The Mechanism Of Autophagy Activation Induced By Sphingosine Kinase 2 In Neural Cells

Aim:To study the effects of SPK2 on ischemic injury and autophagy activation in neural cells,and to explore whether the mechanism of autophagy activation by SPK2 is dependent on its BH3 domain and the interaction with Bcl-2.Methods:Stable infected HT22 cells were established by SPK2 overexpressed lentivirus（LV-HA-SPK2）and mutant lentivirus（LV-HA-SPK2-L219A）.Immunoblotting was used to examine the protein expression of SPK2,HA-tag and LC3.Sphingosine kinase activity was also examined.NH₄Cl,a lysosomal inhibitor was used to examine autophagy flux.Transmission electron microscope（TEM）was used to observe the formation of autophagosomes.Co-immunoprecipitation and GST pull down was used to determine the interaction between SPK2 and Bcl-2.Alex flour 488-conjuated streptavidin was used to determine whether Tat-SPK2 peptide and control peptide（Tat-L219A）could enter neural cells.HT22 cells and primary cortical neurons were treated with Tat-SPK2 peptide and its control peptide,and the oxygen glucose deprivation（OGD）model was established.The protection of SPK2 on OGD/R induced neural cells injury was detected by Cell Counting Kit-8 assay.3-MA and Atg5 siRNA were used to determine the protection of autophagy induced by SPK2.Immunoblotting was used to examine the protein expression of LC3 and NH₄Cl was used to examine autophagy flux after the treatment with Tat-SPK2 peptide.Results:In LV-SPK2-HT22 cells,the HA-tag protein was expressed and the expression of SPK2 was upregulated as well.Overexpression of SPK2 increased LC3II level and enhanced the autophagy flux,and the autophagosome formation increased.Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2.Notably,an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy.Tat-SPK2 peptide,but not the L219A mutated BH3domain of SPK2 activated autophagy in neural cells.The Tat-SPK2 peptide also protected HT22 cells and cortical neurons against OGD injury through autophagy activation,while the autophagy inhibitor 3-MA abolished the neuroprotection induced by Tat-SPK2 peptide.Conclusions:SPK2,via its BH3 domain,could dissociate complex between Beclin-1and Bcl-2 by interacting with Bcl-2 to activate autophagy.Tat-SPK2 peptide designed from the BH3 domain of SPK2 could activate autophagy and protect neural cells against OGD injury.