Juan P. Bolaños

The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C–Cdh1

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 (F2,6P2), 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.

Glycolysis: a bioenergetic or a survival pathway?

Following inhibition of mitochondrial respiration neurons die rapidly, whereas astrocytes utilize glycolytically-generated ATP to increase their mitochondrial membrane potential, thus becoming more resistant to pro-apoptotic stimuli. Neurons are unable to increase glycolysis due to the lack of activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, isoform 3 (PFKFB3). In neurons, PFKFB3 is degraded constantly via the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)- CDH1. Glucose metabolism in neurons is directed mainly to the pentose phosphate pathway, leading to regeneration of reduced glutathione. In addition to their relevance to brain physiology and pathophysiology, these observations suggest that APC/C-CDH1 might link activation of glycolysis and cell proliferation as it is also involved in the regulation of cell cycle proteins.

E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3—either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1—or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

Persistent mitochondrial damage by nitric oxide and its derivatives: neuropathological implications.

Approximately 15 years ago we reported that cytochrome c oxidase (CcO) was persistently inhibited as a consequence of endogenous induction and activation of nitric oxide (•NO) synthase-2 (NOS2) in astrocytes. Furthermore, the reactive nitrogen species implicated was peroxynitrite. In contrast to the reversible inhibition by •NO, which occurs rapidly, in competition with O2, and has signaling regulatory implications, the irreversible CcO damage by peroxynitrite is progressive in nature and follows and/or is accompanied by damage to other key mitochondrial bioenergetic targets. In purified CcO it has been reported that the irreversible inhibition occurs through a mechanism involving damage of the heme a3-CuB binuclear center leading to an increase in the Km for oxygen. Astrocyte survival, as a consequence of peroxynitrite exposure, is preserved due to their robust bioenergetic and antioxidant defense mechanisms. However, by releasing peroxynitrite to the neighboring neurons, whose antioxidant defense can, under certain conditions, be fragile, activated astrocytes trigger bioenergetic stress leading to neuronal cell death. Thus, such irreversible inhibition of CcO by peroxynitrite may be a plausible mechanism for the neuronal death associated with neurodegenerative diseases, in which the activation of astrocytes plays a crucial role.

Cdk5 phosphorylates Cdh1 and modulates cyclin B1 stability in excitotoxicity

Anaphase‐promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that destabilizes cell cycle proteins, is activated by Cdh1 in post‐mitotic neurons, where it regulates axonal growth, synaptic plasticity and survival. The APC/C–Cdh1 substrate, cyclin B1, has been found to accumulate in degenerating brain areas in Alzheimers disease and stroke. This highlights the importance of elucidating cyclin B1 regulation by APC/C–Cdh1 in neurons under stress conditions relevant to neurological disease. Here, we report that stimulation of N‐methyl‐D‐aspartate receptors (NMDARs) that occurs in neurodegenerative diseases promoted the accumulation of cyclin B1 in the nuclei of cortical neurons; this led the neurons to undergo apoptotic death. Moreover, we found that the Ser‐40, Thr‐121 and Ser‐163 triple phosphorylation of Cdh1 by the cyclin‐dependent kinase‐5 (Cdk5)–p25 complex was necessary and sufficient for cyclin B1 stabilization and apoptotic death after NMDAR stimulation. These results reveal Cdh1 as a novel Cdk5 substrate that mediates cyclin B1 neuronal accumulation in excitotoxicity.

Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/Akt neuroprotective signaling pathway.

