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.

GPCR-interacting proteins (GIPs): nature and functions.

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7.

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.

The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways.

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.

Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer.

G-protein-coupled receptors (GPCRs) transduce signals from extracellular transmitters to the inside of the cell by activating G proteins. Mutation and overexpression of these receptors have revealed that they can reach their active state even in the absence of agonist, as a result of a natural shift in the equilibrium between their inactive and active conformations. Such agonist-independent (constitutive) activity has been observed for the glutamate GPCRs (the metabotropic glutamate receptors mGluR1a and mGluR5) when they are overexpressed in heterologous cells. Here we show that in neurons, the constitutive activity of these receptors is controlled by Homer proteins, which bind directly to the receptors' carboxy-terminal intracellular domains. Disruption of this interaction by mutagenesis or antisense strategies, or expression of endogenous Homer1a (H1a), induces constitutive activity in mGluR1a or mGluR5. Our results show that these glutamate GPCRs can be directly activated by intracellular proteins as well as by agonists.

AMPA receptor activation induces association of G-beta protein with the alpha subunit of the sodium channel in neurons.

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine-nucleotide binding- (G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), which has been shown to be physically associated with the alpha-subunit of Gi1 protein and activates this G-protein. Here, we report that, in addition to a Ca2+ influx, AMPA induces the mobilization of Ca2+ from the mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes required the activation of tetrodotoxin-sensitive Na+ channels. AMPA receptor activation modified the gating properties of the Na+ channel, independently of the AMPA current, suggesting a G-protein-mediated process. Indeed, co-immunoprecipitation experiments indicated that AMPA receptor activation induced the association of Gbeta with the alpha-subunit of the Na+ channel. These results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G-proteins and that this novel metabotropic function is involved in the control of neuronal excitability.

Selective blockade of P/Q-type calcium channels by the metabotropic glutamate receptor type 7 involves a phospholipase C pathway in neurons.

Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca(2+) channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca(2+) channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca(2+) channels. The effect was mimicked by GTPgammaS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein alphao subunit, indicating the involvement of a G(o)-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein betagamma subunits and the alpha1A Ca(2+) channel subunit, but was abolished by quenching betagamma subunits with specific intracellular peptides. Intracellular dialysis of G-protein betagamma subunits did not mimic the action of mGluR7, suggesting that both G-protein betagamma and alphao subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein betagamma with alphao subunits was required. The Ca(2+) chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP(3)) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP(3) formation. These results indicated that IP(3)-mediated intracellular Ca(2+) release was required for PKC-dependent inhibition of the Ca(2+) channels. Possible control of synaptic transmission by the present mechanisms is discussed.

Imidazoline-induced neuroprotective effects result from blockade of NMDA receptor channels in neuronal cultures.

Imidazolines have been shown to be neuroprotective in focal and global ischemia in the rat. However, their mechanism of action is still unclear. We have studied the neuroprotective effects of imidazolines against NMDA-induced neuronal death and hypoxic insult in cerebellar and striatal neuronal cultures. All of the imidazolines tested decreased the NMDA-mediated neurotoxicity in a non-competitive manner. Antazoline was the most effective (IC(50) of 5 microM, maximal neuroprotection reaching 90% at 100 microM). The neuroprotective effects were still present when the imidazolines were applied during the post-insult period. Antazoline, idazoxan and guanabenz also showed neuroprotective effects against hypoxia-induced neuronal death (neuroprotection reaching 95% for antazoline at 100 microM). Antazoline was still active if applied during the reoxygenation period (15% neuroprotection). To determine the mechanism of the neuroprotective effects, the possible interaction of imidazolines with NMDA receptors was studied. Imidazolines dose-dependently and non-competitively inhibited NMDA currents. As found for the neuroprotective effects, antazoline was the most effective imidazoline, with an IC(50) of 4 microM and a maximal inhibition of 90% at 100 microM. This blockade was rapid, reversible and voltage-dependent. We compared these effects to those of the classical non-competitive antagonist of NMDA channels, MK-801. In contrast to imidazolines, blockade of the NMDA current by MK-801 was voltage-independent and reversible only at positive potentials. When co-applied with MK-801, antazoline prevented the long lasting blockade of the NMDA current by MK-801. These results are consistent with the existence of overlapping binding sites for these drugs on the NMDA receptor channel. They indicate that imidazolines exert a strong neuroprotective effect against excitotoxicity and hypoxia in cerebellar and striatal primary neuronal cultures by inhibiting NMDA receptors. Since these effects were non-competitive, imidazolines appear to be interesting new drugs with therapeutic potential.

