Elsevier

Neuropharmacology

Volume 41, Issue 4, September 2001, Pages 421-432
Neuropharmacology

Excitotoxic profiles of novel, low-affinity kainate receptor agonists in primary cultures of murine cerebellar granule cells

https://doi.org/10.1016/S0028-3908(01)00086-7Get rights and content

Abstract

The involvement of low-affinity kainate (KA) receptors in neuronal injury was investigated by employing a variety of agonists active at GluR5–7. Their excitotoxic profiles were determined in primary cultures of cerebellar granule cells, which abundantly expressed low-affinity KA receptors, and in the absence of any AMPA receptor-mediated neurotoxicity. Neurotoxicity induced by these compounds was analysed by phase contrast microscopy, a cell viability assay, the TUNEL technique (apoptosis), and by employing propidium iodide (PI; necrosis). All agonists induced concentration-dependent neurotoxicity, with rank order (EC50 values; μM): (S)-iodowillardiine (IW) 0.2>(2S,4R)-4-methylglutamate (4-MG) 36>(2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) 46>KA 74>(RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4yl)propanoic acid (ATPA) 88. IW exposure resulted in apoptosis at lower concentrations (<30 μM) and necrosis at higher concentrations, both of which were attenuated by CNQX (50 μM), but not MK-801 (10 μM). ATPA-mediated neurotoxicity was purely apoptotic and was attenuated by the non-NMDA receptor antagonists. Both IW and ATPA induced injury with the morphological characteristics of apoptosis shown by the presence of TUNEL-positive neurones. LY339434-mediated neuronal injury was only attenuated by MK-801 and was necrotic in nature. Similarly, 4-MG (>30 μM) exposure caused necrosis that was partially attenuated by MK-801 (10 μM) and CNQX (50 μM). The patterns of neurotoxicity possessed a complex pharmacological profile, demonstrated an apoptotic–necrotic continuum and were inconsistent with past findings, further outlining the importance of characterizing novel compounds at native receptors. ATPA and to a lesser extent IW appear to be suitable drugs for low-affinity KA receptors. Since toxicity-mediated by low-affinity KA receptors seem likely to contribute to neurodegenerative conditions, our study importantly examines the excitotoxic profile of these novel agonists.

Introduction

l-glutamate (Glu) receptor-mediated neurotoxicity, termed excitotoxicity, is well documented, and is likely to be involved in numerous neurological diseases (Lipton and Rosenberg, 1994, Leist and Nicotera, 1998). Glu receptor-mediated excitotoxicity appears to involve two forms of neuronal injury, necrosis and apoptosis (Ankarcrona et al., 1995, Cheung et al., 1998b). Necrosis is a passive process characterized by cellular swelling (Clarke, 1990), whereas apoptosis involves complex mechanisms requiring energy and protein synthesis (Dessi et al., 1994). Morphological changes induced by apoptosis are characterized by cell shrinkage, nuclear condensation, oligonucleosomal fragmentation of DNA and neurite degeneration (Clarke, 1990), and are mediated by various pathways involving caspases and various families of kinases (Sastry and Rao, 2000).

Glu acts at two types of receptors the G-protein coupled metabotropic receptors and the ion-channel gated ionotropic receptors. The ionotropic Glu receptors (iGluRs), which are named after their preferring agonists, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate (KA) (Hollmann and Heinemann, 1994), are heavily implicated in numerous neurodegenerative and acute neurological conditions (Lipton and Rosenberg, 1994, Leist and Nicotera, 1998). Cloning experiments have played an essential role in the understanding of the various Glu receptor subunits and their ion permeabilities, and have revealed a number of families of subunits consisting of GluR1-4 (AMPA), NR1 and NR2A-D (NMDA), KA1-2 (high affinity kainate receptors) and GluR5-7 (low-affinity kainate receptors) (Hollmann and Heinemann, 1994). Low-affinity KA receptors are widely distributed throughout the rodent and primate brain (Wisden and Seeburg, 1993, Carroll et al., 1998, Bernard et al., 1999) and have been associated with various disease states including Alzheimer's disease (Aronica et al., 1998), schizophrenia (Breese et al., 1995), amyotropic lateral sclerosis, cerebral ischaemia and epilepsy (Paschen et al., 1996, Bernard et al., 1999). Other evidence indicates that low-affinity KA receptors are likely to play significant physiological roles in synaptic transmission, with involvements in long term potentiation (Clarke et al., 1997, Vignes and Collingridge, 1997, Bortolotto et al., 1999) and pain processing (Procter et al., 1998). Recently novel Glu analogues have been developed and studies employing recombinant receptor model systems have demonstrated their selectivity for low-affinity KA receptors subunits (Jane et al., 1997, Zhou et al., 1997, Bleakman and Lodge, 1998). These compounds include ATPA, IW, LY339434 and 4-MG (also commonly termed SYM 2081) (Bleakman and Lodge, 1998).

