The distinct role of mGlu1 receptors in post-ischemic neuronal death

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The distinct role of mGlu1 receptors in post-ischemic neuronal death Domenico E. Pellegrini-Giampietro Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy

Metabotropic glutamate receptors of the mGlu1 and mGlu5 subtypes exhibit a high degree of sequence homology and are both coupled to phospholipase C and intracellular Ca21 mobilization. However, functional differences have been detected for these receptor subtypes when they are coexpressed in the same neuronal populations. Experimental evidence indicates that mGlu1 and mGlu5 receptors play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted studies that have provided a new viewpoint on the neuroprotective mechanism of mGlu1 receptor antagonists. The hypothesis predicts that these pharmacological agents attenuate post-ischemic injury by enhancing GABA-mediated neurotransmission. Glutamate exerts its modulatory effects on neuronal excitability and synaptic transmission by interacting with a family of G-protein-coupled receptors (GPCRs) termed metabotropic glutamate (mGlu) receptors [1,2]. Like all GPCRs, mGlu receptors have a seven-transmembrane domain (7TMD) region that appears to be structurally related to rhodopsin, although the sequence similarity with rhodopsin is very low. These receptors are, however, unique because unlike rhodopsin-like receptors their agonist binding site is not located in the 7TMD but instead is located in the large N-terminal extracellular domain (ECD), which is connected to the 7TMD by a cysteine-rich linker (Figure 1). Residues within intracellular loops 2 and 3 appear to be crucial for G-protein coupling selectivity and activation, respectively, whereas the C-terminal tail contains residues that are involved in the protein kinase C (PKC)-induced desensitization of the receptor and in the interaction with intracellular proteins such as calmodulin, Homer and PDZ domain-containing proteins. Eight mGlu receptor subtypes and multiple splice variants have been cloned to date from mammalian brain, and they have been subdivided into three groups on the basis of their structural homology, coupling mechanisms and agonist pharmacology [3,4] (Figure 1). Group I mGlu receptors include mGlu1 and mGlu5 receptors, are coupled to phospholipase C (PLC) and are Corresponding author: Domenico E. Pellegrini-Giampietro (domenico.pellegrini@ unifi.it).

selectively activated by (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG]. Group II (mGlu2 and mGlu3) and group III (mGlu4, mGlu6, mGlu7 and mGlu8) receptors are coupled via Gi/o proteins to adenylyl cyclase inhibition and can be pharmacologically distinguished because only members of group III are selectively stimulated by L -(þ)-2amino-4-phosphonobutyrate. Several splice variants of group I and III mGlu receptors have been identified: they differ mainly in their C-terminal domains and thus in their ability to interact with intracellular proteins [5]. Although members of each of the mGlu receptor groups can be located at presynaptic and postsynaptic sites in the brain, group I receptors have been shown to display a characteristic perisynaptic localization at the postsynaptic membrane of glutamatergic synapses [6], where they can regulate neuronal excitability by modulating a variety of Kþ channels and ionotropic glutamate (iGlu) receptors of the N-methyl-D -aspartic acid (NMDA) or a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtypes. Conversely, group II (which can also be postsynaptic) and group III mGlu receptors are expressed at presynaptic terminals, where they inhibit the release of glutamate and other neurotransmitters [7]. Some subtypes, and in particular mGlu3 and mGlu5 receptors, have also been detected in non-neuronal cells, including astrocytes and microglia. Their expression in these cells is dynamically regulated in response to brain stimulation and injury, which is suggestive of a mechanism for the modulation of glial function and glial – neuronal communication [8]. Following the initial observation that in native tissue mGlu5 receptors exist almost exclusively as homodimers stabilized by disulfide bonds, the concept has been extended to other mGlu subtypes and GPCRs (Figure 1). The mGlu1 receptor ECDs have been shown to form dimeric structures that are dramatically modified in their conformation following glutamate binding, thus indicating that the dimerization process might play a crucial role in mGlu receptor activation [9]. Evidence has accumulated in the past few years that mGlu receptors contribute to the neurotoxic effects of glutamate (also known as ‘excitotoxicity’) and are implicated in the mechanisms that lead to neurodegeneration in models of cerebral ischemia [8]. Because of their presynaptic inhibitory effects, group II and III mGlu receptor subtypes, which are negatively coupled to adenylyl cyclase, have also been proposed as potential targets for neuroprotective drugs in various disease models. However, mGlu1 and mGlu5 receptors, which are coupled to PLC

