Motoki Tanaka

Selective blockade of astrocytic glutamate transporter GLT-1 with dihydrokainate prevents neuronal death during ouabain treatment of astrocyte/neuron cocultures

Glutamate (Glu) is a major excitatory neurotransmitter of the mammalian central nervous system and under normal conditions plays an important role in information processing in the brain. Therefore, extracellular Glu is subject to strong homeostasis. Astrocytes in the brain have been considered to be mainly responsible for the clearance of extracellular Glu. In this study, using mixed neuron/astrocyte cultures, we investigated whether astrocytic Glu transporter GLT‐1 is crucial to the survival of neurons under various conditions. Treatment of the mixed cultures with a low concentration of Glu did not produce significant death of neurons. However, cotreatment with dihydrokainate (DHK), a specific blocker of GLT‐1, resulted in significant neuronal death that was suppressed by an antagonist of N‐methyl‐D‐aspartate (NMDA) receptors. These results suggested that astrocytic GLT‐1 participated in the clearance of extracellular Glu and protected neurons from NMDA receptor‐mediated toxicity. When the cultures were treated with ouabain, an inhibitor of Na+/K+‐ATPase, a low concentration of Glu resulted in massive neuronal death that was also suppressed by cotreatment with an antagonist of NMDA receptors. In this case, however, cotreatment with DHK significantly protected neurons from death, suggesting that GLT‐1 was responsible for the death of neurons. The present study provides evidence suggesting that astrocytes use their Glu transporter GLT‐1 to protect neurons from Glu toxicity, but, ironically, also use GLT‐1 to kill neurons through Glu toxicity depending on their status. GLIA 40:337–349, 2002.

Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures

Sublethal ischemia leads to increased tolerance against subsequent prolonged cerebral ischemia in vivo. In the present study, we investigated the roles of the astrocytic glutamate (Glu) transporter GLT‐1 in preconditioning (PC)‐induced neuronal ischemic tolerance in cortical neuron/astrocyte co‐cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) increased the survival rate of neurons significantly when cultures were exposed to a lethal OGD 24 h later. The extracellular concentration of Glu increased significantly during PC, and treatment with an inhibitor of N‐methyl‐D‐actetate (NMDA) receptors significantly reversed the PC‐induced ischemic tolerance of neurons, suggesting that the increase in extracellular concentration of Glu during PC was critical to the development of PC‐induced neuronal ischemic tolerance via the activation of NMDA receptors. Treatment with a GLT‐1 blocker during PC suppressed this increase in Glu significantly, and antagonized the PC‐induced neuronal ischemic tolerance. This study suggested that the reversed operation of GLT‐1 was crucial to the development of neuronal ischemic tolerance.

Nitric Oxide Produced During Sublethal Ischemia Is Crucial for the Preconditioning-Induced Down-Regulation of Glutamate Transporter GLT-1 in Neuron/Astrocyte Co-Cultures

In the brain, prior sublethal ischemia (preconditioning, PC) produces tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating whether and how nitric oxide (NO) produced during PC is involved in the PC-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. The rise in the extracellular concentration of glutamate during ischemia caused by the reversed uptake of glutamate (Glu) by the astrocytic Glu transporter GLT-1 was markedly suppressed by the prior PC treatment, but the suppression was reversed by treatment with an inhibitor of nitric oxide synthase (NOS) during PC. Immunocytochemical and Western blot analyses demonstrated that the expression of GLT-1 was down-regulated after the PC insult, and this down-regulation was also antagonized by treatment with NOS inhibitors during PC. Here we show that nNOS-derived NO produced during PC was crucial for the down-regulation of astrocytic GLT-1, and this down-regulation coincided with an increased survival rate of neurons.

