Inhibition Of mPT Research Articles

Melatonin modulates permeability transition pore and 5-hydroxydecanoate induced KATP channel inhibition in isolated brain mitochondria

There is increasing recognition of the magnitude of mitochondria in neurodegenerative disorders. Mitochondria play a key role in apoptotic and necrotic cell death. Melatonin (Mel), an indoleamine produced in several organs including the pineal gland has been known for its neuroprotective actions. In our study, we have investigated whether the mitochondrial ATP sensitive potassium (mtKATP) channel blocker 5-hydroxydecanoate (5-HD) and calcium (Ca2+) affects permeability transition pore (PTP) alterations in isolated brain mitochondria treated with melatonin (Mel) and cyclosporin A (CsA). Mitochondrial swelling, mitochondrial membrane potential (Δψm), ROS measurement and mitochondrial respiration were evaluated in isolated brain mitochondria. In our results, mitochondrial swelling stimulated by exposing Ca2+ ions and 5-HD associated by mPTP opening as depicted by modulation of CsA and Mel. In addition, Ca2+ and 5-HD decreased Δψm, depleted intracellular ROS, and inhibition of mitochondrial respiration (state 3 and state 4) in isolated brain mitochondria. Addition of Mel and CsA has shown significant restoration in mitochondrial swelling, Δψm, intracellular ROS measurement and mitochondrial respiration in isolated brain mitochondria. Therefore, we speculate the modulatory effect of Mel and CsA in mitochondria treated with 5-HD and Ca2+ hinders the mPTP-mediated mitochondrial dysfunction and cellular oxidative stress. We conclude that inhibition of mPT is one likely mechanism of CsA's and its neuroprotective actions. Development of neuroprotective agents including Mel targeting the mPTP therefore bears hope for future treatment of severe neurodegenerative diseases.

Ketone bodies mediate antiseizure effects through mitochondrial permeability transition.

Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.

Functional and Pharmacological Characteristics of Permeability Transition in Isolated Human Heart Mitochondria

The objective of the present study was to validate the presence and explore the characteristics of mitochondrial permeability transition (mPT) in isolated mitochondria from human heart tissue in order to investigate if previous findings in animal models of cardiac disorders are translatable to human disease. Mitochondria were rapidly isolated from fresh atrial tissue samples obtained from 14 patients undergoing Maze surgery due to atrial fibrillation. Human heart mitochondria exhibited typical mPT characteristics upon calcium overload such as swelling, evaluated by changes in light scattering, inhibition of respiration and loss of respiratory coupling. Swelling was a morphologically reversible event following transient calcium challenge. Calcium retention capacity (CRC), a quantitative measure of mPT sensitivity assayed by following extramitochondrial [Ca2+] and changes in respiration during a continuous calcium infusion, was significantly increased by cyclophilin D (CypD) inhibitors. The thiol-reactive oxidant phenylarsine oxide sensitized mitochondria to calcium-induced mPT. Release of the pro-apoptotic intermembrane protein cytochrome c was increased after, but not before, calcium discharge and respiratory inhibition in the CRC assay. From the present study, we conclude that adult viable heart mitochondria have a CypD- and oxidant-regulated mPT. The findings support that inhibition of mPT may be a relevant pharmacological target in human cardiac disease and may underlie the beneficial effect of cyclosporin A in reperfusion injury.

What makes you can also break you: mitochondrial permeability transition pore formation by the c subunit of the F1F0 ATP-synthase?

What makes you can also break you: mitochondrial permeability transition pore formation by the c subunit of the F1F0 ATP-synthase?

