SPHK1 adenovirus

Sirtuin-3 (Sirt3) deacetylates multiple mitochondrial proteins associated with cerebral ischemia/reperfusion (I/R) injury. The mitochondrial unfolded protein response (UPR^mt) favors mitochondrial proteostasis under various stressors. Here, researchers evaluated the molecular basis of Sirt3 regulation of UPR^mt during post-ischemic dysfunction using Sirt3 transgenic mice and transient middle cerebral artery occlusion models. The study showed that Sirt3 abundance in the brain was suppressed after brain ischemic abnormalities. In vivo Sirt3 overexpression suppressed infarct size and reduced neuroinflammation after cerebral ischemia/reperfusion injury. Sirt3 overexpression restored neuronal viability by reducing mitochondrial ROS synthesis, maintaining mitochondrial potential, and promoting mitochondrial ATP synthesis. Sirt3 overexpression triggered UPR^mt via the forkhead box O3 (Foxo3)/sphingosine kinase 1 (Sphk1) pathway, protecting neuronal mitochondria from post-ischemic dysfunction. Inhibition of either UPR^mt or Foxo3/Sphk1 pathway attenuated the beneficial effects of Sirt3 on neuronal function and mitochondrial behavior. Conversely, Sphk1 overexpression was sufficient to reduce infarct size, attenuate neuroinflammation, maintain neuronal viability, and prevent mitochondrial abnormalities during post-ischemic dysfunction. Thus, UPR^mt protects neuronal viability and mitochondrial homeostasis, and the Sirt3/Foxo3/Sphk1 pathway is a promising therapeutic candidate for ischemic stroke.

Here, researchers evaluated whether Sphk1 overexpression alleviates cerebral ischemia-reperfusion dysfunction by normalizing UPR^mt. Data showed that adenoviral overexpression of Sphk1 (Ad-Sphk1) suppressed the OGD/R-mediated decrease in cell viability (Figure 1A). However, treatment with the UPR^mt inhibitor AEBSF abolished the prosurvival effect of Ad-Sphk1. Ad-Sphk1 also suppressed the release of LDH in OGD/R-treated N2a cells, and AEBSF inhibited this effect (Figure 1B). In terms of mitochondrial function, Ad-Sphk1 maintained the mitochondrial membrane potential of N2a cells under OGD/R stress, while AEBSF administration abolished the protective effect of Ad-Sphk1 (Figure 1C and D). In addition, Ad-Sphk1 transfection suppressed the generation of mitochondrial ROS in OGD/R-stressed N2a cells, while AEBSF abolished the antioxidant capacity of Ad-Sphk1 (Figure 1E). Finally, ELISA results showed that the activity levels of caspase-3 and caspase-9 were enhanced after OGD/R treatment and decreased to near normal levels after Ad-Sphk1 transfection; however, Ad-Sphk1 failed to inhibit the activation of caspase-3/9 in N2a cells treated with AEBSF (Figure 1F and G). Therefore, inhibition of UPR^mt would counteract the beneficial effects of Sphk1 overexpression on neuronal function and mitochondrial integrity.

Figure 1. Inhibiting the UPR^mt abolishes the protective effects of Sphk1 overexpression on neurons and their mitochondria. (Xiaowei X, et al., 2023)