Brain Poster Session: Mitochondria

454. Mitochondrial ros preconditioning protects rat brain endothelial cells against glucotoxicity through HIF-1 activation

S. Correia, M.S. Santos and P. Moreira

Center for Neuroscience and Cell Biology, Coimbra, Portugal

Endothelial dysfunction is a critical feature in the onset of the type 2 diabetes-induced vascular complications. It has been proposed that hyperglycemia, a hallmark of type 2 diabetes, is a major causal factor in the development of endothelial dysfunction in diabetic patients, which predisposes to several pathologies including vascular dementia and Alzheimer's disease. Accumulating evidence demonstrates that hypoxic preconditioning protects the brain against several types of deleterious/lethal insults through the induction of the transcription factor hypoxia inducible factor 1 (HIF-1). Furthermore, it has been reported that mitochondrial reactive oxygen species (ROS) are required for HIF-1 activation and stabilization. Thus, the aim of this study was to evaluate if mitochondrial ROS are able to protect brain endothelial cells against hyperglycemia through the activation of HIF-1. For this purpose, we pre-incubated rat brain endothelial cells (RBE4) with several mitochondrial modulators (rotenone, antimycin A and potassium cyanide). Using fluorimetric methods we observed that these mitochondrial modulators were able to increase mitochondrial ROS production without affecting cell viability (mitochondrial preconditioning). Furthermore, immunocytochemistry and Western blotting analyses revealed that these mitochondrial modulators also activated HIF-1 and its target genes, the vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1). In addition, mitochondrial preconditioning protected RBE4 cells against deleterious effects induced by high levels of glucose (30 mmol/L). Altogether, our results show that mitochondrial ROS preconditioning protects brain endothelial cells against high glucose levels through the activation of HIF-1. Exploring how mitochondrial manipulation activate adaptive responses mediated by HIF-1, may offer new avenues for the treatment of vascular endothelial dysfunction associated to several pathologies including neurodegenerative conditions.

This work is supported by the European Foundation for the Study of Diabetes/Servier.

Sónia Correia has a PhD fellowship from the Fundação para a Ciência e a Tecnologia (SFRH/BD/40702/2007).

680. NS1619 Induces immediate preconditioning in neurons against glutamate ecitotoxicity via ROS generation but not BK_Ca channel activation

D. Busija¹, T. Gáspár¹, L. Lenti², P. Katakam¹, J. Snipes¹, F. Domoki² and F. Bari²

¹Physiology and Pharmacology, Wake Forest U Health Sciences, Winston-Salem, North Carolina, USA; ²Department of Physiology, University of Szeged, Szeged, Hungary

Objectives: Activation of ATP-sensitive potassium (K_ATP) channels in the inner mitochondrial membrane has been shown to protect cultured neurons and brain against lethal insults by inducing immediate and delayed preconditioning (Stroke 30:2713, 1999; Brain Res. Rev. 56:89, 2007; Adv. Drug Del. Rev. 16:1471, 2008). It has been reported recently that the large conductance calcium activated potassium (BK_Ca) channel is present in the mitochondria of neurons and its activation with NS1619 has been reported to induce preconditioning. NS1619 is the most commonly used BK_Ca agonist. However, our recent study has questioned the role of functional mitochondrial BK_Ca channels in delayed neuronal preconditioning (J. Neurochem. 105:1115, 2008). On the other hand, BK_Ca channels might be involved in immediate preconditioning, but this possibility has never been explored. The objectives of our present experiments were:

• (1)

  to determine whether the BK_Ca channel opener NS1619 induces immediate preconditioning in cultured rat cortical neurons; and

• (2)

  to elucidate the role of BK_Ca channels in immediate preconditioning.

Methods: Primary cortical neurons were isolated from E18 Sprague-Dawley rat fetuses and studied at 7 to 9 days in culture.

Results: NS1619 (25 to 200 μmol/L) increased reactive oxygen species (ROS) generation and depolarized mitochondria in a dose dependent manner, but neither was inhibited by BK_Ca channel antagonists. Furthermore, we could not detect the presence of the pore forming subunit of the BK_Ca channel in isolated mitochondria using commercially available antibodies while this subunit was present in brain lysates. One-hour treatment with NS1619 followed by washout of the drug induced protection against glutamate excitotoxicity (200 μmol/L for 60 mins). Neuronal viability 24 h after glutamate exposure was 58.45±0.95% for untreated cells. However, NS1619 at 50 μmol/L increased viability to 78.99%±0.90%*; NS1619 at 100 μmol/L increased viability to 86.89%±1.20%*; and NS1619 at 150 μmol/L increased viability to 93.23%±1.23%*; mean±s.e.m.; *P<0.05 vs. untreated cells; n = 12 to 24). Viability of neurons exposed to only NS1619 was ∼100%. Administration of the PI3-kinase inhibitor wortmannin, the PKC blocker chelerythrine, the MAP kinase antagonist PD98059, the ryanodine receptor inhibitor dantrolene, and the BK_Ca channel blockers iberiotoxin and paxilline were unable to antagonize the neuroprotective effects. In contrast, eliminating ROS with a scavenger during NS1619 application effectively blocked the development of preconditioning. Finally, treatment with NS1619 reduced the calcium load and ROS surge upon glutamate exposure.

