-mediated-ERK(1/2) phosphorylation. In conclusion, the activation of GABA(B) receptor leads to ERK(1/2) phosphorylation via the coupling of GABA(B2) to G(i/o) and by releasing Gbetagamma subunits which in turn induce the activation of CREB. These findings suggest a role of GABA(B) receptor in long-term change in the central nervous system.

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.

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.

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.

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G‐protein‐mediated inhibition of voltage‐sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two‐hybrid screen of a new‐born rat brain cDNA library using the cytoplasmic C‐terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine‐rich c‐kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull‐down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G‐protein‐mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G‐proteins.

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

Background: Rich2 is a synaptic Rho-GAP (Rho-GTPase-activating protein) the target of which was unknown. Results: We found that Rich2 controls dendritic spine morphogenesis by inhibiting Rac1 activity. Conclusion: Rac1 is the target of Rich2 in spines. Significance: We identified for the first time Rich2 as a Rac1-GAP protein that plays an important role in spine formation. 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.