Maryse Paquet

Group I Metabotropic Glutamate Receptor Signalling and its Implication in Neurological Disease

Stimulation of Group I metabotropic glutamate receptors (mGluR1 and mGluR5) leads to activation of a wide variety of signalling pathways. mGluRs couple to Gα(q/)₁₁ proteins, activating phospholipase Cβ1 resulting in both diacylglycerol and inositol-1,4,5-triphosphate formation followed by the activation of protein kinase C. In addition, mGluR activation can lead to modulation of a number of ion channels, such as different types of calcium and potassium channels. Group I mGluRs can also activate other downstream protein kinases, such as ERK1/2 and AKT, which are implicated in cellular growth, differentiation, and survival. Moreover, Group I mGluRs interact with a variety of different proteins that are important for the regulation of synaptic signalling, such as Homer and PDZ domain containing proteins, such as Tamalin. A role for mGluR1/5 in a number of disease states has also been proposed. As mGluR1/5 signal transduction is complex and involves multiple partners, a better understanding of alterations in mGluR signalling in brain disorders will be required in order to discern the molecular and cellular basis of these pathologies. This review will highlight recent findings concerning mGluR signaling alterations in brain pathologies, such as stroke, fragile X syndrome, Alzheimers disease, Parkinsons disease, Huntingtons disease, epilepsy, and drug addiction.

Presynaptic NMDA receptor subunit immunoreactivity in GABAergic terminals in rat brain.

N‐methyl‐D‐aspartate (NMDA) receptors are commonly found post‐synaptically; they mediate fast excitatory neurotransmission in the central nervous system. In this study, we provide immunocytochemical data supporting the existence of presynaptic NMDA receptors in GABAergic terminals using polyclonal antisera raised against the C‐terminus of the NMDAR1 subunit. At the light microscope level, rich plexuses of NMDAR1‐positive varicose fibers were found in various nuclei in the basal forebrain (bed nucleus of stria terminalis, septum, parastrial nucleus, vascular organ of the lamina terminalis), thalamus (paraventricular nucleus, midline nuclei), and hypothalamus (parvocellular paraventricular nucleus, arcuate nucleus, preoptic nucleus, suprachiasmatic nucleus). In the brainstem, labeled fibers were much less abundant and were confined to the ventral tegmental area, periaqueductal gray, parabrachial nucleus, and locus coeruleus. At the electron microscope level, NMDAR1‐immunoreactive terminals examined in the bed nucleus of stria terminalis, parvocellular paraventricular hypothalamic nucleus, and arcuate nucleus formed symmetric synapses, contained darkly stained large dense‐core vesicles, and displayed γ‐aminobutyric acid (GABA) immunoreactivity. Terminals with similar ultrastructural features were found in the paraventricular thalamic nucleus. These findings demonstrate the existence of NMDAR1 subunit immunoreactivity in subsets of GABAergic terminals, which raises questions about the potential roles and mechanisms of activation of presynaptic NMDA heteroreceptors in the rat central nervous system. The pattern of distribution and ultrastructural features of these boutons suggest that they may arise from local GABAergic projections interconnecting a group of brain structures mediating stress responses and/or other endocrine, autonomic, and limbic functions. J. Comp. Neurol. 423:330–347, 2000.

Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase

Cocaine and amphetamine regulated transcript (CART) encodes a novel brain‐enriched protein whose features are reminiscent of a neurotransmitter propeptide. We have now localized CART peptide(s) in the gastrointestinal tract by immunohistochemical methods. Polyclonal antisera raised to CART peptide 106–129 stained neuronal cell bodies and fibers in rat ileum myenteric plexus‐longitudinal muscle tissue preparations. Electron microscopic analysis of thin sections showed immunopositive axon terminals in close apposition to CART‐labelled and unlabelled neuronal cell bodies as well as to the longitudinal muscle. CART peptide‐immunoreactive terminals contained numerous ovoid electron‐lucent vesicles and a few dark‐stained dense‐core vesicles. Light microscopic double labelling studies revealed CART peptide immunoreactivity in a subpopulation of choline acetyltransferase (ChAT)‐immunoreactive neurons. This combined light microscopic and ultrastructural examination of CART peptides in the gastrointestinal tract suggests a role of CART peptides as transmitters or neuromodulators in the peripheral nervous system. Synapse 30:1–8, 1998.