Conclusions: Our results indicate that NS1619 is a potent inducer of immediate (current study) and delayed (previous study) neuronal preconditioning, and the protective effect is dependent on ROS generation but independent of the activation of BK_Ca channels. Since NS1619 has been shown to inhibit the mitochondrial electron transport chain, it seems likely that this effect may be the source of increased ROS and the resultant preconditioning. Our collective studies indicate that mitochondrial depolarization and subsequent protein kinase activation without ROS generation following selective activation of mitochondrial K_ATP channels (J. Cereb. Blood Flow Metab 2008;28:1090) as well enhanced mitochondrial ROS release independent of potassium channel activation are both capable of protecting neurons against a variety of lethal stresses through distinct mechanisms. Suppported by NIH grants.

808. Estrogen receptor-mediated protection of astrocytes and cerebral endothelial cells during ischemic conditions in vitro

D.N. Krause¹, J. Guo^1,2, J.H. Weiss³ and S.P. Duckles¹

¹Pharmacology, University of California, Irvine, Irvine, California, USA; ²Pharmacology, Peking University, Beijing, China; ³Neurology, University of California, Irvine, Irvine, California, USA

We recently demonstrated that 17β-estradiol (E) improves mitochondrial efficiency and reduces mitochondrial free radical formation in brain blood vessels and endothelial cells. We hypothesized that during ischemic insult, E would protect mitochondrial function and enhance cell viability in two key supportive cells in the brain: endothelial cells and astrocytes. To test this, oxygen-glucose deprivation (OGD)/reperfusion was applied in culture to either primary neonatal mouse astrocytes or immortalized mouse cerebral endothelial cells (bEnd.3). In both cases, cell death induced by 6 h OGD was prevented by long-term (24, 48 h), but not short-term (<12 h) pretreatment with E (10 nmol/L). E also suppressed OGD-induced translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei. E inhibited lactate dehydrogenase (LDH) leakage during OGD and reperfusion and abrogated OGD-induced ATP depletion. The latter effects were blocked by the estrogen receptor (ER) antagonist, ICI-182,780 and mimicked by an ERalpha-selective agonst but not an ERbeta agonist. E treatment also suppressed free radical formation that was evident after only 1 h OGD and preserved the integrity of the mitochondrial membrane potential. Interestingly, E stimulated mitochondrial protein expression in endothelial cells but not astrocytes, suggesting that suppression of free radical formation, not mitochondrial biogenesis, may be a common protective mechanism of E in both cell types.

[Supported by NIH Grant R01 HL-50775 and Chinese Government Scholarship 20073020].

1057. Impaired mitochondrial energy metabolism and neuronal apoptotic cell death after chronic aluminum exposure in rat brain

P. Khanna and B. Nehru

Biophysics Dept, Panjab University, Chandigarh, India

The present study elucidates a possible mechanism by which chronic aluminum exposure (100 mg/kg b.wt. p.o. for 8 weeks daily) causes neuronal degeneration. Mitochondria, the primary site of cellular energy generation and oxygen consumption might represent a likely target for aluminum toxicity. Therefore, the objective of the current study was to investigate the effect of chronic aluminum exposure on mitochondrial energy metabolism, oxidative stress generation and its implication in the induction of neuronal apoptosis in rat model. Mitochondrial fractions were isolated from cerebral cortex, mid brain and cerebellar region of rat brain and the complexes were estimated biochemicaly in the same fractions. Our results indicated significant decrease in the activity of complex I, II and IV in all the three regions, though the changes were more pronounced in mid brain region. The MTT assay which indirectly shows the activity of complex III did not register any significant change except for mid brain. Mitochondria generate ROS that are thought to augment intracellular oxidative stress. The alterations in the mitochondrial electron transfer enzyme activities in turn might have caused an increase in malondialdehyde which is observed in all the three regions. Further the decreased levels of GSH and also decreased MnSOD activity in the mitochondrial fraction of rat brain enhanced oxidative stress. Thus, chronic aluminum exposure increases the oxidative stress both by mitochondrial damage as well as due to the failure in the ROS removal system which in turn causes oligonucleosomal DNA fragmentation and formation of comet, a hallmark of apoptosis. The present study provides an evidence of impaired mitochondrial bioenergetics and apoptotic neuronal degeneration after chronic exposure of aluminum.