María Delgado-Esteban

Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex‐I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose–phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.

Neuroprotective Role of Antidiabetic Drug Metformin Against Apoptotic Cell Death in Primary Cortical Neurons

Oxidative damage has been reported to be involved in the pathogenesis of diabetic neuropathy and neurodegenerative diseases. Recent evidence suggests that the antidiabetic drug metformin prevents oxidative stress-related cellular death in non-neuronal cell lines. In this report, we point to the direct neuroprotective effect of metformin, using the etoposide-induced cell death model. The exposure of intact primary neurons to this cytotoxic insult induced permeability transition pore (PTP) opening, the dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c release, and subsequent death. More importantly, metformin, together with the PTP classical inhibitor cyclosporin A (CsA), strongly mitigated the activation of this apoptotic cascade. Furthermore, the general antioxidant N-acetyl-l-cysteine also prevented etoposide-promoted neuronal death. In addition, metformin was shown to delay CsA-sensitive PTP opening in permeabilized neurons, as triggered by a calcium overload, probably through its mild inhibitory effect on the respiratory chain complex I. We conclude that (1) etoposide-induced neuronal death is partly attributable to PTP opening and the disruption of ΔΨm, in association with the emergence of oxidative stress, and (2) metformin inhibits this PTP opening-driven commitment to death. We thus propose that metformin, beyond its antihyperglycemic role, can also function as a new therapeutic tool for diabetes-associated neurodegenerative disorders.

D-glucose prevents glutathione oxidation and mitochondrial damage after glutamate receptor stimulation in rat cortical primary neurons

Abstract: The possible neuroprotective effect of D‐glucose against glutamate‐mediated neurotoxicity was studied in rat cortical neurons in primary culture. Brief (5‐min) exposure of neurons to glutamate (100 μM) increased delayed (24‐h) necrosis and apoptosis by 3‐ and 1.8‐fold, respectively. Glutamate‐mediated neurotoxicity was accompanied by a D‐(‐)‐2‐amino‐5‐phosphonopentanoate (100 μM) and Nω‐nitro‐L‐arginine methyl ester (1 mM)‐inhibitable, time‐dependent ATP depletion (55% at 24 h), confirming the involvement of NMDA receptor stimulation followed by nitric oxide synthesis in this process. Furthermore, the presence of D‐glucose (20 mM), but not its inactive enantiomer, L‐glucose, fully prevented glutamate‐mediated delayed ATP depletion, necrosis, and apoptosis. Succinate‐ cytochrome c reductase activity, but not the activities of NADH‐coenzyme Q1 reductase or cytochrome c oxidase, was inhibited by 32% by glutamate treatment, an effect that was abolished by incubation with D‐glucose. Lactate accumulation in the culture medium was unmodified by any of these treatments, ruling out the possible involvement of the glycolysis pathway in either glutamate neurotoxicity or D‐glucose neuroprotection. In contrast, D‐glucose, but not L‐glucose, abolished glutamate‐mediated glutathione oxidation and NADPH depletion. Our results suggest that NADPH production from D‐glucose accounts for glutathione regeneration and protection from mitochondrial dysfunction. This supports the notion that the activity of the pentose phosphate pathway may be an important factor in protecting neurons against glutamate neurotoxicity.

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.

Tetrahydrobiopterin deficiency increases neuronal vulnerability to hypoxia

Tetrahydrobiopterin (BH4) is an essential co‐factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH4 levels were depleted by pre‐incubating neurones with 5 mm 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia‐induced cellular damage, BH4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH4‐deficient neurones. All these effects were counteracted whenneuronal BH4 levels were restored by incubating cells with exogenous BH4 during the hypoxic period. Moreover, hypoxia‐induced damage in BH4‐deficient neurones was prevented when Nω‐nitro‐l‐arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH4, NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability tohypoxia‐induced mitochondrial damage and necrosis.

Inhibition of mitochondrial respiration by nitric oxide: Its role in glucose metabolism and neuroprotection

There is an increasing body of evidence demonstrating that inhibition of cytochrome c oxidase by nitric oxide (NO) may be one more step in a signaling cascade involved in the physiologic regulation of cell functions. For example, in both astrocytes and neurons the inhibition of mitochondrial respiration by endogenously produced NO induces transient and modest decreases in cellular ATP concentrations. This mitochondrial impairment may serve as a cellular sensor of energy charges, hence modulating metabolic pathways, such as glycolysis, through AMP‐activated protein kinase (AMPK) in astrocytes. In neurons, the NO derivative peroxynitrite anion triggers signaling pathways leading to glucose oxidation through the pentose‐phosphate pathway to form reducing equivalents in the form of NADPH. The modulation of these metabolic pathways by nitric oxide or its derivatives may be important for understanding the mechanisms by which this free radical affects neuronal death or survival.

The human Tp53 Arg72Pro polymorphism explains different functional prognosis in stroke

Poor prognosis after ischemic stroke or intracerebral hemorrhage is linked to a particular polymorphism in the human gene encoding p53.

APC/C-Cdh1 coordinates neurogenesis and cortical size during development

The morphology of the adult brain is the result of a delicate balance between neural progenitor proliferation and the initiation of neurogenesis in the embryonic period. Here we assessed whether the anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1--which regulates mitosis exit and G1-phase length in dividing cells--regulates neurogenesis in vivo. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

Human neuroblastoma cells with MYCN amplification are selectively resistant to oxidative stress by transcriptionally up-regulating glutamate cysteine ligase

J. Neurochem. (2010) 113, 819–825.

Regulation of Bcl-xL–ATP Synthase Interaction by Mitochondrial Cyclin B1–Cyclin-Dependent Kinase-1 Determines Neuronal Survival

The survival of postmitotic neurons needs continuous degradation of cyclin B1, a mitotic protein accumulated aberrantly in the damaged brain areas of Alzheimers disease and stroked patients. Degradation of cyclin B1 takes place in the proteasome after ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C)–cadherin 1 (Cdh1), an E3 ubiquitin ligase that is highly active in neurons. However, during excitotoxic damage—a hallmark of neurological disorders—APC/C–Cdh1 is inactivated, causing cyclin B1 stabilization and neuronal death through an unknown mechanism. Here, we show that an excitotoxic stimulus in rat cortical neurons in primary culture promotes cyclin B1 accumulation in the mitochondria, in which it binds to, and activates, cyclin-dependent kinase-1 (Cdk1). The cyclin B1–Cdk1 complex in the mitochondria phosphorylates the anti-apoptotic protein B-cell lymphoma extra-large (Bcl-xL), leading to its dissociation from the β subunit of F1Fo–ATP synthase. The subsequent inhibition of ATP synthase activity causes complex I oxidative damage, mitochondrial inner membrane depolarization, and apoptotic neuronal death. These results unveil a previously unrecognized role for mitochondrial cyclin B1 in the oxidative damage associated with neurological disorders.