Abstract
The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.
Highlights
Glycolysis is widely considered a pro-survival metabolic pathway because it meets the energy needs of cells during mitochondrial bioenergetic stress[1]
We used an enzymatic cell-free assay, which revealed that AZ67 inhibited the enzymatic activity of PFKFB3 with an IC50 of 0.018 μM (Fig. 1c), a value that is in accordance with previously published results[18]
We show that pharmacological inhibition of PFKFB3 activity, by preventing glycolytic activation, protects neurons against excitotoxicity both in the NMDA and glutamate receptor activation models and in the oxygen-glucose deprivation/reperfusion model
Summary
Glycolysis is widely considered a pro-survival metabolic pathway because it meets the energy needs of cells during mitochondrial bioenergetic stress[1]. Under certain neuropathological conditions, such as during excitotoxicity, the activity of APC/C-Cdh[1] in neurons is inhibited[12], which allows PFKFB3 protein stabilization in these cells[13]. Active neuronal PFKFB3 stimulates glucose consumption through glycolysis, which results in a concomitant decreased PPP to cause redox stress and, eventually, apoptotic death[13]. We reasoned whether pharmacological inhibition of PFKFB3, by preventing the redox stress associated with glycolytic activation, would protect neurons from the apoptotic death upon excitotoxic insults. We report that small molecule inhibitor of PFKFB3 is able to protect against the apoptotic death caused by excitotoxic stimuli and an oxygen-glucose deprivation (OGD)/reoxygenation model in mouse primary cortical neurons. We show that in vivo administration of this PFKFB3 inhibitor protects against motor discoordination, neurological deficiency and brain damage in a mouse model of brain ischemia/reperfusion
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