Thomas F.W. Horn

Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin

Melatonin, the secretory product of the pineal gland, is known to be neuroprotective in cerebral ischemia, which is so far mostly attributed to its antioxidant properties. Here we show that melatonin directly inhibits the mitochondrial permeability transition pore (mtPTP). mtPTP contributes to the pathology of ischemia by releasing calcium and cytochrome c (cyt c) from mitochondria. Consistently, NMDA‐induced calcium rises were diminished by melatonin in cultured mouse striatal neurons, similar to the pattern seen with cyclosporine A (CsA). When the mouse striatal neurons were subjected to oxygen‐glucose deprivation (OGD), melatonin strongly prevented the OGD‐induced loss of the mitochondrial membrane potential. To assess the direct effect of melatonin on the mtPTP activity at the single channel level, recordings from the inner mitochondrial membrane were obtained by a patch‐clamp approach using rat liver mitoplasts. Melatonin strongly inhibited mtPTP currents in a dose‐dependent manner with an IC50 of 0.8 µM. If melatonin is an inhibitor of the mtPTP, it should prevent mitochondrial cyt c release as seen in stroke models. Rats underwent middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. Melatonin (10 mg/kg ip) or vehicle was given at the time of occlusion and at the time of reperfusion. Indeed, infarct area in the brain sections of melatonin‐treated animals displayed a considerably decreased cyt c release along with less activation of caspase‐3 and apoptotic DNA fragmentation. Melatonin treatment diminished the loss of neurons and decreased the infarct volume as compared with untreated MCAO rats. Our findings suggest that the direct inhibition of the mtPTP by melatonin may essentially contribute to its anti‐apoptotic effects in transient brain ischemia.

Oxyresveratrol (trans-2,3′,4,5′-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia

Oxidative stress is one of the major pathological factors in the cascade that leads to cell death in cerebral ischemia. Here, we investigated the neuroprotective effect of a naturally occurring antioxidant, oxyresveratrol, to reduce brain injury after cerebral stroke. We used the transient rat middle cerebral artery occlusion (MCAO) model of brain ischemia to induce a defined brain infarction. Oxyresveratrol was given twice intraperitoneally: immediately after occlusion and at the time of reperfusion. Oxyresveratrol (10 or 20 mg/kg) significantly reduced the brain infarct volume by approximately 54% and 63%, respectively, when compared to vehicle-treated MCAO rats. Also, the neurological deficits as assessed by different scoring methods improved in oxyresveratrol-treated MCAO rats. Histological analysis of apoptotic markers in the ischemic brain area revealed that oxyresveratrol treatment diminished cytochrome c release and decreased caspase-3 activation in MCAO rats. Also, staining for apoptotic DNA showed that the number of apoptotic nuclei in ischemic brain was reduced after oxyresveratrol treatment as compared to the vehicle-treated MCAO rats. This dose-dependent neuroprotective effect of oxyresveratrol in an in vivo stroke model demonstrates that this drug may prove to be beneficial for a therapeutic strategy to limit brain injury in acute brain ischemia.

Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide.

4,5‐diaminofluorescein diacetate (DAF‐2DA) is widely used as a fluorescent probe to detect endogenously produced nitric oxide (NO). Recent reports that refer to the high sensitivity of DAF‐2 toward NO prompted us to test its efficiency and specificity in a mixed murine primary glial culture model, in which the NO‐synthesizing enzyme inducible nitric oxide synthase (iNOS) is expressed by stimulation with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). Cultures were loaded with DAF‐2DA and the fluorescence was measured using confocal microscopy. NO production in the cultures was determined using the ozone/chemiluminescence technique. Due to the extremely high photosensitivity of DAF‐2, low laser intensities were used to avoid artifacts. No difference in DAF‐2 fluorescence was observed in NO‐producing cultures compared to control cultures, whereas the NO/peroxynitrite‐sensitive dye 2,7‐dihydrodichlorofluorescein (DCF) showed a significant fluorescence increase specifically in microglia cells. A detectable gain in fluorescence was seen when NO‐containing buffer was added to the DAF‐2DA–loaded cells with a minimum NO concentration at 7.7 μM. An additional gain of DAF‐2 fluorescence was obtained when the cells were depleted of glutathione (GSH) with L‐buthionine S,R‐sulfoximine (BSO). Hence, we monitored the change in DAF‐2 fluorescence intensity in the presence of NO and O  −·2 in a cell‐free solution. The fluorescence due to NO was indeed larger when O  −·2 was added, implying a higher sensitivity of DAF‐2 for peroxynitrite. Nevertheless, our results also indicate that measurement of DCF fluorescence is a better tool for monitoring intracellular changes in the levels of NO and/or peroxynitrite than DAF‐2. GLIA 38:103–114, 2002.

Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons

Overproduction of nitric oxide by NMDA receptor stimulation is implicated in calcium deregulation and neurodegeneration of striatal neurons. We investigated the involvement of nitric oxide (NO) in inducing intracellular calcium release and in modifying calcium transients evoked by NMDA. NO application (4–10 fM) reversibly and repeatedly increased the intracellular calcium concentration [Ca2+]i in Fura‐2‐or fluo‐3‐loaded cultured mouse striatal neurons. NO‐induced [Ca2+]i responses persisted in the absence of extracellular calcium, indicating that Ca2+ was released from intracellular stores. The source of calcium was distinct from [Ca2+]i‐activated (ruthenium red and ryanodine sensitive) or IP3‐activated (thapsigargin‐sensitive) Ca2+ stores and was not dependent on cGMP production because a cell permeant analog, 8‐bromo‐cGMP, did not increase basal [Ca2+]i. Glucose removal potentiated the NO‐induced release of [Ca2+]i. In contrast, pretreatment with either the mitochondrial uncoupler carbonyl cyanide m‐chlorophenylhydrazone or cyclosporin A, a blocker of the mitochondrial permeability transition pore, prevented the [Ca2+]i increase after NO. The rise in [Ca2+]i during NO exposure was preceded by a decrease in mitochondrial membrane potential that was partly reversible during washout. Repeated applications of NMDA induced irreversible [Ca2+]i responses in a subpopulation of striatal cells that were greatly reduced by the NOS inhibitor NW‐nitro‐L‐arginine. Calcium transients were prolonged by conjoint application of NMDA and NO. We conclude that NMDA‐evoked [Ca2+]i transients are modulated by endogenous NO production, which leads to release of calcium from the mitochondrial pool. An NO‐activated mitochondrial permeability transition pore may lead to cell death after overstimulation of NMDA receptors.—Horn, T. F. W., Wolf, G., Duffy, S., Weiss, S., Keilhoff, G., MacVicar, B. A. Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons. FASEB J. 16, 1611–1622 (2002)

Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia

Nitric oxide (NO) is produced in mam‐ mals by different isoforms of NO synthase (NOS), in‐ cluding the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochon‐ dria. We show that mitochondrially derived NO medi‐ ates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the im‐ paired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L‐NAME. These observations suggest that mitochon‐ dria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochon‐ drial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.— Schild, L., Reinheckel, T., Reiser, M., Horn, T. F. W., Wolf, G., Augustin, W. Nitric oxide produced in rat liver mitochondria causes oxidative stress and impair‐ ment of respiration after transient hypoxia. FASEB J. 17, 2194‐2201 (2003)

Lack of mitochondrial nitric oxide production in the mouse brain

Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF‐fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark‐type NO‐sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5‐ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS‐dependent mechanism.

Natural and newly synthesized hydroxy-1-aryl-isochromans: A class of potential antioxidants and radical scavengers

We investigated the antioxidant and radical scavenging activity of polyphenolic isochromans. To assess the relation between structure and scavenging properties the natural occurring 1-(3′-methoxy-4′-hydroxy)phenyl-6,7-dihydroxy-isochroman (ISO-3, three OH groups) was compared with three newly synthesized derivatives that differ in their degree of hydroxylation by substitution with methoxy-groups (ISO-4: four OH groups; ISO-2: two OH groups and ISO-0: fully methoxylated). We found that ISO-4 is a 2-fold better scavenger for the artificial radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, 100 μM) with an EC50=10.3 μM compared to the natural ISO-3 (EC50=22.4 μM) and to ISO-2 (EC50=25.1 μM), while ISO-0 did not react with DPPH. The scavenging capacity for superoxide enzymatically generated in a hypoxanthin-xanthinoxidase reaction was the highest for ISO-4 (EC50=34.3 μM) compared to those of ISO-3 (EC50=84.0 μM) and ISO-2 (EC50=91.8 μM), while ISO-0 was inactive. In analogy, ISO-4 scavenged peroxynitrite (ONOO−, EC25=23.0 μM) more effective than ISO-3, ISO-2 and ISO-0. When C6 rat glioma cells loaded with the reactive oxygen/nitrogen (ROS/RNS)-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, were exposed to hydrogen peroxide, the lowest stress level as indicated by the fluorescence signal was detected when the cells were pretreated with ISO-4 or ISO-2 but to a much lesser extent with ISO-3, while ISO-0 did not show any effect. All tested hydroxyisochromans superceded the scavenging effect of trolox. The excellent radical and ROS/RNS scavenging features of the hydroxy-1-aryl isochromans and their simple synthesis let these compounds appear to be interesting candidates for pharmaceutical interventions that protect against the deleterious action of ROS/RNS.

Mitochondria produce reactive nitrogen species via an arginine-independent pathway

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.

Minocycline, a possible neuroprotective agent in Leber’s hereditary optic neuropathy (LHON): Studies of cybrid cells bearing 11778 mutation

Lebers hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca(2+) imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.

Release patterns of excitatory and inhibitory amino acids within the hypothalamic supraoptic nucleus in response to direct nitric oxide administration during forced swimming in rats.

The effect of direct intrahypothalamic nitric oxide (NO) administration on the release of selected amino acids in the hypothalamic supraoptic nucleus (SON) with and without concomitant forced swimming was investigated. Adult male Wistar rats were chronically fitted with U-shaped microdialysis probes in the SON and forced to swim for 10-min in 20-degree C warm water. Thirty-min microdialysis samples were collected before, during and after the forced swimming period while NO was administered into the SON via microdialysis. The results show that NO administration in combination with forced swimming affects the release of aspartate, glutamate, taurine, and gamma aminobutyric acid (GABA) in different patterns. Whereas the release of the excitatory amino acids aspartate and glutamate was triggered only during NO administration and forced swimming, the inhibitory amino acids GABA and taurine were found in the extracellular fluid in increased concentrations also after the treatment. These data indicate that NO administration differently affects the release of excitatory and inhibitory amino acids within the SON. Thus, SON neurons which contain high concentrations of neuronal NO synthase, might contribute to the regulation of their own secretory activity by releasing NO that controls the orchestrated release of excitatory and inhibitory amino acids from axon terminals of afferences and interneurons as well as release from glial cells.