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(a)

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NH2

NH2 –S-S–

ECD

LB1 1

LB1

Glu

Glu

LB2

LB2

Cys-rich region 7TMD

1

2

3

4 α

Gq protein

AM

5 β

6

7

AM

1

2

γ

3 α

4 β

5

Receptor group

I

COOH

Receptor subtypes

Signal transduction

mGlu1α mGlu1β mGlu1d mGlu1e mGlu5a mGlu5b

7

γ

COOH

(b)

6

PtdIns(4,5)P2 Gq

PLC

+

DAG PKC + Ins(1,4,5)P3 Ca2+

mGlu2 II mGlu3 mGlu4a mGlu6

III

mGlu7a mGlu7b mGlu7c mGlu7d mGlu7e

Gi

AC cAMP

– ATP

mGlu8a mGlu8b TRENDS in Pharmacological Sciences

Fig. 1. Proposed secondary structure and classification of metabotropic glutamate (mGlu) receptors. (a) Schematic representation of a homodimeric group I mGlu receptor stabilized by a disulfide bond. Sharing structural similarities with bacterial perisplasmic binding proteins, the large N-terminal extracellular domain (ECD) of each protomer consists of two globular ligand binding (LB1 and LB2) lobes linked by a hinge region where the agonist glutamate (Glu) binds. Agonists are thought to stabilize an active closed conformation by making contacts with residues of both lobes, whereas competitive antagonists bind at the same site as agonists but are unable to stabilize the active conformation. The ECD is connected to the seven-transmembrane domain (7TMD) by a cysteine-rich region. Gq proteins are associated with intracellular loops 2 and 3, whereas allosteric modulators (AMs) interact with TMD 6 and 7 (the coexisting tridimensional interaction with TMD 3 is not shown). (b) The features of each mGlu receptor family group are shown. Greek letters are used for mGlu1a and mGlu1b splice variants, according to their original designation. Splice variants mGlu1c and mGlu4b are not included because data from different laboratories strongly suggest that they do not exist as full-length transcripts but probably represent recombination artifacts ([97] and F. Ferraguti, pers. commun.). Group I mGlu receptors are coupled to phospholipase C (PLC) via Gq proteins: their stimulation promotes the breakdown of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] into inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG). Ins(1,4,5)P3 releases Ca2þ from intracellular stores, whereas DAG activates protein kinase C (PKC). Group II and III mGlu receptors are negatively coupled via Gi/o proteins to adenylyl cyclase (AC): their stimulation inhibits the formation of cAMP from its precursor ATP. http://tips.trends.com

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activation and are associated with several potentially detrimental events, have received particular attention for their possible role in post-ischemic neuronal death [10,11]. The results of these studies indicate that group I mGlu receptor subtypes play a different role in models of cerebral ischemia and that mGlu1 rather than mGlu5 receptors are implicated in the pathways leading to post-ischemic neuronal injury. Group I mGlu receptor subtypes are attractive targets for anti-ischemic drugs According to the World Health Organization, stroke is the second leading cause of death and the most common cause of disability in the world. Unfortunately, clinical trials of neuroprotective drugs for the treatment of stroke have been generally unsuccessful so far. Therefore, together with fine-tuning of the design of these trials and of the development of more appropriate experimental animal models, drugs that possess a better therapeutic index and are aimed at alternative targets in the excitotoxic cascade that is responsible for neuronal death are required for the development of new and effective neuroprotective therapies for cerebral ischemia. Group I mGlu receptor antagonists represent a valid alternative as anti-ischemic drugs. In addition to the classical activation of PLC and release of Ca2þ from intracellular stores, stimulation of mGlu1 and mGlu5 receptors can trigger a variety of signaling cascades and modulate the activity of ion and ligand-gated channels through functional coupling with other transduction pathways such as adenylyl cyclase, phospholipase A2, phospholipase D, tyrosine kinase and mitogen-activated protein kinase [12,13]. Activation of mGlu1 and mGlu5 receptors might thus promote multiple processes that are known to participate in the pathological cascade leading to post-ischemic neuronal death, including: (i) an increase in neuronal excitability caused by the activation of inward cationic currents or the reduction of Kþ conductances; (ii) a rise in cytosolic free Ca2þ via a facilitatory coupling between ryanodine receptors and L-type Ca2þ channels or direct Ca2þ influx from the extracellular space through NMDA receptors and L-type channels [14]; (iii) an enhancement of the release of glutamate that correlates with the neurotoxic effects of group I mGlu receptor agonists [15]; (iv) a potentiation of NMDA and AMPA receptor responses that has been observed in a large number of brain areas; and (v) activation of the mitogenactivated protein kinase pathway via PKC [16]. mGlu1 but not mGlu5 receptor antagonists are neuroprotective in models of cerebral ischemia The assumption that pharmacological stimulation of group I mGlu receptors invariably leads to neurotoxic effects is too simplistic [11]. The poor selectivity of ligands (Table 1), the possible coupling of these receptors to alternative signaling pathways, and the observed neuroprotective effects of elevated cytosolic free Ca2þ [17] and PKC activation [18] under certain conditions are all factors that could explain the conflicting results observed with mGlu1 and/or mGlu5 receptor agonists. It has been proposed that the capability of group I mGlu receptors to http://tips.trends.com