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.

Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by homer1 proteins and neuronal excitation.

The physiological actions of neurotransmitter receptors are intimately linked to their proper neuronal compartment localization. Here we studied the effect of the metabotropic glutamate receptor (mGluR)-interacting proteins, Homer1a, b, and c, in the targeting of mGluR5 in neurons. We found that mGluR5 was exclusively localized in cell bodies when transfected alone in cultured cerebellar granule cells. In contrast, mGluR5 was found also in dendrites when coexpressed with Homer1b or Homer1c, and in both dendrites and axons when cotransfected with Homer1a. In dendrites, cotransfected mGluR5 and Homer1b/c formed clusters that colocalized with the synaptic marker synaptophysin. Interestingly when transfected alone, the Homer proteins were also translocated to neurites but did not form such clusters. Depolarization of the neurons with a mixture of ionotropic glutamate receptor agonists, NMDA and kainate, or potassium channel blockers, tetraethylammonium and 4-aminopyridine, induced transient expression of endogenous Homer1a and persistent neuritic localization of transfected mGluR5 even long after degradation of Homer1a. These results suggest that Homer1a/b/c proteins are involved in the targeting of mGluR5 to dendritic synaptic sites and/or axons and that this effect can be regulated by neuronal activity. Because the activity-dependent effect of endogenous Homer1a was also long-lasting, the axonal targeting of mGluR5 by this protein is likely to play an important role in synaptic plasticity.

A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of the mGlu5 receptor in cerebellar granule cells.

We describe a method to transfer cDNA into neuronal primary cultures with a commercialised cationic lipid, Transfast. Cultures were transfected at a rate of about 5% with green fluorescent protein (GFP) cDNA. Comparing Transfast to other transfection reagents, we found this compound to be the most efficient. GFP-transfected mouse cerebellar granule cells displayed normal whole-cell voltage-sensitive and unitary big K+ channel currents. We also used this transfection method with success to transfer GFP cDNA into primary cultures of striatum and colliculus. Transfast was then used to cotransfect cultured cerebellar cells with GFP cDNA, in conjunction with cDNA coding for the metabotropic glutamate receptor type 5 (mGlu5 receptor). Ninety percent of the cells expressing GFP also expressed mGlu5 receptor. Though neurones were best transfected one day after plating, they still expressed both GFP and mGlu5 receptor proteins 2 weeks after plating, i.e. after full differentiation. A functional test of the expressed mGlu5 receptor was thus performed in GFP-transfected neurones. Stimulation of mGlu5 receptor induced single big K+ channel activity, as it was the case for the native mGlu1 receptor. This indicated that the transfected mGlu5 receptor plasmid was functionally expressed and that both mGlu1 and mGlu5 receptors may share common coupling mechanisms to big K+ channels in neurones.

Inositol-1,4,5-trisphosphate-mediated rescue of cerebellar long-term depression in subtype 1 metabotropic glutamate receptor mutant mouse.

Recent reports have outlined that cerebellar long-term depression requires the activation of subtype 1 metabotropic glutamate receptors, since long-term depression is impaired in subtype 1 metabotropic glutamate receptor (mGluR1) knockout mice. In order to better define the role of mGluR1-activated signal transduction pathways, we attempted to rescue cerebellar long-term depression in mGluR1 knockout mice by direct activation of subsequent intracellular cascades. The present results demonstrate that the inositol-1,4,5-trisphosphate signal transduction pathway remains functional in mGluR1 knockout mice, that calcium release from internal stores evoked by the combined photolytic release of inositol- 1,4,5-trisphosphate/pairing protocol is sufficient to rescue long-term depression in these mutants, and that this long-term depression is sensitive to a protein kinase C inhibitor. Therefore, our results provide compelling evidence that the impairment of long-term depression observed in mGluR1 knockout mice is not a consequence of developmental abnormalities, but is directly due to mGluR1 gene inactivation.

mGluR7-like metabotropic glutamate receptors inhibit NMDA-mediated excitotoxicity in cultured mouse cerebellar granule neurons.

