Conclusion
AZD2014 was detrimental to neurons that underwent ischemia. AZD2014 appeared to reduce hamartin, a known neuroprotective mediator, thereby preventing any beneficial effects of mTOR inhibition. Further characterization of the role of individual mTOR complexes (mTORC1 and mTORC2) and their upstream and downstream regulators are necessary to reveal whether these pathways are neuroprotective targets for stroke.
Methods
Primary neuronal cultures from E18 Wistar rat embryos were exposed to 2 h OGD or normoxia. AZD2014 was administered either during OGD, 24 h prior to OGD or for 24 h following OGD. Cell death was quantified by lactate dehydrogenase assay. We characterized the expression of mTOR pathway proteins following exposure to AZD2014 using western blotting.
Results
Following 2 h OGD +24 h recovery, AZD2014 increased neuronal death when present during OGD. Rapamycin, the archetypal mTOR inhibitor, had no effect on cell death. Treatment with AZD2014 24 h prior to OGD and 24 h after OGD also enhanced cell death. While Western blotting showed a trend towards decreased expression levels of phospho-Akt relative to total Akt with increasing AZD2014 concentration, hamartin expression was also significantly decreased leading to activation of mTOR.