We have previously shown that
sphingosine kinase 2 (SPK2) interacts with Bcl-2 via its BH3 domain, activating autophagy by inducing the dissociation of
Beclin-1/Bcl-2 complexes, and that a TAT-SPK2
peptide containing the BH3 domain of SPK2 protects neurons against ischemic injury. The goals of the present study were to establish the functional significance of these findings, by testing whether TAT-SPK2 was effective in a mouse model of
ischemic stroke, and to explore potential underlying mechanisms. Mice were administered with TAT-SPK2 by
intraperitoneal injection before or after transient
middle cerebral artery occlusion (tMCAO).
Infarct volume, neurological deficit and brain water content were assessed 24 h after reperfusion. Mitophagy inhibitor
Mdivi-1 and BNIP3 siRNAs were used to examine the involvement of BNIP3-dependent mitophagy in the neuroprotection of TAT-SPK2. Mitophagy was quantified by immunoblotting, immunofluorescence and electron microscopy. The interaction between TAT-SPK2 and Bcl-2, Bcl-2 and BNIP3 was detected by co-immunoprecipitation. In the tMCAO model, pre-treatment with TAT-SPK2 significantly reduced
infarct volume, improved neurological function and decreased
brain edema. Neuroprotection by TAT-SPK2 was still seen when the
peptide was administered 3 h after reperfusion. TAT-SPK2 also significantly improved functional recovery and reduced long-term brain
atrophy of the ischemic hemisphere 30 days after administration. Our studies further showed that TAT-SPK2 directly binds to Bcl-2 and disrupts Bcl-2/
Beclin-1 or Bcl-2/BNIP3 complexes to induce mitophagy. These results suggest that TAT-SPK2 protects neurons against
ischemia reperfusion injury by activating BNIP3-mediated mitophagy. Agents exploiting this molecular mechanism are potential candidates for the treatment of
ischemic stroke.