Both hypoxia and bilirubin are common risk factors in newborns, which may act synergistically to produce anatomical and functional disturbances of the CNS. Using primary cultures of neurons from the ...fetal rat brain, it was recently reported that neuronal apoptosis accounts for the deleterious consequences of these two insults. To investigate the influence of hypoxia, bilirubin, or their combination on the outcome of neuronal cells of the immature brain, and delineate cellular mechanisms involved, 6-d-old cultured neurons were submitted to either hypoxia (6 h), unconjugated bilirubin (0.5 microM), or to combined conditions. Within 96 h, cell viability was reduced by 22.7% and 24.5% by hypoxia and bilirubin, respectively, whereas combined treatments decreased vital score by 34%. Nuclear morphology revealed 13.4% of apoptotic cells after hypoxia, 16.2% after bilirubin, and 22.6% after both treatments. Bilirubin action was specifically blocked by the glutamate receptor antagonist MK-801, which was without effect on the consequences of hypoxia. Temporal changes in (3)Hleucine incorporation rates as well as beneficial effects of cycloheximide reflected a programmed phenomenon dependent upon synthesis of selective proteins. The presence of bilirubin reduced hypoxia-induced alterations of cell energy metabolism, as reflected by 2-D-(3)Hdeoxyglucose incorporation, raising the question of free radical scavenging. Measurements of intracellular radical generation, however, failed to confirm the antioxidant role of bilirubin. Taken together, our data suggest that low levels of bilirubin may enhance hypoxia effects in immature neurons by facilitating glutamate-mediated apoptosis through the activation of N:-methyl-D-aspartate receptors.
Many catechol derivatives are currently used as drugs, even if they produce reactive oxygen species that may cause tissue damage. Among them, apomorphine, a potent dopamine agonist, displays ...efficient anti-parkinsonian properties, but the consequences of its oxidant and toxic properties have been poorly investigated on
in vitro models. In the present work, we investigated apomorphine cytotoxicity by incubating cultures of rat glioma C6 cells and primary cultures of neurons with different concentrations of the drug. Apomorphine-promoted cell death was proportional to its concentration and was time-dependent. The
ed
50 of apomorphine on C6 cell death after 48 hr was about 200 μM. The cytotoxic effects induced by apomorphine were correlated to its autoxidation, which leads to the formation of reactive oxygen species, semiquinones, quinones, and a melanin-like pigment. C6 cells that underwent treatment with 400 μM apomorphine for 6 hr displayed features of necrosis, including loss of membrane integrity, degeneration of mitochondria, and DNA fragmentation. Thiols, such as cysteine,
N-acetyl-
l-cysteine, and glutathione, significantly protected cultured neurons and C6 cells against apomorphine-induced cytotoxicity. Thiols also inhibited apomorphine autoxidation. These data strongly suggest that apomorphine cytotoxicity towards neurons and C6 cells results from an intracellular oxidative stress.
A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid β-peptide (Aβ) may be the proximate effectors of neuronal injuries and death in the early stages of ...Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble Aβ remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437–3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble Aβ-mediated neuronal apoptosis. The data presented here show that soluble Aβ oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Aβ-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble Aβ. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble Aβ-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.
A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid β-peptide (Aβ) may be the proximate
effectors of neuronal injuries and death in the early stages of ...Alzheimer disease. However, the molecular mechanisms associated
with neuronal apoptosis induced by soluble Aβ remain to be elucidated. We recently demonstrated the involvement of an early
reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel,
V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437â3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and
dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable
us to explain the microtubule perturbation involved in soluble Aβ-mediated neuronal apoptosis. The data presented here show
that soluble Aβ oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca 2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a,
MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Aβ-mediated activation of caspase-3
and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species
generation in the perturbation of the microtubule network induced by soluble Aβ. These data clearly demonstrate the impact
of cytoskeletal perturbations on soluble Aβ-mediated cell death and support the notion of microtubule-stabilizing agents as
effective Alzheimer disease drugs.
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