Nitric Oxide Produced During Ischemia Is Toxic but Crucial to Preconditioning-Induced Ischemic Tolerance of Neurons in Culture

The present study investigated the roles of nitric oxide (NO) in preconditioning (PC)-induced neuronal ischemic tolerance in cortical cultures. Ischemia in vitro was simulated by subjecting cultures to both oxygen and glucose deprivation (OGD). A sublethal OGD (PC) significantly increased the survival rate of neurons when cultures were exposed to a lethal OGD 24 h later. Both the inhibition of nitric oxide synthase (NOS) and scavenging of NO during PC significantly attenuated the PC-induced neuronal tolerance. In addition, exposure to an NO donor emulated the PC. In contrast, the inhibition of NOS and the scavenging of NO during lethal OGD tended to increase the survival rate of neurons. This study suggested that NO produced during ischemia was fundamentally toxic, but critical to the development of PC-induced neuronal tolerance.

Functional significance of the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures.

In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating what alterations were induced in neurons and/or astrocytes by PC treatment. The rise in the extracellular concentration of glutamate during ischemia was markedly suppressed by the prior PC treatment. Immunocytochemical and Western blot analyses demonstrated that the expression of the astrocytic glutamate transporter GLT-1 was transiently down-regulated after the PC insult. The PC insult possibly suppressed the neuron-derived factors up-regulating GLT-1. Here we show that PC-induced down-regulation of GLT-1 is crucial for the increased neuronal resistance to subsequent severe ischemic insult.

Changes in the Spontaneous Calcium Oscillations for the Development of the Preconditioning-Induced Ischemic Tolerance in Neuron/Astrocyte Co-culture

Spontaneous Ca2+ oscillations are believed to contribute to the regulation of gene expression. Here we investigated whether and how the dynamics of Ca2+ oscillations changed after sublethal preconditioning (PC) for PC-induced ischemic tolerance in neuron/astrocyte co-cultures. The frequency of spontaneous Ca2+ oscillations significantly decreased between 4 and 8xa0h after the end of PC in both neurons and astrocytes. Treatment with 2-APB, an inhibitor of IP3 receptors, decreased the oscillatory frequency, induced ischemic tolerance and a down-regulation of glutamate transporter GLT-1 contributing to the increase in the extracellular glutamate during ischemia. The expression of GLT-1 is known to be up-regulated by PACAP. Treatment with PACAP38 increased the oscillatory frequency, and antagonized both the PC-induced down-regulation of GLT-1 and ischemic tolerance. These results suggested that the PC suppressed the spontaneous Ca2+ oscillations regulating the gene expressions of various proteins, especially of astrocytic GLT-1, for the development of the PC-induced ischemic tolerance.

Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP‐induced morphologic changes

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N‐ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP‐treated and non‐treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP‐treated astrocytes after stimulation with ATP by high‐performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1‐43 imaging, appeared in cyclic AMP‐treated astrocytes in a punctiform fashion, but not in non‐treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP‐induced morphologic changes, and in particular, a v‐SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP‐treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP‐induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP‐treated astrocytes in response to ATP released from neighboring neurons and astrocytes.

Increased resistance to nitric oxide cytotoxicity associated with differentiation of neuroblastoma-glioma hybrid (NG108-15) cells.

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.

Neuron is the primary target of Ca2+ paradox-type insult-induced cell injury in neuron/astrocyte co-cultures

Ca(2+) paradox is the phenomenon whereby the intracellular concentration of Ca(2+) paradoxically increases during reperfusion with normal Ca(2+)-containing media after brief exposure to a Ca(2+)-free solution. The present study aims to characterize the Ca(2+) paradox induced cell injury in neuron/astrocyte co-cultures. Prior exposure of the co-cultures to a low Ca(2+) solution for 60 min significantly injured only neurons after reperfusion with a normal Ca(2+) medium for 24h, but astrocytes remained intact. An analysis of the Ca(2+) paradox-induced changes in the intracellular concentration of Na(+) revealed that the concentration in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic Na(+)-dependent glutamate transporter GLT-1. These results suggested that Ca(2+) paradox-induced accumulation of Na(+) in astrocytes was crucially involved in the excitotoxic neuronal injury resulting from the reversed astrocytic GLT-1 during the reperfusion episode. Previous studies have suggested that Ca(2+) paradox-induced injury in the brain occurs first in astroglial cells and only later in neurons resulting from the prior damage of astrocytes. Here we show that if Ca(2+) paradox occurs in the brain, neurons would be the primary target of Ca(2+) paradox-induced cell injury in the central nervous system.