Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Abstract

Cultured cerebellar granule cells were co-loaded with Ca2+-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (ΔΨm). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca2+]i. This sMD was insensitive to a removal of external Ca2+ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca2+ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6–8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]c) and ΔΨm changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]c and ΔΨm. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ΔΨm despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca2+ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.