Laurent Fagni

Nitric oxide-induced blockade of NMDA receptors

Abstract We studied the effects of nitric oxide (NO)-producing agents on N-methyl-d-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholinosydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca 2+ . The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine(an NO-containing drug) and with NO released from NaNO 2 . Pretreatment with hemoglobil potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.

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 GluR2s 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.

Complex interactions between mGluRs, intracellular Ca2+ stores and ion channels in neurons

Metabotropic glutamate receptors (mGluRs) can increase intracellular Ca2+ concentration via Ins(1,4,5)P3- and ryanodine-sensitive Ca2+ stores in neurons. Both types of store are coupled functionally to Ca2+-permeable channels found in the plasma membrane. The mGluR-mediated increase in intracellular Ca2+ concentration can activate Ca2+-sensitive K+ channels and Ca2+-dependent nonselective cationic channels. These mGluR-mediated effects often result from mobilization of Ca2+ from ryanodine-sensitive, rather than Ins(1,4, 5)P3-sensitive, Ca2+ stores, suggesting that close functional interactions exist between mGluRs, intracellular Ca2+ stores and Ca2+-sensitive ion channels in the membrane.

Shank Expression Is Sufficient to Induce Functional Dendritic Spine Synapses in Aspiny Neurons

Shank proteins assemble glutamate receptors with their intracellular signaling apparatus and cytoskeleton at the postsynaptic density. Whether Shank plays a role in spinogenesis and synaptogenesis remained unclear. Here, we report that knock-down of Shank3/prolinerich synapse-associated protein-2 by RNA interference reduces spine density in hippocampal neurons. Moreover, transgene expression of Shank 3 is sufficient to induce functional dendritic spines in aspiny cerebellar neurons. Transfected Shank protein recruits functional glutamate receptors, increases the number and size of synaptic contacts, and increases amplitude, frequency, and the AMPA component of miniature EPSCs, similar to what is observed during synapse developmental maturation. Mutation/deletion approaches indicate that these effects require interactions of Shank3 with the glutamate receptor complex. Consistent with this observation, chronic treatment with glutamate receptor antagonists alters maturation of the Shank3-induced spines. These results strongly suggest that Shank proteins and the associated glutamate receptors participate in a concerted manner to form spines and functional synapses.

The ‘magic tail’ of G protein-coupled receptors: an anchorage for functional protein networks

All cell types express a great variety of G protein‐coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross‐talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross‐talk. New methodologies (mostly yeast two‐hybrid screens and proteomics) have been used to discover more than 50 GPCR‐associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine‐tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C‐terminus are the most abundant and are the focus of this review.

SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies.

Activation of a Large‐conductance Ca2+‐Dependent K+ Channel by Stimulation of Glutamate Phosphoinositide‐coupled Receptors in Cultured Cerebellar Granule Cells

Trans‐1‐amino‐cyclopentyl‐1,3‐dicarboxylic acid (trans‐ACPD), a specific agonist of the glutamate phosphoinositide‐coupled receptor (Qp receptor), increased the amplitude of the outward K+ current recorded in the whole‐cell configuration of the patch‐clamp technique in mouse cultured cerebellar granule cells. This effect was abolished by buffering internal Ca2+ with BAPTA [1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid]. Activation of a large‐conductance K+ channel was observed when trans‐ACPD or quisqualic acid (QA), another Qp receptor agonist, was applied outside the cell‐attached patch pipettes. No activation was observed with alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), a specific agonist of ionotropic non‐N‐methyl‐d‐aspartate (non‐NMDA) receptors. The effects of trans‐ACPD or QA were potentiated in the presence of external Ca2+. The channel was also directly activated by both micromolar concentrations of internal Ca2+ and membrane depolarization. Its unitary conductance was 100–115 pS under asymmetrical K+ and 195–235 pS under high symmetrical K+ conditions. In the absence of agonist, the channel was blocked by 1 mM external tetraethylammonium. This is the first description of a large conductance Ca2+‐activated K+ channel in cultured cerebellar granule cells. It possesses properties similar to those of the so‐called ‘big K+ channel’ described in other preparations. Our cell‐attached experiments demonstrated an indirect coupling between Qp receptors and this channel. The most likely hypothesis is that the second messenger system inositol 1,4,5‐triphosphate (IP3)‐Ca2+ was involved in the coupling process. This hypothesis was further strengthened by our whole‐cell experiments. On the basis of the voltage‐ and Ca2+‐sensitivities of the studied channel, we estimated an increase of 350 to 570 nM in internal Ca2+ concentration when Qp receptors were stimulated by 100 μM trans‐ACPD. Under physiological conditions, stimulation of Qp receptors by the endogenous neurotransmitter should lead to similar K+ channel activation and therefore would tend to reduce the efficacy of ionotropic glutamate synaptic receptor stimulation responsible for cell excitation.

Homer-dependent cell surface expression of metabotropic glutamate receptor type 5 in neurons.

The metabotropic glutamate (mGlu) receptors are a family of receptors involved in the tuning of fast excitatory synaptic transmission in the brain. Experiments performed in heterologous expression systems suggest that cell surface expression of group I mGlu receptors is controlled by their auxiliary protein, Homer. However, whether or not this also applies to neurons remains controversial. Here we show that in cultured cerebellar granule cells, the group I mGlu receptor subtype, mGlu5, transfected alone is functionally expressed at the surface of these neurons. Transfected Homer1b caused intracellular retention and clustering of this receptor at synaptic sites. Recombinant Homer1a alone did not affect cell surface expression of the receptor, but in neurons transfected with Homer1b, excitation-induced expression of native Homer1a reversed the intracellular retention of mGlu5 receptors, resulting in the receptor trafficking to synaptic membranes. Transfected Homer1a also increased the latency and amplitude of the mGlu5 receptor Ca2+ response. These results indicate that Homer1 proteins regulate synaptic cycling and Ca2+ signaling of mGlu5 receptors, in response to neuronal activity.

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.

Homer as Both a Scaffold and Transduction Molecule

Increasing evidence shows that scaffold proteins not only control membrane assembly of receptors and channels, but also modulate intracellular signaling by assembled receptors. The Homer family of proteins act as scaffolds to bind clusters of proteins and glutamate receptors at postsynaptic sites. We review results of cloning and gene expression of this protein family, and summarize roles in glutamate receptor function and intracellular signaling in neurons. Homer proteins trigger the localization of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) to the postsynaptic plasma membrane. They can also alter the kinetics and peak amplitude of the intracellular Ca2+ responses of mGlu1 and mGlu5 receptors. Homer proteins can either prevent or promote spontaneous activation of these receptors, depending on the type of Homer protein isoform expressed. Glutamate is the major excitatory neurotransmitter of the mammalian brain. It activates two types of synaptic receptors: ionotropic receptor-channels and metabotropic heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The ionotropic glutamate receptors generate fast postsynaptic responses, whereas the metabotropic glutamate (mGlu) receptors modulate these fast responses, generate slower postsynaptic potentials, or both. Glutamatergic synaptic transmission depends on the adequate localization and intracellular signaling of these postsynaptic receptors and channels. This is achieved by scaffolding proteins that assemble glutamate receptors into functional complexes at postsynaptic membranes. However, little is known about the role of these intracellular proteins in the receptor signaling. A new family of proteins that interact with mGlu receptors has been cloned from rat brain and named Homer or Ves1 proteins. We review recent data indicating that Homer proteins not only control expression and localization of mGlu receptors and channels, but also participate in signaling of glutamate receptors in neurons.