G. Casabona

Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders?

Metabotropic glutamate (mGlu) receptors are a large, heterogeneous family of G-protein coupled receptors, which modulate excitatory synaptic transmission through various transduction pathways. Evidence is now accumulating that individual mGlu-receptor subtypes mediate distinct, facilitatory (group I subtypes) or inhibitory (group II and group III subtypes), actions on neurodegenerative processes. Drugs interacting with mGlu receptors are expected to influence both the induction and progression of neuronal degeneration without hampering the efficiency of fast excitatory synaptic transmission. For these reasons, mGlu receptors can be considered as promising drug targets in the experimental therapy of acute or chronic neurodegenerative diseases.

Nootropic Drugs Positively Modulate α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-Sensitive Glutamate Receptors in Neuronal Cultures

Abstract: Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage‐sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor‐mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N‐methyl‐d‐aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for [3H]AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA‐sensitive glutamate receptors in neurons.

Growth Conditions Differentially Regulate the Expression of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate (AMPA) Receptor Subunits in Cultured Neurons

We have studied the expression of a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K± (10, 25, or 40 mM) with or without 100 μM N‐methyl‐D‐aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K± (K10) and increased dramatically in cultures grown in 25 mM K± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and ‐3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA‐stimulated 4SCa2± influx and 40‐[3H]phorbol 12, 13‐dibutyrate binding in intact cells. Both functional parameters increased along with the K± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.

The metabotropic glutamate receptor mGlu5 controls the onset of developmental apoptosis in cultured cerebellar neurons

Cultured cerebellar granule cells grown in medium containing 10 mm K+ undergo apoptosis after 4–5 days in vitro (DIV), and, at that time, the activity of metabotropic glutamate (mGlu) receptors coupled to polyphosphoinositide (PI) hydrolysis begins to decline. In granule cells at 4 DIV, the mGlu receptor subtype mGlu5 was expressed at high levels. The expression of another PI‐coupled mGlu receptor, the mGlu1a, was low at 4 DIV but increased during the following days. In cultures at 4–5 DIV, the few cells that already showed an apoptotic phenotype were devoid of mGlu5 receptors, but they all expressed mGlu1a receptors. The development of apoptosis was accelerated after treating the cultures with: (i) mGlu5 antisense oligonucleotides; (ii) the mixed mGlu receptor antagonist, (+)‐α‐methyl‐4‐carboxyphenylglycine; or (iii) the glutamate depleting enzyme, alanine aminotransferase. In contrast, an induced overexpression of mGlu5 receptors protected cultured granule cells against apoptotic death. We suggest that the activity of mGlu5 receptors supports cell survival, and a decline in the expression of mGlu5 receptors gives access to programmed cell death in cerebellar granule cells developing in primary cultures.

Developmental changes in the modulation of cyclic AMP formation by the metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid in brain slices

Abstract: Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide‐binding regulatory protein‐coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (1S, 1R‐ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin‐stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R‐ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N‐methyl‐d‐aspartate, kainate, and α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate and was antagonized by l‐2‐amino‐3‐phosphonopro‐pionate (l‐AP‐3). l‐AP‐3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R‐ACPD on forskolin‐stimulated cAMP formation. In hippocampal slices from 1‐, 8‐, or 15‐day‐old rats, 1S,3R‐ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar developmental pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R‐ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin‐stimulated cAMP formation by 1S,3R‐ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.

Metabotropic Glutamate Receptors and Neurodegeneration

Metabotropic glutamate (mGlu) receptors, which exert a modulatory effect on excitatory synaptic transmission, are considered as potential targets for neuroprotective drugs and the advent of potent and centrally available subtype-selective ligands has lead to an extensive investigation of the role of individual mGlu receptor subtypes in neurodegeneration. Pharmacological blockade of mGlu1 or -5 receptors or pharmacological activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of NMDA receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes, suggesting a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s disease (AD). MGlu2/3 receptor agonists inhibit glutamate release, and also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of eNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders.