P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

P2Y1 purinergic receptors (P2Y1Rs) mediate rises in intracellular Ca2+ in response to ATP, but the duration and characteristics of this Ca2+ response are known to vary markedly in distinct cell types. We screened the P2Y1R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95/Drosophila Discs large/ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na+/H+ exchanger regulatory factor type 2). Furthermore, we found that P2Y1R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y1R to key downstream effectors such as phospholipase Cβ. Finally, we found that coexpression of P2Y1R with NHERF-2 in glial cells prolongs P2Y1R-mediated Ca2+ signaling, whereas disruption of the P2Y1R–NHERF-2 interaction by point mutations attenuates the duration of P2Y1R-mediated Ca2+ responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y1R and may therefore determine cell-specific differences in P2Y1R-mediated signaling.

Ionotropic and metabotropic GABA and glutamate receptors in primate basal ganglia.

The functions of glutamate and GABA in the CNS are mediated by ionotropic and metabotropic, G protein-coupled, receptors. Both receptor families are widely expressed in basal ganglia structures in primates and nonprimates. The recent development of highly specific antibodies and/or cDNA probes allowed the better characterization of the cellular localization of various GABA and glutamate receptor subtypes in the primate basal ganglia. Furthermore, the use of high resolution immunogold techniques at the electron microscopic level led to major breakthroughs in our understanding of the subsynaptic and subcellular localization of these receptors in primates. In this review, we will provide a detailed account of the current knowledge of the localization of these receptors in the basal ganglia of humans and monkeys.

GABAB and group I metabotropic glutamate receptors in the striatopallidal complex in primates

Glutamate and GABA neurotransmission is mediated through various types of ionotropic and metabotropic receptors. In this review, we summarise some of our recent findings on the subcellular and subsynaptic localisation of GABAB and group I metabotropic glutamate receptors in the striatopallidal complex of monkeys. Polyclonal antibodies that specifically recognise GABABR1, mGluR1a and mGluR5 receptor subtypes were used for immunoperoxidase and pre‐embedding immunogold techniques at the light and electron microscope levels. Both subtypes of group I mGluRs were expressed postsynaptically in striatal projection neurons and interneurons where they aggregate perisynaptically at asymmetric glutamatergic synapses and symmetric dopaminergic synaptic junctions. Moreover, they are also strongly expressed in the main body of symmetric synapses established by putative intrastriatal GABAergic terminals. In the globus pallidus, both receptor subtypes are found postsynaptically in the core of striatopallidal GABAergic synapses and perisynaptically at subthalamopallidal glutamatergic synapses. Finally, extrasynaptic labelling was commonly seen in the globus pallidus and the striatum.

Metabotropic Glutamate Receptor-Mediated Cell Signaling Pathways Are Altered in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (Htt). Group I metabotropic glutamate receptors (mGluRs) are coupled to Gαq and play an important role in neuronal survival. We have previously demonstrated that mGluRs interact with Htt. Here we used striatal neuronal primary cultures and acute striatal slices to demonstrate that mGluR-mediated signaling pathways are altered in a presymptomatic mouse model of HD (HdhQ111/Q111), as compared to those of control mice (HdhQ20/Q20). mGluR1/5-mediated inositol phosphate (InsP) formation is desensitized in striatal slices from HdhQ111/Q111 mice and this desensitization is PKC-mediated. Despite of decreased InsP formation, (S)-3,5-dihydroxylphenylglycine (DHPG)-mediated Ca2+ release is higher in HdhQ111/Q111 than in HdhQ20/Q20 neurons. Furthermore, mGluR1/5-stimulated AKT and extracellular signal-regulated kinase (ERK) activation is altered in HdhQ111/Q111 mice. Basal AKT activation is higher in HdhQ111/Q111 neurons and this increase is mGluR5 dependent. Moreover, mGluR5 activation leads to higher levels of ERK activation in HdhQ111/Q111 than in HdhQ20/Q20 striatum. PKC inhibition not only brings HdhQ111/Q111 DHPG-stimulated InsP formation to HdhQ20/Q20 levels, but also causes an increase in neuronal cell death in HdhQ111/Q111 neurons. However, PKC inhibition does not modify neuronal cell death in HdhQ20/Q20 neurons, suggesting that PKC-mediated desensitization of mGluR1/5 in HdhQ111/Q111 mice might be protective in HD. Together, these data indicate that group I mGluR-mediated signaling pathways are altered in HD and that these cell signaling adaptations could be important for striatal neurons survival.