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elicit neuroprotection rather than toxicity might depend on the expression of the NR2C subunit of NMDA receptors [19]. Activation of group I mGlu receptors in cerebellar granule cells attenuates the rise in intracellular Ca2þ and neuronal death driven by NMDA receptors, but when the expression of NR2C subunits is knocked down by antisense oligonucleotides the stimulation of both group I mGlu receptors and PKC enhances the effects of NMDA. Interestingly, the expression of the NR2C subunit is highest in the cerebellum and barely detectable in the hippocampus, striatum and neocortex [20], the regions that are most sensitive to ischemic damage. Another possible explanation has been suggested following studies in mixed cortical cells: two consecutive applications of the group I receptor agonist (S)-3,5-DHPG have been shown to induce a PKC-dependent functional switch of the receptors from a ‘neurotoxic’ to a ‘neuroprotective’ mode [21]. Contrary to the experimental observations with agonists, the use of mGlu1 receptor antagonists (Table 1) has consistently generated neuroprotective results, indicating that the endogenous activation of this receptor subtype is likely to play a major role in neurodegeneration. In the past few years, competitive and noncompetitive antagonists displaying increasing degrees of selectivity for mGlu1 receptors have been shown to reduce NMDA receptormediated neurotoxicity in vitro and in vivo [15,22 – 24] in addition to neuronal injury in a variety of models of cerebral ischemia (Table 2). Neuroprotection in cultured neocortical cells and in hippocampal slices exposed to oxygen– glucose deprivation (OGD) has been observed following the application of both mGlu1 receptor-preferring antagonists that are known to interact with other mGlu receptor subtypes, such as (S)-3-carboxy-4-hydroxyphenylglycine [(S)-4C3HPG], 1-aminoindan-1,5-dicarboxylic acid (AIDA) or (S)-(þ)-2-(30 -carboxybicyclo[1.1.1]pentyl)-glycine [(S)-CBPG] [25 –27], and more selective and potent compounds such as LY367385 (see Chemical names), 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) or 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) [28,29]. In these in vitro models, the neuroprotective effects are evident even when Chemical names BAY367620: (3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on LY344545: (2S)-2-amino-2-((1R,2R)-2-carboxycycloprop-1-yl)3-(9-xanthyl)propanoic acid LY367366: (RS)-a-thioxanthylmethyl-4-carboxyphenylglycine LY367385: (S)-2-methyl-4-carboxyphenylglycine LY393675: (S)-cis-a-thioxanthylmethyl-3-carboxycyclobutylglycine Ro016128: diphenylacetyl-carbamic acid ethyl ester Ro674853: 9H-xanthene-9-carbonyl-carbamic acid butyl ester Ro677476: (S)-2-(4-fluoro-phenyl)-1-(toluene-4-sulfonyl)-pyrrolidone SIB1757: 6-methyl-2-(phenylazo)pyridin-3-ol SIB1893: (E)-2-methyl-6-styrylpyridine SKF89976A: 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid

