Corrado Corti

The Use of Knock-Out Mice Unravels Distinct Roles for mGlu2 and mGlu3 Metabotropic Glutamate Receptors in Mechanisms of Neurodegeneration/Neuroprotection

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (−)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2−/−, or mGlu3−/− mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2−/− mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2−/− mice but not in mGlu3−/− mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

Activation of the extracellular signal-regulated kinase 2 by metabotropic glutamate receptors

Activation of metabotropic glutamate receptors (mGluRs) leads to modulation of a variety of second messenger pathways probably including the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated protein kinases (ERK). MAPK play a key role in the control of cellular responses to changes in the external environment by regulating transcriptional activity and the phosphorylation state of several cytoplasmic targets. In this study, Chinese hamster ovary (CHO) cells permanently transfected with rat mGluR1a, mGluR2 and mGluR4 were employed as a model to examine the activation of MAPK by glutamate through mGluRs. All three mGluR subtypes rapidly stimulated ERK activation. In particular, mGluR1a and mGluR2 preferentially mediated phosphorylation and activation of ERK2 in a pertussis toxin (PTX)‐sensitive and concentration‐dependent manner. The activation was blocked completely by pretreatment with the antagonist (rs)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) or with the MEK inhibitor PD098059. Furthermore, mGluR1a‐mediated ERK activation was suppressed by the depletion of endogenous protein kinase C (PKC) activity and by the PKC inhibitors staurosporine and calphostin C, but not chelerythrine. When cAMP was elevated in mGluR2‐expressing cells, by forskolin or dibutyryl‐cAMP, slight elevation of ERK activity was observed. However, glutamate‐stimulated ERK activation remained unaffected. In these cells, the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin produced a significant, albeit only partial, inhibition of mGluR2‐mediated ERK activation. These findings raise the possibility of a MAPK cascade involvement in glutamate‐dependent neuronal plasticity mediated through stimulation of mGluRs.

CLONING AND CHARACTERIZATION OF ALTERNATIVE MRNA FORMS FOR THE RAT METABOTROPIC GLUTAMATE RECEPTORS MGLUR7 AND MGLUR8

Novel mRNA isoforms for two members of the group III metabotropic glutamate receptors (mGluRs), called mGluR7b and mGluR8b, were identified from rat brain cerebral cortex and hippocampus. In both cases, the alternative splicing is generated by a similar out‐of‐frame insertion in the carboxyl‐terminus that results in the replacement of the last 16 amino acids of mGluR7 and mGluR8 by 23 and 16 different amino acids, respectively. Distribution analysis for mGluR7 and mGluR8 isoforms revealed that the two splice variants are generally coexpressed in the same brain areas. The few exceptions were the olfactory bulb, in which only the mGluR7a form could be detected by reverse transcription–polymerase chain reaction, and the lateral reticular and ambiguus nuclei, which showed only mGluR8a labelling. Despite expression in the same regions, different mRNA abundance for the two variants of each receptor were observed. When transiently coexpressed in HEK 293 cells with the phospholipase C‐activating chimeric Gαqi9‐G‐protein, the a and b forms for both receptor subtypes showed a similar pharmacological profile. The rank order of potencies for both was: dl‐amino‐4‐phosphonobutyrate > l‐serine‐O‐phosphate > glutamate. However, the agonist potencies were significantly higher for mGluR8a, b compared with mGluR7a,b. In Xenopus oocytes, glutamate evoked currents only with mGluR8 when coexpressed with Kir 3.1 and 3.4. Glutamate‐induced currents were antagonized by the group II/III antagonist (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine. In conclusion, the two isoforms of each receptor have identical pharmacological profiles when expressed in heterologous systems, despite structural differences in the carboxyl‐terminal domains.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca2+ channels encoded by the CaV3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three CaV3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. CaV3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic CaV3.3−/− mouse line and demonstrate that CaV3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of CaV3.3 channels prevented oscillatory bursting in the low-frequency (4–10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing CaV3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in CaV3.3−/− mice were <30% compared with those in WT mice, and the remaining current, carried by CaV3.2 channels, generated dendritic [Ca2+]i signals insufficient to provoke oscillatory bursting that arises from interplay with Ca2+-dependent small conductance-type 2 K+ channels. Finally, naturally sleeping CaV3.3−/− mice showed a selective reduction in the power density of the σ frequency band (10–12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for CaV3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

Activated astrocytes in areas of kainate-induced neuronal injury upregulate the expression of the metabotropic glutamate receptors 2/3 and 5

