Neurons are known to have a lower glycolytic rate than astrocytes and when stressed they are unable to upregulate glycolysis because of low Pfkfb3 (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3) activity. This enzyme generates fructose-2,6-bisphosphate (F2,6P(2)), the most potent activator of 6-phosphofructo-1-kinase (Pfk1; ref. 4), a master regulator of glycolysis. Here, we show that Pfkfb3 is absent from neurons in the brain cortex and that Pfkfb3 in neurons is constantly subject to proteasomal degradation by the action of the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1. By contrast, astrocytes have low APC/C-Cdh1 activity and therefore Pfkfb3 is present in these cells. Upregulation of Pfkfb3 by either inhibition of Cdh1 or overexpression of Pfkfb3 in neurons resulted in the activation of glycolysis. This, however, was accompanied by a marked decrease in the oxidation of glucose through the pentose phosphate pathway (a metabolic route involved in the regeneration of reduced glutathione) resulting in oxidative stress and apoptotic death. Thus, by actively downregulating glycolysis by APC/C-Cdh1, neurons use glucose to maintain their antioxidant status at the expense of its utilization for bioenergetic purposes.

After inhibition of cytochrome c oxidase by nitric oxide, astrocytes maintain energy production by upregulating glycolysis--a response which does not seem to be available to neurons. Here, we show that in astrocytes, after inhibition of respiration by nitric oxide, there is a rapid, cyclic GMP-independent increase in the activity of 6-phosphofructo-1-kinase (PFK1), a master regulator of glycolysis, and an increase in the concentration of its most powerful positive allosteric activator, fructose-2,6-bisphosphate (F2,6P(2)). In neurons, nitric oxide failed to alter F2,6P(2) concentration or PFK1 activity. This failure could be accounted for by the much lower amount of 6-phosphofructo-2-kinase (PFK2, the enzyme responsible for F2,6P(2) biosynthesis) in neurons. Indeed, full activation of neuronal PFK1 was achieved by adding cytosol from nitric oxide-treated astrocytes. Furthermore, using the small interfering RNA (siRNA) strategy, we demonstrated that the rapid activation of glycolysis by nitric oxide is dependent on phosphorylation of the energy charge-sensitive AMP-activated protein kinase, resulting in activation of PFK2 and protection of cells from apoptosis. Thus the virtual absence of PFK2 in neurons may explain their extreme sensitivity to energy depletion and degeneration.

It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (⌬m) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes-cell types that differ greatly in their glycolytic capacity. Continuous and significant (Ϸ85%) inhibition of respiration by NO (1.4 M at 175 M O2) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by Ϸ25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in ⌬m and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F1F0-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in ⌬m and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in ⌬m and cell death, NO and staurosporin synergized in decreasing ⌬m and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.glycolysis ͉ mitochondrial membrane potential ͉ apoptosis ͉ oxygen consumption ͉ neurodegeneration N itric oxide (NO) is a physiological messenger (1) that has been reported either to cause or prevent cellular apoptotic death (2, 3). The mitochondrion is now emerging as a key organelle playing a role in the NO-mediated apoptotic pathway. Disruption of the membrane potential across the mitochondrial inner membrane (⌬ m ) dissipates the electrochemical gradient necessary for ATP synthesis (4). Moreover, ⌬ m collapse is associated with mitochondrial swelling, disruption of the outer mitochondrial membrane, and the release of proapoptotic factors such as cytochrome c, apoptosis-inducing factor, and some procaspases (5, 6) from the intermembrane space. NO inhibits the activity of certain components of the mitochondrial respiratory chain, including cytochrome c oxidase (reviewed in ref. 7), and it has recently been shown that endogenous NO formation in primary neurons triggers a rapid and transient ATP depletion associated with ⌬ m collapse and apoptosis (8).Beltrán et al. (9), however, have shown in Jurkat cells that inhibition of mitochondrial respiration by NO induces mitochondrial hyperpolarization. They suggested that this phenomenon may be a...

Neurons depend on oxidative phosphorylation for energy generation, whereas astrocytes do not, a distinctive feature that is essential for neurotransmission and neuronal survival. However, any link between these metabolic differences and the structural organization of the mitochondrial respiratory chain is unknown. Here, we investigated this issue and found that, in neurons, mitochondrial complex I is predominantly assembled into supercomplexes, whereas in astrocytes the abundance of free complex I is higher. The presence of free complex I in astrocytes correlates with the severalfold higher reactive oxygen species (ROS) production by astrocytes compared with neurons. Using a complexomics approach, we found that the complex I subunit NDUFS1 was more abundant in neurons than in astrocytes. Interestingly, NDUFS1 knockdown in neurons decreased the association of complex I into supercomplexes, leading to impaired oxygen consumption and increased mitochondrial ROS. Conversely, overexpression of NDUFS1 in astrocytes promoted complex I incorporation into supercomplexes, decreasing ROS. Thus, complex I assembly into supercomplexes regulates ROS production and may contribute to the bioenergetic differences between neurons and astrocytes.redox | brain | bioenergetics | lactate | glycolysis T he brain is a metabolically demanding organ (1) that requires tight cooperation between neurons and astrocytes (2). Astrocytes provide crucial metabolic and structural support (3, 4) and are key players in neurotransmission (5-7) and behavior (8). The status of many major redox couples in the brain is also regulated by astrocytes (9), through their high content of antioxidant compounds and enzymes (10) and by the constitutive stabilization of the master antioxidant transcriptional activator, nuclear factor erythroid 2-related factor 2 (Nrf2) (11). Thus, astrocytes are equipped to protect themselves when exposed to excess reactive oxygen species (ROS) (12) and reactive nitrogen species (13,14). Moreover, astrocytes also provide nearby neurons with protective antioxidant precursors through a cell-signaling mechanism involving glutamate receptor activation by neurotransmission (11,15,16). The tight coupling between astrocytes and neurons therefore helps in energy and redox metabolism during normal brain function.Intriguingly, the ATP used by neurons is supplied by oxidative phosphorylation, whereas most energy needs of astrocytes are met by glycolysis (17). In fact, the survival of neurons requires oxidative phosphorylation (18,19). The different energy metabolisms of the two cell types are closely coupled, with astrocytes releasing the glycolytic end product, lactate, which is used by neighboring neurons to drive oxidative phosphorylation (20)(21)(22). As the molecular mechanisms underlying the markedly different modes of ATP production in the two cell types are not understood, we investigated whether the organization of the mitochondrial respiratory chain in brain cells could contribute. Here, we report that the extent of supercomplex f...