Abstract 2584: Inhibition of Mitochondrial Permeability Transition with NIM811 Improves Functional Recovery after Focal Cerebral Ischemia

Introduction: Mitochondrial dysfunction plays a seminal role in acute ischemic neuronal degeneration, the signature injury in stroke. A selective inhibitor of mitochondrial permeability transition (mPT) may prevent collapse of the mitochondrial membrane potential, thereby inhibiting the intrinsic pathway to neuronal death. Hypothesis: A novel mPT inhibitor, NIM811, will reduce infarct volume and improve functional recovery after focal cerebral ischemia. Methods: NIM811 was obtained as a gift from Novartis Pharma Ltd. (Basel, Switzerland). Focal cerebral ischemia was induced by unilateral carotid and middle cerebral artery occlusion for 2 hrs in male spontaneously hypertensive rats (SHRs). During post-ischemic reperfusion, animals received NIM811 (50 mg/kg at 1- and 24-hrs of post-ischemic reperfusion) or drug-free vehicle. Infarct volume was measured at 24 hrs or 7 days. Functional performance was assessed with 2 measures, fall latency time (FLT) in a Rotarod device and Removal Delay (RD) in a tape removal task, over 28 days. Results: When given after 1 hr of post-ischemic reperfusion, 50 mg/kg NIM811 reduced 24-hr infarct volume (63.6 ± 18.7 [SD] v. 89.3 ± 12.5 mm 3 in controls; n = 8 - 10/group; p ≤ 0.01; unpaired t -test). Serial dosing of NIM811 lowered 7-day infarct volume compared to controls (51.9 ± 7.4 v. 61.9 ± 10.3 mm 3 ; n = 13 - 16/group; p ≤ 0.01; unpaired t -test). In the Rotarod task, NIM811-treated animals had prolonged FLT over controls at all examination times up to 28 days (n = 7 per group; p ≤ 0.0001; ANOVA/Scheffe’s test). NIM811-treated SHRs also had shorter RD for discarding tape applied to the paretic forelimb, compared to controls (n = 7 per group; p ≤ 0.002; ANOVA/Scheffe’s test). Conclusions: Inhibition of mPT with NIM811 significantly reduced infarct volume, when given within a clinically relevant post-ischemic interval. Serial dosing of NIM811 achieved neuroprotective resilience at 7 days. Reductions in infarct volume by NIM811 were complemented by improved functional performance up to 28 days after ischemia. These promising results highlight the potential of NIM811 for neuroprotective treatment of stroke in man.

Cyclophilin D-Sensitive Mitochondrial Permeability Transition in Adult Human Brain and Liver Mitochondria

The mitochondrial permeability transition (mPT) is considered to be a major cause of cell death under a variety of pathophysiological conditions of the central nervous system (CNS) and other organs. Pharmacological inhibition or genetic knockout of the matrix protein cyclophilin D (CypD) prevents mPT and cell degeneration in several models of brain injury. If these findings in animal models are translatable to human disease, pharmacological inhibition of mPT offers a promising therapeutic target. The objective of this study was to validate the presence of a CypD-sensitive mPT in adult human brain and liver mitochondria. In order to perform functional characterization of human mitochondria, fresh tissue samples were obtained during hemorrhage or tumor surgery and mitochondria were rapidly isolated. Mitochondrial calcium retention capacity, a quantitative assay for mPT, was significantly increased by the CypD inhibitor cyclosporin A in both human brain and liver mitochondria, whereas thiol-reactive compounds and oxidants sensitized mitochondria to calcium-induced mPT. Brain mitochondria underwent swelling upon calcium overload, which was reversible upon calcium removal. To further explore mPT of human mitochondria, liver mitochondria were demonstrated to exhibit several classical features of the mPT phenomenon, such as calcium-induced loss of membrane potential and respiratory coupling, as well as release of the pro-apoptotic protein cytochrome c. We concluded that adult viable human brain and liver mitochondria possess an active CypD-sensitive mPT. Our findings support the rationale of CypD and mPT inhibition as pharmacological targets in acute and chronic neurodegeneration.

Evaluation of putative inhibitors of mitochondrial permeability transition for brain disorders — Specificity vs. toxicity