Neuro-transmitters in the central nervous system & their implication in learning and memory processes.

This review article gives an overview of a number of central neuro-transmitters, which are essential for integrating many functions in the central nervous system (CNS), such as learning, memory, sleep cycle, body movement, hormone regulation and many others. Neurons use neuro-transmitters to communicate, and a great variety of molecules are known to fit the criteria to be classified as such. A process shared by all neuro-transmitters is their release by excocytosis, and we give an outline of the molecular events and protein complexes involved in this mechanism. Synthesis, transport, inactivation, and cellular signaling can be very diverse when different neuro-transmitters are compared, and these processes are described separately for each neuro-transmitter system. Here we focus on the most well known neuro-transmitters: acetyl-choline, catechol-amines (dopamine and nor-adrenalin), indole-amine (serotonin), glutamate, and gamma-amino-butyric acid (GABA). Glutamate is the major excitatory neuro-transmitter in the brain and its actions are counter-balanced by GABA, which is the major inhibitory substance in the CNS. A balance of neuronal transmission between these two neuro-transmitters is essential to normal brain function. Acetyl-choline, serotonin and catechol-amines have a more modulatory function in the brain, being involved in many neuronal circuits. Apart from summarizing the current knowledge about the synthesis, release and receptor signaling of these transmitters, some disease states due to alteration of their normal neuro-transmission are also described.

Phosphorylation-independent regulation of metabotropic glutamate receptor 5 desensitization and internalization by G protein-coupled receptor kinase 2 in neurons.

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Gαq/11 binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.

The PDZ Scaffold NHERF-2 Interacts with mGluR5 and Regulates Receptor Activity

The two members of the group I metabotropic glutamate receptor family, mGluR1 and mGluR5, both couple to Gq to mediate rises in intracellular calcium. The alternatively spliced C termini (CT) of mGluRs1&5are known to be critical for regulating receptor activity and to terminate in motifs suggestive of potential interactions with PDZ domains. We therefore screened the CTs of both mGluR1a and mGluR5 against a PDZ domain proteomic array. Out of 96 PDZ domains examined, the domain that bound most strongly to mGluR5-CT was the second PDZ domain of the Na+/H+ exchanger regulatory factor 2 (NHERF-2). This interaction was confirmed by reverse overlay, and a single point mutation to the mGluR5-CT was found to completely disrupt the interaction. Full-length mGluR5 robustly associated with full-length NHERF-2 in cells, as assessed by co-immunoprecipitation and confocal microscopy experiments. In contrast, mGluR1a was found to bind NHERF-2 in vitro with a weaker affinity than mGluR5, and furthermore mGluR1a did not detectably associate with NHERF-2 in a cellular context. Immunohistochemical experiments revealed that NHERF-2 and mGluR5 exhibit overlapping patterns of expression in mouse brain, being found most abundantly in astrocytic processes and postsynaptic neuronal elements. In functional experiments, the interaction of NHERF-2 with mGluR5 in cells was found to prolong mGluR5-mediated calcium mobilization and to also potentiate mGluR5-mediated cell death, whereas coexpression of mGluR1a with NHERF-2 had no evident effects on mGluR1a functional activity. These observations reveal that NHERF-2 can selectively modulate mGluR5 signaling, which may contribute to cell-specific regulation of mGluR5 activity.