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Table 1. Pharmacological tools to explore the function of group I mGlu receptorsa Ligand Agonists Nonselective L -Glutamate L -Quisqualate (1S-3R)-ACPD (S)-3HPG (S)-3,5-DHPG mGlu1-selective Ro016128b Ro677476b Ro674853b mGlu5-selective (RS)-CHPG (Z)-CBQA Antagonists Nonselective (S)-MCPG 4-BuHIB LY367366b LY393675b mGlu1-selective (S)-4C3HPG (S)-4CPG AIDA (S)-Homoquisqualate (S)-ACUDA (S)-TBPG (S)-CBPG LY367385b 3-MATIDA CPCCOEt BAY367620b EM-TBPC Dicarboxy-pyrrole n. 3 mGlu5-selective LY344545b SIB1757b SIB1893b MPEP MTEP

mGlu1

mGlu5

Notes

Refse

EC50 (mM) 1 – 184 0.03 –17 5 – 214 18 –600 6 – 60

1 –11 0.05 –0.3 5 –122 14 –35 0.7 –20

Endogenous, mGlu and iGlu receptor agonist AMPA receptor agonist Group II and III mGlu receptor agonist Weak group II mGlu receptor agonist –

0.21 0.17 0.07

. 10 .1 10

Positive allosteric modulatorc Positive allosteric modulatorc Positive allosteric modulatorc

. 10000 . 1000 IC50

750 11 (mM)

[90] [90] [90]

– –

40 –300 110 6.6 0.35

100 –500 97 5.6 0.48

Group II mGlu receptor antagonist – – –

11 –393 4 – 163 3 – 214 184 76 –232 69 25 –65 1.4 –12 6.3 7 – 23 0.16 0.015 –0.13 0.016

Partial agonist? Partial agonist? . 100 Agonist . 1000 . 1000 Partial agonist . 100 . 300 . 100 . 100 .1 . 16

mGlu2 receptor agonist Weak mGlu2 receptor agonist Weak mGlu2 receptor agonist – – – – – – Noncompetitive Noncompetitive, inverse agonistd Noncompetitive Noncompetitive

39.5 . 100 . 100 . 100 NP

5.5 0.37 0.29 0.032 0.005

Group II mGlu receptor antagonist Noncompetitive Noncompetitive Noncompetitive, inverse agonistd Noncompetitive, inverse agonistd

[91]

[29] [92] [93] [94] [95]

[96]

Abbreviations: (1S-3R)-ACPD, (1S-3R)-1-aminocyclopentane-1,3-dicarboxylic acid; (S)-ACUDA, (S)-2-(40 -carboxycubyl)glycine; (S)-AIDA, 1-aminoindan-1,5-dicarboxylic acid; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; 4-BuHIB, (S)-4-n-butylhomoibotenic acid; (S)-CBPG, (S)-(þ)-2-(30 -carboxybicyclo[1.1.1]pentyl)-glycine; (Z)-CBQA, 1-amino-3-[20 -(30 ,50 -dioxo-10 ,20 ,40 -oxadiazolidinyl)]cyclobutane-1-carboxylic acid; (S)-4C3HPG, (S)-3-carboxy-4-hydroxyphenylglycine; (RS)-CHPG, (RS)-2-chloro5-hydroxyphenylglycine; CPCCOEt, 7-hydroxyiminocyclopropan[b ]chromen-1a-carboxylic acid ethyl ester; 4CPG, (S)-4-carboxyphenylglycine; (S)-3,5-DHPG, (S)-3,5dihydroxyphenylglycine; dicarboxy-pyrrole n. 3, 3,5-dimethyl-pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester; EM-TBPC, 1-ethyl-2-methyl-6-oxo-4(1,2,4,5-tetrahydro-benzo[d ]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile; (S)-3HPG, (S)-3-hydroxyphenylglycine; iGlu receptor, ionotropic glutamate receptor; 3-MATIDA, 3-methyl-aminothiophene dicarboxylic acid; (S)-MCPG, (S)-a-methyl-4-carboxyphenylglycine; mGlu receptor, metabotropic glutamate receptor; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine; NP, not published; (S)-TBPG, 2-(30 -(1H-tetrazol-5-yl)bicyclo[1.1.1]pentyl-1-yl)-glycine. b See Chemical names. c Positive allosteric modulators have no effect per se but potentiate the action of agonists by increasing their affinity, potency and efficacy. d Inverse agonists are compounds that inhibit agonist-independent constitutive activity of mGlu receptors in hetereologous cells. e Except where noted, data are from [4] and references therein. a

mGlu1 receptor antagonists are added to the incubation medium up to 60 min after OGD [29,30]. In addition, different laboratories have reported CA1 pyramidal cell neuroprotection using (S)-4C3HPG [31], LY367385 [22] and AIDA [26] in the transient two-vessel occlusion (2VO) gerbil model of global ischemia. Despite the initial observation that the targeted disruption of the gene encoding the mGlu1a receptor does not affect the extent of neuronal injury following permanent middle cerebral artery occlusion (MCAO) in mice [32], possibly as a result of compensatory mechanisms provided by other splice variants of mGlu1 receptors or perhaps mGlu5 receptors, more recent studies in the rat using selective and, in some cases, systemically active competitive and noncompetitive http://tips.trends.com

