Tatsuro Kosugi

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.

Reversed Actrocytic GLT-1 during Ischemia is Crucial to Excitotoxic Death of Neurons, but Contributes to the Survival of Astrocytes themselves

During ischemia, the operation of astrocytic/neuronal glutamate transporters is reversed and glutamate and Na+ are co-transported to the extracellular space. This study aims to investigate whether this reversed operation of glutamate transporters has any functional meanings for astrocytes themselves. Oxygen/glucose deprivation (OGD) of neuron/astrocyte co-cultures resulted in the massive death of neurons, and the cell death was significantly reduced by treatment with either AP5 or DHK. In cultured astrocytes with little GLT-1 expression, OGD produced Na+ overload, resulting in the reversal of astrocytic Na+/Ca2+-exchanger (NCX). The reversed NCX then caused Ca2+ overload leading to the damage of astrocytes. In contrast, the OGD-induced Na+ overload and astrocytic damage were significantly attenuated in PACAP-treated astrocytes with increased GLT-1 expression, and the attenuation was antagonized by treatment with DHK. These results suggested that the OGD-induced reversal of GLT-1 contributed to the survival of astrocytes themselves by releasing Na+ with glutamate via reversed GLT-1.

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.