Melittin enhances excitatory amino acid release and AMPA-stimulated 45Ca2+ influx in cultured neurons

Melittin, a potent activator of phospholipase A2, enhanced both spontaneous and depolarization-induced release of D-[3H]aspartate in primary cultures of cerebellar granule cells. The action of melittin was concentration-dependent (EC50 value = 300 ng/ml) and did not require the presence of extracellular Ca2+. Melittin also stimulated the release of glutamate and aspartate, in addition to other endogenous amino acids (taurine, alanine and gamma-aminobutyric acid). These effects were accompanied by an enhanced influx of 45Ca2+, which was in part mediated by the activation of excitatory amino acid receptors by endogenous agonists. Low concentrations of melittin (50 ng/ml) potentiated the efficacy of AMPA in stimulating 45Ca2+ influx without affecting stimulation by kainate or by glutamate added in the absence of extracellular Mg2+ (a condition that favors the activation of NMDA receptors). These results indicate that activation of phospholipase A2 evokes both an enhanced glutamate release and an increased sensitivity of AMPA receptors, two events that may support synaptic facilitation and LTP formation.

Chapter 14 Metabotropic glutamate receptors and neurodegeneration

Metabotropic glutamate (mGlu) receptors, which exert a modulatory effect on excitatory synaptic transmission, are considered as potential targets for neuroprotective drugs and the advent of potent and centrally available subtype-selective ligands has lead to an extensive investigation of the role of individual mGlu receptor subtypes in neurodegeneration . Pharmacological blockade of mGlu I or -5 receptors or pharmacologica l activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlul receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of NMDA receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes, suggesting a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimers disease (AD). MGlu2/3 receptor agonists inhibit glutamate release, and also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of eNS disorders . Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders.

Amyloid β protein does not interact with tachykinin receptors coupled to inositol phospholipid hydrolysis in human astrocytoma cells

We have tested the interaction between amyloid beta protein (A beta P) and tachykinin receptors in cultured UC-11MG astrocytoma cells, which express high affinity substance P receptors and respond to substance P with an unusually large stimulation of polyphosphoinositide hydrolysis. Both the full-length A beta P (A beta P1-40) and the fragment 25-35 (A beta P25-35) did not affect the stimulation of [3H]inositolmonophosphate (InsP) formation by substance P. A beta P25-35 was also inactive when applied to the cultures 18 or 72 h prior to the assay. In addition, A beta P25-35 did not displace specifically bound [3H]SarMet substance P from its recognition sites in intact UC-11MG cells. These results suggest that, at least in this specific cell type, amyloid peptides do not interact with substance P receptors.

Metabotropic Glutamate Receptors as a Drug Target in Brain Ischemia

This chapter examines whether metabotropic glutamate receptors can serve as drug target in brain ischemia. Glutamate that is released in large amounts in ischemic brain tissue activates both ionotropic receptors (NMDA, AMPA, or kainate receptors) and metabotropic receptors (mGluRs). The mGluRs form a family of eight subtypes (named mGluR1 to 8) that are tentatively classified into groups I (mGluR1 and 5), II (mGluR2 and 3), and III (mGluR4, 6, 8) on the basis of sequence homology and transduction pathways. Group I mGluRs facilitate the activation of NMDA receptors by either activating protein kinase C or producing membrane depolarization. A role for group I mGluRs in excitotoxicity have been revealed by using selective agonists for mGluR1/5. Groups II and III mGluRs are negatively coupled to either adenylyl cyclase or N-type voltage-sensitive Ca 2+ channels in heterologous expression systems. The chapter also discusses the Group II mGluR agonists that are promising drugs for the experimental therapy of stroke in which the treatment is usually initiated several hours after the onset of the ischemic insult. The results also indicate that mGluRs are promising “targets” for the development of novel neuroprotective agents which may satisfy some of the major criteria to be taken into consideration for clinical practice.