antagonists have shown that activation of mGlu1 receptors might also contribute to brain damage in models of focal ischemia [29,33,34]. Although mGlu5 receptors are physically and functionally associated with NMDA receptors [35,36] and their blockade has been demonstrated to be protective against NMDA- or b-amyloid-induced neurotoxicity [23,37], the results obtained using selective mGlu5 receptor antagonists in models of cerebral ischemia are scarce and not particularly encouraging (Table 2). Muralikrishna Rao and co-workers first demonstrated that the mGlu5 receptor-selective noncompetitive antagonist 2-methyl-6(phenylethynyl)-pyridine (MPEP) was more effective than AIDA in reducing CA1 pyramidal cell loss following

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Table 2. Effects of group I mGlu receptor antagonists in models of cerebral ischemia in vitro and in vivoa Drug mGlu1 receptor antagonists Oxygen –glucose deprivation (S)-4C3HPG AIDA, (S)-CBPG AIDA, (S)-CBPG (S)-4CPG LY367385b LY367385b CPCCOEt CPCCOEt 3-MATIDA 3-MATIDA Transient global ischemia (S)-4C3HPG LY367385b AIDA (S)-CBPG Focal ischemia (S)-4C3HPG BAY367620b LY367385b 3-MATIDA

Model

Effect

Refs

þ þ þ þ þ þ þ 2 þ þ

[25] [26 –28,30,60] [26,28,30] [27,60] [28,29] [28,29] [28] [28] [29] [29,57]

Gerbil 2VO Gerbil 2VO Gerbil 2VO Gerbil 2VO

þ þ þ þ

[31] [22] [26,28,30,57] [26,28,30,57]

pMCAO pMCAO pMCAO pMCAO

þ ^ þ þ

[33] [34] [29] [29]

Cortical cells Cortical cells Hippocampal Cortical cells Cortical cells Hippocampal Cortical cells Hippocampal Cortical cells Hippocampal

slices

slices slices slices

Drug

Model

Effect

Refs

mGlu5 receptor antagonists Oxygen –glucose deprivation MPEP MPEP

Cortical cells Hippocampal slices

2 2

[28] [28]

Transient global ischemia MPEP MPEP

Gerbil 2VO Gerbil 2VO

þ 2

[38] [28]

Focal ischemia MPEP MPEP

tMCAO pMCAO

þc 2

[39] [2]

Abbreviations: AIDA, 1-aminoindan-1,5-dicarboxylic acid; (S)-CBPG, (S)-(þ)-2-(30 -carboxybicyclo[1.1.1]pentyl)-glycine; (S)-4C3HPG, (S)-3-carboxy-4-hydroxyphenylglycine; CPCCOEt, 7-hydroxyiminocyclopropan[b ]chromen-1a-carboxylic acid ethyl ester; (S)-4CPG, (S)-4-carboxyphenylglycine; 3-MATIDA, 3-methyl-aminothiophene dicarboxylic acid; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; pMCAO, permanent middle cerebral artery occlusion; tMCAO, transient middle cerebral artery occlusion; 2VO, two-vessel occlusion. Symbols: þ , neuroprotective;-, not neuroprotective; ^ , trend for a neuroprotective effect. b See Chemical names. c MPEP was protective only at a high dose, presumably via NMDA receptors. a

