Tamás Gáspár

Mitochondrial-mediated suppression of ROS production upon exposure of neurons to lethal stress : Mitochondrial targeted preconditioning

Preconditioning represents the condition where transient exposure of cells to an initiating event leads to protection against subsequent, potentially lethal stimuli. Recent studies have established that mitochondrial-centered mechanisms are important mediators in promoting development of the preconditioning response. However, many details concerning these mechanisms are unclear. The purpose of this review is to describe the initiating and subsequent intracellular events involving mitochondria which can lead to neuronal preconditioning. These mitochondrial specific targets include: 1) potassium channels located on the inner mitochondrial membrane; 2) respiratory chain enzymes; and 3) oxidative phosphorylation. Following activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels and/or increased production of reactive oxygen species (ROS) resulting from the disruption of the respiratory chain or during energy substrate deprivation, morphological changes or signaling events involving protein kinases confer immediate or delayed preconditioning on neurons that will allow them to survive otherwise lethal insults. While the mechanisms involved are not known with certainty, the results of preconditioning are the enhanced neuronal viability, the attenuated influx of intracellular calcium, the reduced availability of ROS, the suppression of apoptosis, and the maintenance of ATP levels during and following stress.

A Mutation in the Inner Mitochondrial Membrane Peptidase 2-Like Gene (Immp2l) Affects Mitochondrial Function and Impairs Fertility in Mice

Abstract The mitochondrion is involved in energy generation, apoptosis regulation, and calcium homeostasis. Mutations in genes involved in mitochondrial processes often result in a severe phenotype or embryonic lethality, making the study of mitochondrial involvement in aging, neurodegeneration, or reproduction challenging. Using a transgenic insertional mutagenesis strategy, we generated a mouse mutant, Immp2lTg(Tyr)979Ove, with a mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. The mutation affected the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The inefficient processing of mitochondrial membrane proteins perturbed mitochondrial function so that mitochondria from mutant mice manifested hyperpolarization, higher than normal superoxide ion generation, and higher levels of ATP. Homozygous Immp2lTg(Tyr)979Ove females were infertile due to defects in folliculogenesis and ovulation, whereas mutant males were severely subfertile due to erectile dysfunction. The data suggest that the high superoxide ion levels lead to a decrease in the bioavailability of nitric oxide and an increase in reactive oxygen species stress, which underlies these reproductive defects. The results provide a novel link between mitochondrial dysfunction and infertility and suggest that superoxide ion targeting agents may prove useful for treating infertility in a subpopulation of infertile patients.

Contribution of poly(ADP-ribose) polymerase to postischemic blood-brain barrier damage in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood–brain barrier (BBB) disruption and edema formation after ischemia–reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia–reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170 ± 9, saline: 760 ± 95, PJ34: 472 ± 61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction.

The Mitochondrial KATP Channel Opener BMS-191095 Reduces Neuronal Damage after Transient Focal Cerebral Ischemia in Rats

Activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels protects the brain against ischemic or chemical challenge. Unfortunately, the prototype mitoKATP channel opener, diazoxide, has mitoKATP channel-independent actions. We examined the effects of BMS-191095, a novel selective mitoKATP channel opener, on transient ischemia induced by middle cerebral artery occlusion (MCAO) in rats. Male Wister rats were subjected to 90 mins of MCAO. BMS-191095 (25 μg; estimated brain concentration of 40 μmol/L) or vehicle was infused intraventricularly before the onset of ischemia. In addition, the effects of BMS-191095 on plasma and mitochondrial membrane potentials and reactive oxygen species (ROS) production in cultured neurons were examined. Finally, we determined the effects of BMS-191095 on cerebral blood flow (CBF) and potassium currents in cerebrovascular myocytes. Treatment with BMS-191095 24 h before the onset of ischemia reduced total infarct volume by 32% and cortical infarct volume by 38%. However, BMS-191095 administered 30 or 60 mins before MCAO had no effect. The protective effects of BMS-191095 were prevented by co-treatment with 5-hydroxydecanoate (5-HD), a mitoKATP channel antagonist. In cultured neurons, BMS-191095 (40μmol/L) depolarized the mitochondria without affecting ROS levels, and this effect was inhibited by 5-HD. BMS-191095, similar to the vehicle, caused an unexplained but modest reduction in the CBF. Importantly, BMS-191095 did not affect either the potassium currents in cerebrovascular myocytes or the plasma membrane potential of neurons. Thus, BMS-191095 afforded protection against cerebral ischemia by delayed preconditioning via selective opening of mitoKATP channels and without ROS generation.

Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels

1,3‐Dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BKCa) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen‐glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3‐kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase‐3/7. NS1619 dose‐dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BKCa channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BKCa channel blockers. NS1619 inhibited the activation of capase‐3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BKCa channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.

ROS-independent preconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095.

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoKATP) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3β. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K—Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

Depolarization of Mitochondria in Endothelial Cells Promotes Cerebral Artery Vasodilation by Activation of Nitric Oxide Synthase

Objective—Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. Approach and Results—Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca2+]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca2+]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. Conclusions—Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca2+]i as well as by phosphoinositide-3 kinase-protein kinase B–induced eNOS phosphorylation. Both mitochondrial reactive oxygen species–dependent and –independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Acute treatment with rosuvastatin protects insulin resistant (C57BL/6J ob/ob) mice against transient cerebral ischemia

The purpose of this study was to investigate the short-term effects of rosuvastatin (RSV), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on transient, focal cerebral ischemia in C57BL/6J ob/ob mice with insulin resistance (IR). Male ob/ob, lean, or wild-type (WT) mice were treated with RSV (10 mg/kg per day, i.p.) or vehicle for 3 days. Ischemia was induced by 60 mins of middle cerebral artery occlusion (MCAO) and cortical blood flow (CBF) was monitored by laser-Doppler flowmetry. Infarct volumes were measured 24 h after reperfusion. IR mice exhibited a higher infarct volume compared with Lean or WT mice, and RSV reduced infarct volume only in obese mice (40% ± 3% versus 32% ± 3%, P < 0.05). Blood cholesterol and insulin levels were elevated in ob/ob mice but were unaffected by RSV. The CBF reductions during MCAO were similar in all groups and were not affected by RSV. Although RSV did not increase cortical endothelial NO synthase (eNOS) levels in the ob/ob mice, it attenuated the increased cortical expression of intracellular adhesion molecule-1 (ICAM-1) after MCAO from ob/ob mice. Thus, RSV protects against stroke in IR mice by a mechanism independent of effects on the lipid profile, CBF, or eNOS but dependent on suppression of post-MCAO ICAM-1 expression.

Cloning of cyclooxygenase-1b (putative COX-3) in mouse.

Abstract.ObjectivesTo clone and sequence cyclooxygenase-1b (COX-1b, also known as COX-3) mRNA and to generate an antibody against the mouse COX-1b protein and to demonstrate its existence in vivo in mouse tissues.Animals10 C57BL/6 mice, 4 COX-1 knockout mice and 4 COX-1 wild type mice were used.MethodsCOX-1b mRNA sequence was determined by RT-PCR amplification using specific primers followed by DNA sequencing. COX-1b protein expression was determined by Western blotting.ResultsThe mouse COX-1b mRNA is a splice variant of the COX-1 mRNA generated by the retention of intron-1. COX-1b mRNA encodes a 127 amino acid protein with no similarity with known COX sequences. We generated an anti-mouse COX-1b antibody and demonstrated the existence of COX-1b protein in vivo with the highest expression in kidney, heart, and neuronal tissues. We also detected COX-1b mRNA and protein expression in COX-1 knockout mice.ConclusionsIn mouse, COX-1b encodes a protein with a completely different amino acid sequence than COX-1 or COX-2; therefore it is improbable that COX-1b in this species plays a role in prostaglandin-mediated fever and pain. In addition, the COX-1−/− mouse is not a COX-1b−/− mouse, therefore it cannot be used to elucidate the function of the COX-1b protein.

Rosuvastatin induces delayed preconditioning against oxygen-glucose deprivation in cultured cortical neurons

We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.