Abstract. All forms of brain injury induce activation of astrocytes, although different types of injury induce different astrocytic responses. Activated astrocytes are characterised by hypertrophy, proliferation and increased expression of glial fibrillary acidic protein (GFAP). However, neither the process by which astrocytes become reactive nor the consequences are well understood. Recently, the application of specific growth factors to primary astrocytic cultures was shown to regulate dramatically the level of expression of the metabotropic glutamate receptors (mGluR) 5 and 3. In the present study, we have used an intracerebroventricular injection of a subconvulsive dose of kainic acid to produce a lesion of CA3a pyramidal neurones in the mouse hippocampus and to investigate whether mGluR expression was altered in reactive astrocytes in vivo. Immunohistochemical analysis showed strong mGluR5 and mGluR2/3 immunoreactivity in glial cells within the area of neuronal loss possessing the morphological feature of activated astrocytes. Double labelling with GFAP confirmed the expression of mGluRs by reactive astrocytes. The mechanical injury produced by the needle insertion in the cerebral cortex also produced enhanced expression of mGluR5 and mGluR2/3 in activated astrocytes proximal to the area of neuronal injury. Our finding of an increased mGluR expression in reactive astrocytes in vivo suggests that transcriptional regulation by specific growth factors on mGluRs is a phenomenon extendible to specific circumstances in vivo and not limited to in vitro models. Identification of the mechanisms of this adaptive plasticity will be central in the understanding of the events leading to neuronal survival and/or death.

Immunohistochemical localization of the mGluR1β metabotropic glutamate receptor in the adult rodent forebrain: Evidence for a differential distribution of mGluR1 splice variants

Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy‐termini have been described so far and named mGluR1α, mGluR1β and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1α isoform. In the present work, we examined the localization of mGluR1β in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1β immunostaining was compared with that obtained with antibodies specific for mGluR1α or with a pan‐mGluR1 antibody which recognizes all isoforms. mGluR1β‐like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1α and mGluR1β was observed. In the hippocampus, mGluR1α‐LI was restricted to non‐principal neurons in all fields, whereas mGluR1β‐LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1β, with a distribution pattern reflecting the patch‐matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1‐deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission. J. Comp. Neurol. 400:391–407, 1998.

Structure-Activity Studies on Neuropeptide S IDENTIFICATION OF THE AMINO ACID RESIDUES CRUCIAL FOR RECEPTOR ACTIVATION

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1–10); (ii) Phe2, Arg3, and Asn4 are crucial for biological activity; (iii) the sequence Thr8-Gly9-Met10 is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val6-Gly7 acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1–10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.

Altered Dimerization of Metabotropic Glutamate Receptor 3 in Schizophrenia

BACKGROUND Metabotropic glutamate receptors (mGlus) may be involved in the pathophysiology of schizophrenia. Group II mGlus (mGlu2 and mGlu3) have attracted considerable interest since the development of potent specific agonists that exhibit atypical antipsychotic-like activity and reports of a genetic association between the mGlu3 gene and schizophrenia. METHODS In this postmortem study, mGlu3 protein levels in Brodmann area 10 of prefrontal cortex from schizophrenic (n = 20) and control (n = 35) subjects were analyzed by western immunoblotting using a novel specific mGlu3 antibody and an antibody for the vesicular glutamate transporter 1 (VGluT1). RESULTS We report a significant decrease in the dimeric/oligomeric forms of mGlu3 in schizophrenic patients compared with control subjects, whereas total mGlu3 and VGluT1 levels were not altered significantly. CONCLUSIONS This is the first experimental evidence that mGlu3 receptor levels are altered in schizophrenia and supports the hypothesis that neurotransmission involving this particular excitatory amino acid receptor is impaired in schizophrenia.

Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2−/− and mGluR3−/− mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1‐3), as well as the remaining group II mGluR, in the hippocampus of mGluR2−/− and mGluR3−/− mice, compared with wild‐type controls. mGluR2 mRNA was increased in mGluR3−/− mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT‐1) protein, were reduced in mGluR3−/− mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2−/− mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2−/− and mGluR3−/− mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice. Synapse 62:842–850, 2008.

Altered levels of glutamatergic receptors and Na+/K+ ATPase-α1 in the prefrontal cortex of subjects with schizophrenia.

Evidence has accumulated over the past years that dysregulation of glutamatergic neurotransmission maybe implicated in the pathophysiology of schizophrenia. Glutamate acts on two major classes of receptors: ionotropic receptors, which are ligand-gated ion channels, and metabotropic receptors (mGluRs), coupled to heterotrimeric G-proteins. Although several pharmacological evidences point to abnormal glutamatergic transmission in schizophrenia, changes in the expression of glutamatergic receptors in the prefrontal cortex of patients with schizophrenia remains equivocal. In the present work, we have investigated glutamatergic neurotransmission in schizophrenia by assessing the expression in Brodmann Area 10 of mGluR5, the AMPA receptor subunits GluR1 and GluR2, and Na(+)/K(+) ATPase-α1, a potential modulator of glutamate uptake in the brain. Semiquantitative analysis of the expression of these proteins from postmortem brains revealed a particularly prominent reduction of GluR1 and GluR2 expression in patients with schizophrenia vs the control group. Conversely, we observed an up-regulation in the levels of Na(+)/K(+) ATPase-α1 expression. Finally, no change in the protein levels of mGluR5 was observed in schizophrenia. Our findings support and expand the hypothesis of glutamatergic dysfunction in prefrontal cortex in the pathophysiology of schizophrenia.