transient global ischemia in the gerbil [38], but this result could not be confirmed in a subsequent study, in which both the same (intraperitoneal) and a different (intracerebroventricular) route of administration were used [28]. Similarly, MPEP was not neuroprotective following OGD in vitro [28] (Figure 2) nor after permanent MCAO in the rat [2]. Moreover, incubation of cultured cortical cells and hippocampal slices with the nonselective group I mGlu receptor agonist (S)-3,5-DHPG but not with the selective mGlu5 receptor agonist (RS)-2-chloro-5-hydroxyphenylglycine [(S)-CHPG] significantly enhanced the extent of neuronal injury induced by OGD [28]. In rats subjected to transient focal ischemia, MPEP was able to reduce the size of the ischemic infarct when administered intracerebroventricularly at a high dose, but the protective effect was ascribed to a noncompetitive antagonism of NMDA receptors, rather than to an interaction with mGlu5 receptors [39]. Indeed, MPEP significantly reduces steady state NMDA-evoked whole-cell currents at high concentrations (20 –200 mM, 500– 5 000-fold above its IC50 value) that are neuroprotective against excitotoxicity and posttraumatic injury in cultured cortical neurons [40,41]. A differential pathophysiological role for mGlu1 and mGlu5 receptors in the CNS? Although mGlu1 and mGlu5 receptors are highly homologous and are both coupled to Gq proteins and phosphoinositide hydrolysis, they appear to serve different roles in the CNS [13]. Functional differences for mGlu1 and mGlu5 receptors are suggested by their distinct anatomical distribution but have also been detected when they are coexpressed in the same cells. For example, activation of mGlu1 receptors in transfected cells leads to a peaked rise in intracellular Ca2þ, whereas activation of mGlu5 http://tips.trends.com

receptors generates an oscillatory Ca2þ response, which is due to the presence of a threonine residue that is phosphorylated by PKC in the proximal region of the mGlu5 receptor C-terminal tail [42]. In hippocampal CA1 pyramidal cells, mGlu1 receptors mediate the depolarization and intracellular Ca2þ release evoked by the broad

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Fig. 2. CA1 pyramidal cell death induced by oxygen– glucose deprivation (OGD) in cultured hippocampal slices is attenuated by the metabotropic glutamate 1 (mGlu1) receptor antagonist 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) but not by the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Cultured slices were exposed to OGD for 30 min, incubated with propidium iodide (5 mg ml21) for 24 h and then photographed under fluorescence optics. The vertical scale shows different colors used to denote fluorescence intensity, in arbitrary units. Drugs were present in the medium during OGD and the subsequent 24 h recovery period. Scale bar ¼ 1.8 mm. Modified, with permission, from [28,29].

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agonist the (S)-3,5-DHPG, whereas mGlu5 receptors are responsible for the (S)-3,5-DHPG-induced suppression of Ca2þ-activated Kþ currents and potentiation of NMDA receptor activation [43]. Despite a high level of mGlu1 and mGlu5 receptor coexpression, depolarization is evoked only by selective activation of mGlu5 receptors in the subthalamic nucleus [44] and of mGlu1 receptors in the substantia nigra pars reticulata [45] and in type II globus pallidus interneurons [46]. In striatal medium spiny neurons, induction of corticostriatal long-term depression requires the activation of mGlu1 but not mGlu5 receptors [47], whereas potentiation of NMDA receptor responses is mediated exclusively by mGlu5 receptors [48]. In cultured cortical neurons, stimulation of mGlu1 but not mGlu5 receptors induces potentiation of NMDA receptor currents through a Ca2þ – calmodulin-dependent and PKCindependent Pyk2/Src-family kinase pathway [49]. The specificity of mGlu1 and mGlu5 receptor functions in these neurons can also be explained by their diverse coupling to other downstream effectors or scaffolds. Recently, the activity of group I mGlu receptors has been shown to be tightly controlled by dynamic regulatory mechanisms such as the phosphorylation of specific mGlu1 or mGlu5 receptor domains by PKC and G-protein-coupled receptor kinases (GRKs), their interaction with regulators of G-protein signaling (RGS) proteins or their modulation by Homer and related proteins [13]. A distinct role for mGlu1 or mGlu5 receptors has been recognized not only in post-ischemic neuronal death (Table 2) but also in other experimental conditions that mimic human brain disease. For example, the use of antisense oligodeoxynucleotides directed to mGlu1 but not to mGlu5 receptors is neuroprotective in cortical neuronal cultures subjected to mechanical injury or following brain trauma in vivo [50]. In addition, selective mGlu1 receptor antagonists such as AIDA, CPCCOEt and LY367385 reduce traumatic neuronal injury in the same models [51,52], whereas the selective mGlu5 receptor antagonists MPEP and SIB1893 are neuroprotective only at high doses that antagonize NMDA receptors [41]. Studies with selective antagonists have established that mGlu1 and mGlu5 receptors play a complementary pathophysiological role in determining the histological and behavioral outcome following in vitro and in vivo spinal cord injury [53,54]. Although mGlu5 receptor antagonists suppress clonic seizures in vivo [55], a preferential role for mGlu1 receptors has also been shown in sustaining the prolongation of epileptiform bursts in hippocampal slices [56] and in cortical wedges [57] in vitro. By contrast, mGlu5 rather than mGlu1 receptor blockade appears to be beneficial in attenuating neurotoxicity induced by the active fragment of b-amyloid peptide [37,58] or methamphetamine [59]. Taken together, these experimental data show compelling evidence for the involvement of mGlu1 receptors in neurodegeneration but do not point to mGlu5 receptors as potentially important targets for neuroprotection against cerebral ischemia or traumatic injury. Differences in the release of arachidonic acid [38,60], the formation of cAMP [61] or the subtype of PKC [54] that is activated following activation of mGlu1 or mGlu5 receptors are among the possible explanations for http://tips.trends.com