Peroxynitrite is usually considered as a neurotoxic nitric oxide‐derivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1u2003mmol/L) rapidly elicited Akt‐Ser473 phosphorylation. Inhibition of phosphoinositide‐3‐kinase (PI3K)/Akt pathway with wortmannin or Akt small hairpin RNA (shRNA) abolished the ability of peroxynitrite to prevent etoposide‐induced apoptotic death. Endogenous peroxynitrite formation by short‐term incubation of neurons with glutamate stimulated Akt‐Ser473 phosphorylation, whereas Akt shRNA enhanced the vulnerability of neurons against glutamate. We further show that Akt‐Ser473 phosphorylation was consequence of the oxidizing, but not the nitrating properties of peroxynitrite. Peroxynitrite failed to nitrate or phosphorylate neurotrophin tyrosine kinase receptors (Trks), and it did not modify the ability of brain‐derived neurotrophic factor (BDNF), to phosphorylate its cognate receptor, TrkB; however, peroxynitrite enhanced BDNF‐mediated Akt‐Ser473 phosphorylation. Finally, we found that peroxynitrite‐stimulated Akt‐Ser473 phosphorylation was associated with an increased proportion of oxidized phosphoinositide phosphatase, PTEN, in neurons. Moreover, peroxynitrite prevented the increase of apoptotic neuronal death caused by over‐expression of PTEN. Thus, peroxynitrite exerts neuroprotection by inhibiting PTEN, hence activating the anti‐apoptotic PI3K/Akt pathway in primary neurons.

Regulation of glycolysis and pentose-phosphate pathway by nitric oxide: impact on neuronal survival.

Besides its essential role at regulating neural functions through cyclic GMP, nitric oxide is emerging as an endogenous physiological modulator of energy conservation for the brain. Thus, nitric oxide inhibits cytochrome c oxidase activity in neurones and glia, resulting in down-regulation of mitochondrial energy production. The subsequent increase in AMP facilitates the activation of 5-AMP-dependent protein kinase, which rapidly triggers the activation of 6-phosphofructo-1-kinase--the master regulator of the glycolytic pathway--and Glut1 and Glut3--the main glucose transporters in the brain. In addition, nitric oxide activates glucose-6-phosphate dehydrogenase, the first and rate-limiting step of the pentose-phosphate pathway. Here, we review recent evidences suggesting that nitric oxide exerts a fine control of neuronal energy metabolism by tuning the balance of glucose-6-phosphate consumption between glycolysis and pentose-phosphate pathway. This may have important implications for our understanding of the mechanisms controlling neuronal survival during oxidative stress and bioenergetic crisis.

Mitochondria and reactive oxygen and nitrogen species in neurological disorders and stroke: Therapeutic implications

Mitochondria represent both the main source and target of reactive oxygen and nitrogen species (RONS). In view of the large energy expenditure made by neurons during neurotransmission, an intact mitochondrial function is of paramount importance for the correct function of the brain. Accordingly, the search of therapeutic strategies against situations in which there is an abnormal brain function, such as neurological disorders and stroke, should be focused towards mitochondria. Here, we have reviewed the normal and abnormal mitochondrial bioenergetics and dynamics, highlighting the relevance that, for these processes in the brain RONS exert. Evidence suggests that disruption of mitochondrial bioenergetics and dynamics may have a critical role in the pathogenesis of these brain diseases. Drug therapies directed toward providing safer mitochondria are currently under both pre- and clinical investigations.

The pentose-phosphate pathway in neuronal survival against nitrosative stress.

Neurons are thought to be particularly vulnerable cells against reactive oxygen and nitrogen species (RONS) damage (nitrosative stress), due in part to their weak antioxidant defense and low ability to compensate energy homeostasis. Intriguingly, nitrosative stress efficiently stimulates the rate of the antioxidant pentose‐phosphate pathway (PPP), which generates NADPH a necessary cofactor for the reduction of glutathione disulfide. In fact, inhibition of PPP sensitizes cultured neurons to glutathione oxidation and apoptotic death, whereas its stimulation confers resistance to nitrosative stress. Furthermore, we recently described that neurons can preferentially use glucose through the PPP by inhibiting glycolysis, which is achieved by continuously degrading the glycolytic positive‐effector protein, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase‐3 (Pfkfb3) by the action of the E3 ubiquitine ligase anaphase‐promoting complex/cyclosome (APC/C)Cdh1. These results suggest that the antioxidant fragility of neurons may be compensated by the PPP at the expense of inhibiting bioenergetic glycolysis.

Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid

J. Neurochem. (2010) 112, 56–65.