Glutamate-induced glutamate release may be involved in the delayed neuronal death induced by N-methyl-D-aspartate (NMDA). In order to examine a possible modulatory effect of the presynaptic group III mGluRs on glutamate excitotoxicity, the effect of L-2-amino-4-phosphonobutyrate (L-AP4) was examined on NMDA-induced delayed death of mouse cerebellar granule neurons in culture. We found that L-AP4, at high concentration (in the millimolar range), inhibited in a non-competitive manner the NMDA-induced toxicity. This effect was mimicked by high concentration of L-serine-o-phosphate (L-SOP), and was inhibited by pertussis toxin (PTX) indicating the involvement of a Gi/o protein. This suggests the involvement of mGluR7 in the L-AP4 effect, and this was consistent with the detection of both mGluR7 protein and mRNA in these cultured neurons. To examine the mechanism of the L-AP4-induced protection from excitotoxic damage, the effect of L-AP4 on glutamate release was examined. L-AP4 (> or = 1 mM) noncompetitively inhibited by more than 60% the glutamate release induced by NMDA during the insult. We also observed that the 10-min NMDA receptor stimulation resulted in a dramatic increase in the extracellular glutamate concentration reaching 6000% of the control value 24 h after the insult. This large increase was also inhibited when NMDA was applied in the presence of > or = 1 mM L-AP4. Part of the L-AP4-induced protection from excitotoxic damage of granule neurons may therefore result from the inhibition of the vicious cycle: dying cells release glutamate, glutamate induced cell death. The present results add to the hypothesis that presynaptic mGluRs, probably mGluR7, may be the targets of drugs decreasing glutamate release and then neuronal death observed in some pathological situations.

cAMP-mediated long-term modulation of voltage-dependent K+ channels in cultured colliculi neurons.

Previously, we have described prolonged cAMP-induced inhibition of a K+ current in cultured colliculi neurons. The aim of the present study was to characterize the channel responsible for this cAMP-dependent effect. We detected the presence of a non-inactivating voltage-dependent 16-pS K+ channel that displayed long-lasting inhibition upon a brief application of cAMP and greater sensitivity to tetraethylammonium than to 4-aminopyridine. In addition to this channel, colliculi neurons express two other voltage-sensitive, non-inactivating K+ channels (8 and 49 pS) whose activity is facilitated by a brief application of cAMP, the effect of which is also long-lasting. These results suggest the presence of common sustained cAMP-dependent processes responsible for both up- and down-regulation of these channels in the neurons studied. They indicate that the 16-pS, but not the 8-pS or the 49-pS channels, participates in the cAMP-inhibited macroscopic K+ current.

Modulation of big K+ channel activity by ryanodine receptors and L-type Ca2+ channels in neurons.

As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca2+ channels and ryanodine receptors (RyR, Chavis et al., 1996) in cerebellar granule cells, we examined if such a coupling could activate a Ca2+-sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar granule cells. We observed that (+/-)-1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)-alpha-carboxycyclopropyl-glycine (L-CCG-I) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that was independent of L-type Ca2+ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca2+ channels.

Synthesis and pharmacological characterization of aminocyclopentanetricarboxylic acids: new tools to discriminate between metabotropic glutamate receptor subtypes.

