Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation

Abstract

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, l-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.

Introduction

Glutamate, the main excitatory neurotransmitter in the mammalian brain, exerts a dual role in the function of the central nervous system. While normal glutamatergic activity is crucial for the physiological excitatory synaptic transmission, glutamate mediated neuronal death is thought to contribute to all major neurodegenerative disorders. The pivotal point between beneficial or deleterious effects of glutamate may rely on extracellular concentration levels reached (Watkins and Jane, 2006). Therefore extracellular glutamate concentration is tightly regulated by high affinity glutamate transporters named excitatory amino acid transporters (EAATs) (Kanai and Hediger, 1992, Rothstein et al., 1994; for review see Danbolt, 2001). This function is primarily performed by the glial transporter proteins glutamate/aspartate transporter

(GLAST)(Storck et al., 1992, Tanaka, 1993) and glutamate transporter 1 (GLT-1) (Pines et al., 1992). These glial transporters remove glutamate from the synaptic cleft by a sodium/ATP-dependent mechanism (Logan and Snyder, 1972). A neuronal form of these transporters named excitatory amino acid carrier 1 (EAAC1) has been identified (Kanai and Hediger, 1992). However it has been suggested that EAAC1 exerts a preponderant role during the development of central nervous system while its impact on glutamate removal is limited in adults (Nieoullon et al., 2006). Failure of glutamate uptake has been described as a possible mechanism contributing to neuronal death observed in several types of acute injuries. For instance, dysfunction of astrocytic glutamate transporter (Rothstein et al., 1996) has been proposed as a possible mechanism responsible for the well characterized extracellular glutamate accumulation described following cerebral ischemia (Benveniste et al., 1984). Indeed, the deprivation in oxygen and energetic metabolites subsequent to cerebral blood flow interruption leads to a dramatic tissue energy depletion and ionic membranous electrochemical gradient disruption that directly affects astrocyte uptake capacity for glutamate (Madl and Burgesser, 1993). This point was strengthened by the study of Rossi et al. (2000) who showed a significant reduction of ischemic induced-extracellular glutamate efflux by the treatment with selective blockers of glutamate transporters. They suggested that glutamate uptake reversal was partly responsible for the ischemia induced-glutamate efflux. This observation was confirmed by the non vesicular origin of the ischemic induced-extracellular glutamate accumulation (Jabaudon et al., 2000). This elevation of extracellular glutamate concentration results in a sustained activation of glutamatergic receptors leading to neuronal derangements ranging from cell body swelling, dendritic alterations, mitochondrial failure and neuronal death. This “excessive” excitation leading to neuronal death has been named “excitotoxicity” (for review, see Olney, 1990). Excitotoxicity has been described as a mechanism leading to neuronal death observed in acute neuronal injury such as trauma and stroke but also in neurodegenerative diseases (for review see Dirnagl et al., 1999). Excitotoxicity occurs partly because of the overactivation of N-Methyl d-Aspartate receptors (NMDARs) that results in an excessive calcium (Ca²⁺) influx through the receptor's associated ion channel (Choi, 1988). This unique relationship between NMDAR activation and neuronal death is challenged by their parallel role in neuronal plasticity and neurotrophic processes (Bashir et al., 1991, Bliss and Collingridge, 1993, Nicoll and Malenka, 1999). A key determinant of this duality of NMDAR signalling may rely on the cellular location of the activated receptors. Indeed, while NMDARs are mainly found on the postsynaptic membrane at excitatory synapses (O'Brien et al., 1998, Scannevin and Huganir, 2000) they are not fixed at the synapse. It has been described that some synaptic NMDARs can move laterally to extrasynaptic sites (Rao and Craig, 1997, Tovar and Westbrook, 2002, Choquet and Triller, 2003). Recently, it has been observed that, depending on their subcellular localization, NMDAR activation induces Ca²⁺ pathways that could influence differently the neuronal fate suggesting a functional dichotomy between NMDARs located at the synapse (synaptic receptors) and outside the synapse (extrasynaptic receptors) (Hardingham et al., 2002, Hardingham and Bading, 2003, Zhang et al., 2007). Here, we reproduced the pathological conditions in which glutamate uptake is reversed to evaluate which population of NMDARs may be influenced. Glutamate uptake reversal can be mimicked pharmacologically by EAAT substrate inhibitors, such as l-trans-pyrrolidine-2,4-dicarboxylate (PDC). PDC has been described as a potent substrate inhibitor of glutamate transporters and leads to glutamate uptake reversal by triggering Ca²⁺ independent-glutamate release to the extracellular compartment (Volterra et al., 1996). By promoting glutamate efflux through glutamate transporters reversal, PDC reproduces one of the key early steps leading to neuronal damages during acute brain injury. In the present study, we analyzed the impact of PDC treatment on neuronal fate by determining its impact on synaptic and extrasynaptic NMDAR activation. These experiments were performed on murine primary cortical cultures, a cellular model widely used to study neurons/glia interaction (Volterra et al., 1996, Goldberg and Choi, 1993).