Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo Kinga Szydlowska, Malgorzata Zawadzka and Bozena Kaminska Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warsaw, Poland Abstract Neuron–astrocyte interactions are critical for signalling, en- ergy metabolism, extracellular ion and glutamate home- ostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glu- tamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic dis- eases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleot- idyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal micro- scopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cor- tical astrocytes to 50–100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissi- pation of mitochondrial transmembrane potential (DY) and caspase activation. FK506 (1 lM) efficiently inhibits Glu-in- duced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DY. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia. Keywords: apoptosis, astrocyte death, FK506, glutamate, ischaemia, mitochondria. J. Neurochem. (2006) 99, 965–975. Astrocytes provide metabolic support to neurons, produce many trophic factors, and participate in detection, propaga- tion and modulation of excitatory synaptic signals (Chen and Swanson 2003; Privat 2003). Brain damage in ischaemia comprises distinct pathological events, including excitotox- icity, neuronal death, activation of microglia, and reactive gliosis (Dirnagl et al. 1999). Massive release of glutamate (Glu), leading to excitotoxicity, plays a major role in neuronal death. Glutamate uptake by astrocytes may prevent excitotoxic Glu elevations and support neuronal survival in the ischaemic penumbra. Astrocytes might have important tissue-protective functions after CNS injury and ablation of glial scar-forming astrocytes in transgenic animals worsens neuronal injury (Bush et al. 1999; Faulkner et al. 2004). However, some studies demonstrated that astrocytic Glu transporters fail to quickly reduce high concentrations of Glu under pathological conditions and activated astrocytes have a decreased capacity for Glu to glutamine conversion (Ahlem- eyer et al. 2002; Swanson et al. 2004). Moreover, under ischaemic conditions, activated astrocytes produce pro- inflammatory cytokines and toxic factors such as nitric oxide, reactive oxygen species, and form a ‘glial scar’, which blocks expansion of the injury but inhibits regeneration (Ridet et al. 1997; Perez-Capote et al. 2004; Dirnagl et al. 2005). It suggests that astrocytes may differentially influence neuronal survival early during ischaemia and in the post- ischaemic period. Because astrocytes provide trophic and metabolic support to neurons, modulate glutamate homeostasis preventing from excitotoxicity, astrocyte dysfunction may compromise post- ischaemic neuronal survival (Giffard and Swanson 2005; Nedergaard and Dirnagl 2005). Although, astrocytes are more resistant than neurons to anoxic–ischaemic injury, they Received March 22, 2006; revised manuscript received July 14, 2006; accepted July 14, 2006. Address correspondence and reprint requests to Bozena Kaminska, Department Cell Biology, The Nencki Institute of Experimental Biology, 3 Pasteur Str., 02–093 Warsaw, Poland. E-mail: bozenakk@nencki.gov.pl Abbreviations used: DAPI, 4¢,6-diamidino-2-phenylindole; FCCP, carbonyl cyanide p-(trifluoro-methoxy)-phenyl-hydrazone; GFAP, glial fibrillary acidic protein; Glu, L-glutamate; LSC, laser scanning cytometry; MCAo, middle cerebral artery occlusion; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose)polymerase; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase nick-end labelling. Journal of Neurochemistry , 2006, 99, 965–975 doi:10.1111/j.1471-4159.2006.04136.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 965–975 965