The four stereoisomers of 1-aminocyclopentane-1,3,4-tricarboxylic acid {ACPT-I (18) and -II (19), (3R, 4R)-III [(-)-20], and (3S,4S)-III [(+)-20]} have been synthesized and evaluated for their effects at glutamate receptors subtypes. ACPTs are ACPD analogues in which a third carboxylic group has been added at position 4 in the cyclopentane ring. None of the ACPT isomers showed a significant effect on ionotropic NMDA, KA, and AMPA receptors. On the other hand, ACPT-II (19) was found to be a general competitive antagonist for metabotropic receptors (mGluRs) and exhibited a similar affinity for mGluR1a (KB = 115 +/- 2 microM), mGluR2 (KB = 88 +/- 21 microM), and mGluR4a (KB = 77 +/- 9 microM), the representative members of group I, II and III mGluRs, respectively. Two other isomers, ACPT-I (18) and (+)-(3S,4S)-ACPT-III [(+)-20], were potent agonists at the group III receptor mGluR4a (EC50 = 7.2 +/- 2.3 and 8.8 +/- 3.2 microM) and competitive antagonists with low affinity for mGluR1a and mGluR2 (KB > 300 microM). Finally, (-)-(3R,4R)-ACPT-III [(-)-20] was a competitive antagonist with poor but significant affinity for mGluR4a (KB = 220 microM). These results demonstrate that the addition of a third carboxylic group to ACPD can change its activity (from agonist to antagonist) and either increase or decrease its selectivity and/or affinity for the various mGluR subtypes.

Functional coupling between ryanodine receptors and L-type calcium channels in neurons.

In skeletal muscle, L-type Ca2+ channels act as voltage sensors to control ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum. It has recently been demonstrated that these ryanodine receptors generate a retrograde signal that modifies L-type Ca2+ -channel activity. Here we demonstrate a tight functional coupling between ryanodine receptors and L-type Ca2+ channel in neurons. In cerebellar granule cells, activation of the type-1 metabotropic glutamate receptor (mGluR1) induced a large, oscillating increase of the L-type Ba2+ current. Activation occurred independently of inositol 1,4,5-trisphosphate and classical protein kinases, but was mimicked by caffeine and blocked by ryanodine. The kinetics of this blockade were dependent on the frequency of Ba2+ current stimulation. Both mGluR1 and caffeine-induced increase in L-type Ca2+ -channel activity persisted in inside-out membrane patches. In these excised patches, ryanodine suppressed both the mGluR1- and caffeine-activated L-type Ca2+ channels. These results demonstrate a novel mechanism for Ca2+ -channel modulation in neurons.

Effects of nitric oxide on glutamate-gated channels and other ionic channels.

Nitric oxide is an endogenous molecule that plays a role of second messenger in the central and peripheral nervous system. A major action of this molecule is to control ionic channel activity. Because of technical difficulties to use nitric oxide as a gaseous compound, nitric oxide donors are often utilized under controlled experimental conditions. Here we will review the advantages and limitations in using these compounds. Nitric oxide can affect ionic channels through direct interactions or through the production of cGMP. We will describe an example of direct action of nitric oxide on glutamate-gated channels. We will also review indirect actions of nitric oxide on various potassium and calcium channels. Finally, we will discuss the complex physiological consequences of the action of nitric oxide on these ionic channels.

Modulation of calcium channels by metabotropic glutamate receptors in cerebellar granule cells.

We investigated the mechanisms by which metabotropic glutamate receptors (mGluRs) modulate specific Ca2+ channels in cerebellar granule cells. A large fraction of the current in granule cells is carried by L- and Q-type Ca2+ channels (about 26% each), whereas N- and P-type contribute proportionally less to the global current (9 and 15%, respectively). l-Aminocyclopentane-dicarboxylate (t-ACPD), (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCGI) and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG], but not L(+)-2-amino-4-phosphonobutyrate (L-AP4) reduced the Ca2+ current amplitude. The t-ACPD-induced inhibition was fully antagonized by (+/-)-methyl-4-carboxyphenylglycine [(+/-)-MCPG] and blocked by pertussis toxin (PTX). These results are consistent with inhibitory response mediated by mGluR2/R3. The use of specific Ca2+ channel blockers provided evidence that mGluR2/R3 inhibited both L- and N-type Ca2+ currents. In PTX-treated cells, Glu or t-ACPD, but not L-CCGI or L-AP4, increased the Ca2+ current. Consistent with the activation of mGluR1, the antagonists (+)-MCPG and (S)-4C3HPG prevented the facilitation of Ca2+ current produced by t-ACPD. The mGluR1-activated facilitation was completely blocked by nimodipine, indicating that L-type Ca2+ currents were selectively potentiated.