Fig. 2.

Mitochondrial HKII protects neurons from hypoxic cell death. We established an assay for investigating the functional impact of specific gene products (A and B). We cocultivated two independently transfected neuronal populations and used two different fluorescent protein (eGFP and mOrange) encoding plasmids to differentiate the two populations [cotransfected cells were eGFP + gene of interest (GOI), indicated by “G”]. We calculated ratios of the number of green and red fluorescent cells for each condition. A ratio of 1 means that equal numbers of green and red fluorescent cells survived; a ratio of 2 means that twice as many green as red fluorescent cells survived a damaging insult. Using this assay, we analyzed the effect of HKII on neuronal survival after OGD (C). The results demonstrated potent protection from cell death because of cultivation (pre-OGD) and in response to OGD (post-OGD). Mitochondrial binding (D) of HKII is essential for neuroprotection. Deletion of the mitochondrial binding site of HKII (HKII_∆N) and nonphosphorylatable HKII (HKII_T473A) have a detrimental effect on survival. (E) HKII_WT protected neurons from OD, (F) but promoted cell death after GD. (G) Catalytically inactive HKII_S155A/S603A promoted cell death under OD, (H) but protected cells from detrimental GD. N indicates number of independent experiments. *P = 0.05, **P = 0.01, one-way ANOVA, Duncan post hoc.