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these findings. Also, the contribution of glial cells that express either mGlu1 or mGlu5 receptors [53,62] might account for the discrepant results obtained with antagonists in certain experimental models. Because activation of mGlu5 receptors supports neuronal survival during early development [63] and attenuates apoptosis in vitro [27], mGlu5 receptor antagonists might exacerbate apoptotic neuronal death following cerebral ischemia. On the contrary, mGlu5 receptors are likely to play a fundamental role in other CNS disorders, particularly in pain, anxiety, drug dependence and Parkinson’s disease [2,64]. Mechanisms of post-ischemic neuroprotection by mGlu1 receptor antagonists As discussed, activation of mGlu1 receptors might exacerbate post-ischemic neuronal injury through multiple noxious mechanisms, including an increase in intracellular free Ca2þ or the potentiation of NMDA receptor responses. However, the peculiar localization of this receptor subtype has prompted numerous studies in the past few years that have provided a new viewpoint on the neuroprotective mechanisms of mGlu1 receptor antagonists. In the CA1 hippocampal sub-region, the mGlu1a receptor splice variant is not expressed in vulnerable pyramidal cells, but is enriched in somatostatin-positive GABA-containing interneurons of the stratum oriensalveus [65], which appear to be resistant to global ischemia [66]. In a very recent study, mGlu1a receptor immunoreactivity was also identified in other types of interneurons that are located in various CA1 strata and target different pyramidal cell dendritic domains [67]. Similarly, mGlu1a receptors are expressed by GABA-containing interneurons in the neocortex [68], the striatum [69] and the substantia nigra pars reticulata [70]. These anatomical observations suggest that neuroprotection by mGlu1 receptor antagonists might be induced via changes in GABA-mediated transmission. Functional studies have shown that activation of mGlu1 receptors depresses inhibitory transmission in these brain areas [23,70,71] and that potentiation of GABA-mediated transmission is neuroprotective against post-ischemic injury [72]. The hypothesis that mGlu1 receptor antagonists attenuate post-ischemic injury by enhancing GABAmediated neurotransmission was first proposed by studies showing that perfusion with neuroprotective doses of AIDA enhanced the concentrations of GABA in the hippocampal dialysate of gerbils subjected to global ischemia [30]. In hippocampal slices exposed to OGD, the attenuation of CA1 damage observed with another mGlu1 receptor antagonist, 3-MATIDA, was mimicked by GABAA and GABAB receptor agonists and reduced by GABA receptor antagonists [57]. Likewise, LY367385 and CPCCOEt produced an increase in the extracellular levels of GABA in the corpus striatum of freely moving rats that was associated with a reduction in NMDA-mediated neurotoxicity [23]. In cultured neuronal cells exposed to NMDA, the neuroprotective effects of mGlu1 receptor antagonists were occluded by the previous application of GABA and SKF89976A (a GABA transporter inhibitor) and prevented by GABAA and GABAB receptor antagonists [23]. It is interesting to note that the reduction of

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spontaneous epileptiform activity induced by 3-MATIDA was similarly reproduced by a GABA receptor agonist and reverted by an antagonist in mouse cortical wedges [57]. Taken together, these data suggest that a common GABA-mediated mechanism, which involves the release of GABA and stimulation of GABA receptors, might contribute to the neuroprotective effects of mGlu1 receptor antagonists (Figure 3). This hypothesis implies that mGlu1 receptors are located presynaptically and that their activation inhibits the release of GABA. Although most immunocytochemical studies have failed to detect a presynaptic localization of mGlu1 receptors, two recent reports provide electron microscopy evidence for mGlu1a receptors in GABAergic terminals that appear to be responsible for a decrease in