Inhibition of mitochondrial permeability transition (mPT) has emerged as a promising approach for neuroprotection and development of well-tolerated mPT inhibitors with favorable blood-brain barrier penetration is highly warranted. In a recent study, 28 clinically available drugs with a common heterocyclic structure were identified as mPT inhibitors e.g. trifluoperazine, promethazine and nortriptyline. In addition, neuroprotection by structurally unrelated drugs e.g. neurosteroids, 4-hydroxy-tamoxifen and trimetazidine has been attributed to direct inhibition of mPT. The regulation of mPT is complex and highly dependent on the prevailing experimental conditions. Several features of mPT, such as swelling, depolarization or NADH oxidation, can also occur independently of the mPT phenomenon. Here, in isolated rodent brain-derived and human liver mitochondria, we re-evaluate drugs promoted as potent mPT inhibitors. We address the definition of an mPT inhibitor and present strategies to reliably detect mPT inhibition in vitro. Surprisingly, none of the 12 compounds tested displayed convincing mPT inhibition or effects comparable to cyclophilin D inhibition by the non-immunosuppressive cyclophilin inhibitor D-MeAla 3-EtVal 4-Cyclosporin (Debio 025). Propofol and 2-aminoethoxydiphenyl borate (2-APB) inhibited swelling in de-energized mitochondria but did not increase calcium retention capacity (CRC). Progesterone, trifluoperazine, allopregnanolone and 4-hydroxy-tamoxifen dose-dependently reduced CRC and respiratory control and were thus toxic rather than beneficial to mitochondrial function. Interestingly, topiramate increased CRC at high concentrations likely by a mechanism separate from direct mPT inhibition. We conclude that a clinically relevant mPT inhibitor should have a mitochondrial target and increase mitochondrial calcium retention at concentrations which can be translated to human use.

Does Inhibition of Glycogen Synthase Kinase Protect in Mice?

See related article, pages 307–314 Preconditioning (PreC) and postconditioning (PostC) have been shown to initiate a number of signaling cascades that reduce cell death. However, the mechanisms by which these signals reduce cell death have been elusive.1 PreC has been shown to phosphorylate and thereby inhibit glycogen synthase kinase (GSK)-3β, and perfusion with GSK inhibitors has been shown to reduce cell death induced by ischemia/reperfusion, when added before ischemia2 or when added at the start of reperfusion.3–5 These studies are consistent with data in other tissues showing that inhibition of GSK-3β reduces apoptosis. Information regarding the mechanism by which inhibition of GSK protects has been provided by Juhaszova et al,6 who report that inhibition of GSK-3β delays the opening of the mitochondrial permeability transition pore (MPT) (see the Figure). The MPT is a large-conductance pore in the inner mitochondrial membrane which is opened under conditions associated with ischemia/reperfusion, such as high matrix reactive oxygen species and high matrix calcium. Pharmacological inhibitors of the MPT have been shown to reduce ischemia/reperfusion injury, suggesting that activation of MPT might have a role in ischemia/reperfusion-mediated cell death. However the molecular components of the MPT have not been identified.7 Figure. Nishino et al8 raise 2 questions: (1) whether inhibition of GSK is required for protection in mice; and (2) whether inhibition of GSK is protective in mouse hearts. PreC and PostC activate a number of redundant signaling pathways that lead to inhibition of MPT. The relative importance of different pathways may vary depending on the model and species. Previous studies6 have suggested that inhibition of GSK is a major signaling pathway that results in inhibition of MPT; this is illustrated with the large arrow. However, other pathways exist and it is not surprising that under different …

Re-evaluation of mitochondrial permeability transition as a primary neuroprotective target of minocycline

Minocycline has been shown to be neuroprotective in ischemic and neurodegenerative disease models and could potentially be relevant for clinical use. We revisited the hypothesis that minocycline acts through direct inhibition of calcium-induced mitochondrial permeability transition (mPT) resulting in reduced release of cytochrome c (cyt c). Minocycline, at high dosage, was found to prevent calcium-induced mitochondrial swelling under energized conditions similarly to the mPT inhibitor cyclosporin A (CsA) in rodent mitochondria derived from the CNS. In contrast to CsA, minocycline dose-dependently reduced mitochondrial calcium retention capacity (CRC) and respiratory control ratios and was ineffective in the de-energized mPT assay. Further, minocycline did not inhibit calcium- or tBid-induced cyt c release. We conclude that the neuroprotective mechanism of minocycline is likely not related to direct inhibition of mPT and propose that the mitochondrial effects of minocycline may contribute to toxicity rather than tissue protection at high dosing in animals and humans.

A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death

In ischemia–reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.

Brain-derived respiring mitochondria exhibit homogeneous, complete and cyclosporin-sensitive permeability transition.

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.