Recombinant receptor studies have revealed some of the physiological and binding characteristics of these Glu analogues. Amino-3-(hydroxy-5-tert-butylisoxazol-4yl)propanoic acid (ATPA), an analogue of AMPA, is an effective AMPA agonist, with activity at GluR5 but not GluR6 (Clarke et al., 1997, Wahl et al., 1998), while IW, like other willardiines, has some AMPA activity (Patneau et al., 1992), but is also a potent GluR5 receptor agonist (Jane et al., 1997). Binding studies have revealed that 4-MG has higher affinity for GluR6 than NMDA or AMPA receptors and displaces [3H]KA binding more effectively than KA itself (Zhou et al., 1997). While ligand binding studies demonstrated that LY339434 is 1000-fold more selective for GluR5 than GluR6 (Small et al., 1998), functional studies have demonstrated activity at AMPA and NMDA receptors (Small et al., 1998, Moldrich et al., 2000b). Limited studies have been reported on the excitotoxic profiles of 4-MG, ATPA, IW and LY339434, with investigations conducted in primary cultures of cortical neurones demonstrating that these novel compounds exhibited complex excitotoxic profiles, involving actions not only at KA receptors, but also at AMPA and NMDA receptors (Small et al., 1998, Moldrich et al., 2000a, Moldrich et al., 2000b).

The current study examines these putative compounds in primary cultures of cerebellar granule cells (CGCs). CGCs are a particularly useful model system in many respects, as they are homogenous cell preparation containing >95% neurones when maintained under serum-free conditions (Cheung et al., 1998a) and at 8 d in vitro possess no functional AMPA receptors (Hack et al., 1995). CGCs cultured under the present conditions are presumed to be glutamatergic (Fonnum, 1984) since they actively accumulate d[3H]aspartate and not d[3H]GABA (Carroll et al., 1997). In addition, the current model system is particularly sensitive to KA receptor-mediated toxicity (Cheung et al., 1998a, Giardina et al., 1998). This study demonstrates for the first time the excitotoxic profiles of these novel compounds at native KA receptors, in an AMPA receptor-free environment, using primary cultures of murine CGCs.

Section snippets

Reagents

KA, 4-MG, ATPA, AMPA, NMDA and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Cookson (Bristol, UK). Neurobasal™ medium (NBM), B27 nutrients, N2 supplements and Ca2+-free-Hank's balanced salt solution (HBSS) were purchased from GibcoBRL Life Technologies (Melbourne, Australia). 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) were purchased from Sigma RBI

Characterization of receptor expression

Levels of KA2 receptor immunolabelling were much less abundant than that for GluR6/7, with few immunopositive cells present at div 1 (39% ±3.8; Fig. 1A) and a slight increase in expression was evident at div 8 (58% ±4.1; Fig. 1B). Immunocytochemical staining of the cultures revealed extensive expression of the GluR6/7 receptors from div 1 in CGCs (61% ±10; Fig. 1C). Expression of GluR6/7 remained high throughout the period examined up to div 12, (data not shown) and was expressed at high levels

Discussion

The current study is the first to analyse the excitotoxic profile of the putative low-affinity KA receptor agonists ATPA, IW, 4-MG and LY339434 in an essentially AMPA-receptor free environment. CGCs are a particularly useful model system for pharmacological evaluation of compounds as they are an essentially homogenous cell preparation under serum-free conditions, with a negligible glial cell population, and naturally express KA receptor subunits GluR5/6 and high affinity KA subunits in

Acknowledgments

Supported by the National Health and Medical Research Council (Australia), of which P.M. Beart is a Senior Principal Research Fellow, and by grants from the Ramaciotti, Rebecca Cooper and William Buckland Foundations, and Perpetual Trustees.

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