Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage
Federico N. Soria, Alberto Pérez-Samartín, Abraham Martin, Kiran Babu Gona, Jordi Llop, Boguslaw Szczupak, Juan Carlos Chara, Carlos Matute, María Domercq
Federico N. Soria, Alberto Pérez-Samartín, Abraham Martin, Kiran Babu Gona, Jordi Llop, Boguslaw Szczupak, Juan Carlos Chara, Carlos Matute, María Domercq
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Research Article

Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage

Abstract

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.

Authors

Federico N. Soria, Alberto Pérez-Samartín, Abraham Martin, Kiran Babu Gona, Jordi Llop, Boguslaw Szczupak, Juan Carlos Chara, Carlos Matute, María Domercq

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Figure 7

Cystine/glutamate antiporter expression and function are increased in cortical neurons in vitro after chemical ischemia and in vivo after transient focal ischemia.

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Cystine/glutamate antiporter expression and function are increased in co...
(A) xCT mRNA levels in neurons exposed to chemical ischemia (1 hour), as measured by qPCR. Data are expressed as the mean ± SEM (n = 4). (B) xCT protein levels after chemical ischemia (1 hour) plus a 4-hour reoxygenation in cortical neurons. Data were normalized to actin and are expressed as the mean ± SEM (n = 4). Control and OGD lanes were run on the same gel but were noncontiguous. Full, uncut gels are shown in the Supplemental Material. (C) Cl–-dependent [14C] L-cystine uptake by neurons after 1 hour of chemical ischemia plus 4 hours of reoxygenation. Note that cystine/glutamate antagonist AAA (250 μM) almost completely inhibited uptake. Data were normalized to protein concentration and are expressed as the mean ± SEM (n = 3). (D) 18[F]FSPG uptake study by PET imaging in control rats and after MCAO (n = 5 per group). Representative images of control rats and images at 5 minutes and at 5 hours of reperfusion (RPF) following MCAO. (E) Histogram shows 18[F]FSPG signal expressed as the percentage of injected dose per gram (% ID/g) and normalized to the contralateral hemisphere in the total infarct area or in the volumes of interest (VOIs) defined in the striatum and cortex at 5 minutes or at 5 hours after reperfusion. Data are expressed as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control.