autophagy, neuons, neuroprotection, sphingosine-1-phosphate, S1P, stroke, isoflurane, preconditioning, sphingosine kinase
Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 upregulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, while pretreatment with autophagy inhibitors (3-MA or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by western blots for LC3 and p62, LC3 immunostaining and electron microscopy. Isoflurane induced autophagy in mice lacking the SPK1 isoform (SPK1-/-), but not in SPK2-/ - mice. Sphingosine 1-phosphate (S1P) and the S1P receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished ISO-induced disruption of the Beclin1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin1/Bcl-2 interaction.