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inhibitory transmission in the substantia nigra [70,73]. Functional data support the existence of presynaptic mGlu1 receptors modulating neurotransmitter release in the hippocampus [74] and neocortex [75]. Their stimulation could lead to an inhibitory effect by suppressing Ca2þ currents through N- or P/Q-type channels, as observed in cortical neurons [76] and in heterologous systems [77], or by activation of a Ca2þ-dependent Kþ conductance [78]. In opposition to this inhibitory hypothesis, however, it has been reported that activation of mGlu1 receptors results in increased spike frequency and rhythmic firing activity in GABA-containing interneurons [79]. It is also possible that a presynaptic decrease in inhibitory transmission can be indirectly mediated by mGlu1 receptors located postsynaptically in principal

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Fig. 3. Schematic model providing a possible explanation for the neuroprotective effects of metabotropic glutamate 1 (mGlu1) receptor antagonists. Excessive activation of postsynaptic AMPA and NMDA receptors by glutamate (Glu) produces a sustained depolarizing influx of Naþ and Ca2þ in principal neurons, which eventually leads to neurodegeneration. Conversely, activation of postsynaptic GABAA receptors produces an influx of Cl2, hyperpolarization and neuroprotection. GABA can also interact with presynaptic GABAB receptors that negatively control the release of glutamate from afferent terminals, thus leading to reduced excitation of postsynaptic neurons. The release of GABA from interneuron terminals is negatively controlled by mGlu1 receptors and cannabinoid CB1 receptors, via suppression of Ca2þ currents through N-type channels or activation of Kþ channels. Antagonists of mGlu1 receptors can lead to increased release of GABA, and therefore to neuroprotective hyperpolarization of principal neurons, by means of two mechanisms: direct blockade of presynaptic mGlu1 receptors or indirect inhibition of CB1 receptors located on GABAergic terminals. The latter mechanism is promoted by mGlu1 receptors located postsynaptically in principal neurons. Their activation leads to the formation of diacylglycerol (DAG), which can be converted to the cannabinoid 2-arachidonyl glycerol (2-AG) through the action of DAG lipase. 2-AG can diffuse through the membrane and act as a retrograde transmitter to activate CB1 receptors in presynaptic terminals. Abbreviations: PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate. http://tips.trends.com

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neurons. Functional evidence exists for the presence of mGlu1 receptors in CA1 pyramidal cells [43] and, albeit not abundantly in the CA1 region, mGlu1b and mGlu1d splice variants are expressed in hippocampal principal neurons [80,81]. Activation of mGlu1 postsynaptic receptors is known to promote the release of endogenous cannabinoids from CA1 pyramidal cells [82] and cerebellar Purkinje cells [83]. Endocannabinoids could then act as retrograde transmitters to suppress the release of GABA following activation of cannabinoid CB1 receptors located on the presynaptic terminals of interneurons [84 – 87] (Figure 3). Concluding remarks Experimental evidence suggests that selective antagonism of mGlu1 receptors attenuates post-ischemic neuronal injury by enhancing GABA-mediated neurotransmission. This neuroprotective mechanism is unique, in that is not shared by the mGlu5 receptor, which is also coupled to phoshoinositide hydrolysis and Ca2þ mobilization, and resembles the ‘metabotropic’ mode of action of presynaptic kainate receptors [88]. Interestingly, mGlu1 receptor antagonists appear to act by increasing the release of GABA in other models of neurotoxicity, suggesting a more general applicability of this mechanism to other disorders involving glutamate-induced neuronal death. Although antibodies against mGlu1 receptors have been detected in two patients with severe ataxia [89], antagonists of group I mGlu receptors are expected to modulate neuronal excitability and modify the induction and progression of post-ischemic neuronal death without interfering with fast excitatory synaptic transmission, a deleterious mechanism that presumably accounts for most of the clinical sideeffects (e.g. sedation, ataxia and memory loss) that can be observed following the use of NMDA or AMPA receptor antagonists [17]. In addition, the knowledge of the detrimental mechanisms triggered by mGlu1 receptors can be helpful in designing new therapeutic approaches with drugs (including GABA-related drugs or cannabinoids) that intervene along the cascade of events that lead to neuronal death.

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Acknowledgements I wish to thank Flavio Moroni, Guido Mannaioni and Alberto Chiarugi for critical discussions and Elena Meli and Andrea Cozzi for contributing ideas and for coordinating the experiments performed in our laboratory. I am grateful to Francesco Ferraguti for stimulating discussions and for permission to quote unpublished data.

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