Elsevier

Neuroscience

Volume 137, Issue 1, 2006, Pages 231-239
Neuroscience

Neuropharmacology
Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: Role of glutamate uptake and FK506 binding protein 12 kDa

https://doi.org/10.1016/j.neuroscience.2005.08.080Get rights and content

Abstract

Background: The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection.

Methods: Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation.

Results: When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50–500pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects.

Conclusion: The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.

Section snippets

Experimental procedures

The experimental protocols, involving animals and their care, strictly conformed to the guidelines of the French Agriculture and Forestry Ministry (decree 87–848). In all experiments attention was paid to the regulations of local authorities for handling laboratory animals, the European Communities Council Directive (86/609/EEC). Particular efforts were made to minimize animal suffering and to reduce the number of animals used.

Protective effects of tacrolimus against OGD-induced neuronal injury

Neuronal damage was first assessed by cell counting after immunostaining experiments (Table 1). Compared with sham wash, OGD induced a significant decrease in the neuronal count observed in controls 24 h after the injury but did not affect the number of GFAP/DAPI positive cells (underlying glial cells). This decrease was significantly reversed by tacrolimus, MK-801 and GPI1046 but not by rapamycin added during OGD.

LDH release and MTT reduction are closely correlated with cell counting for

Discussion

Using a transient OGD on primary mixed cultures of neurons and astrocytes, we show that tacrolimus reverses OGD-induced neuron death when assessed 24 h after the injury. This protective effect is observed when tacrolimus was added either during OGD or during reoxygenation. Both of these beneficial effects can probably be explained by the binding of tacrolimus to FKBP12. Moreover, the effect of tacrolimus added during OGD may be mediated, at least in part, by the fact that it reverses the

Conclusion

In conclusion, this study demonstrates a neuroprotective effect of tacrolimus in an in vitro model of ischemia/reperfusion. This protective effect was observed both when tacrolimus was added at the time of injury and when added during reoxygenation. Both of these beneficial effects of tacrolimus on neuronal viability may involve the FKBP12 pathway and its preventive action may be mediated, at least in part, by a restoration of the GLT1-mediated activity which is dramatically impaired by OGD,

Acknowledgments

This work was supported by a grant from Fujisawa GmbH (2002) and from DGA (Direction Générale de l’Armement, contract no. 00 34 052 00 470 75 01). We thank Mrs. Marjorie Sweetko for the English version of this manuscript. The institution had the responsibility for maintaining objectivity in research by ensuring that the design, conduct, or reporting of research has not been biased by any conflicting financial interest of investigators responsible for the research.

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