Saori Morota

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

Temporal increase of platelet mitochondrial respiration is negatively associated with clinical outcome in patients with sepsis

IntroductionMitochondrial dysfunction has been suggested as a contributing factor to the pathogenesis of sepsis-induced multiple organ failure. Also, restoration of mitochondrial function, known as mitochondrial biogenesis, has been implicated as a key factor for the recovery of organ function in patients with sepsis. Here we investigated temporal changes in platelet mitochondrial respiratory function in patients with sepsis during the first week after disease onset.MethodsPlatelets were isolated from blood samples taken from 18 patients with severe sepsis or septic shock within 48 hours of their admission to the intensive care unit. Subsequent samples were taken on Day 3 to 4 and Day 6 to 7. Eighteen healthy blood donors served as controls. Platelet mitochondrial function was analyzed by high-resolution respirometry. Endogenous respiration of viable, intact platelets suspended in their own plasma or phosphate-buffered saline (PBS) glucose was determined. Further, in order to investigate the role of different dehydrogenases and respiratory complexes as well as to evaluate maximal respiratory activity of the mitochondria, platelets were permeabilized and stimulated with complex-specific substrates and inhibitors.ResultsPlatelets suspended in their own septic plasma exhibited increased basal non-phosphorylating respiration (state 4) compared to controls and to platelets suspended in PBS glucose. In parallel, there was a substantial increase in respiratory capacity of the electron transfer system from Day 1 to 2 to Day 6 to 7 as well as compared to controls in both intact and permeabilized platelets oxidizing Complex I and/or II-linked substrates. No inhibition of respiratory complexes was detected in septic patients compared to controls. Non-survivors, at 90 days, had a more elevated respiratory capacity at Day 6 to 7 as compared to survivors. Cytochrome c increased over the time interval studied but no change in mitochondrial DNA was detected.ConclusionsThe results indicate the presence of a soluble plasma factor in the initial stage of sepsis inducing uncoupling of platelet mitochondria without inhibition of the electron transfer system. The mitochondrial uncoupling was paralleled by a gradual and substantial increase in respiratory capacity. This may reflect a compensatory response to severe sepsis or septic shock, that was most pronounced in non-survivors, likely correlating to the severity of the septic insult.

Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage.

Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further, we aimed to explore the limits of sample size and the impact of storage as well as to establish a wide range of reference data from different pediatric and adult cohorts. Our results indicate that platelet mitochondria are well suited for ex-vivo analysis with the need for minute sample amounts and excellent reproducibility and stability.

Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition

The mitochondrial permeability transition (mPT) is a potential pathogenic mechanism in neurodegeneration. Varying sensitivity to calcium‐induced mPT has been demonstrated for regions within the CNS possibly correlating with vulnerability following insults. The spinal cord is selectively vulnerable in e.g. amyotrophic lateral sclerosis and increased mPT sensitivity of mitochondria derived from the spinal cord has previously been demonstrated. In this study, we introduce whole‐body hypothermia prior to removal of CNS tissue to minimize the effects of differential tissue extraction prior to isolation of spinal cord and cortical brain mitochondria. Spinal cord mitochondria were able to retain considerably less calcium when administered as continuous infusion, which was not related to a general increased sensitivity of the mPT to calcium, its desensitization to calcium by the cyclophilin D inhibitor cyclosporin‐A, or to differences in respiratory parameters. Spinal cord mitochondria maintained a higher concentration of extramitochondrial calcium during infusion than brain mitochondria possibly related to an increased set‐point concentration for calcium uptake. A hampered transport and retention capacity of calcium may translate into an increased susceptibility of the spinal cord to neurodegenerative processes involving calcium‐mediated damage.

Patients with sepsis exhibit increased mitochondrial respiratory capacity in peripheral blood immune cells

IntroductionIn sepsis, mitochondria have been associated with both initial dysfunction and subsequent upregulation (biogenesis). However, the evolvement of mitochondrial function in sepsis over time is largely unknown, and we therefore investigated mitochondrial respiration in peripheral blood immune cells (PBICs) in sepsis patients during the first week after admission to the intensive care unit (ICU).MethodsPBICs from 20 patients with severe sepsis or septic shock were analyzed with high-resolution respirometry 3 times after admission to the ICU (within 48 hours, days 3 to 4 and days 6 to 7). Mitochondrial DNA (mtDNA), cytochrome c (Cyt c), and citrate synthase (CS) were measured as indicators of cellular mitochondrial content.ResultsIn intact PBICs with endogenous substrates, a gradual increase in cellular respiration reached 173% of controls after 1 week (P = 0.001). In permeabilized cells, respiration using substrates of complex I, II, and IV were significantly increased days 1 to 2, reaching 137%, 130%, and 173% of controls, respectively. In parallel, higher levels of CS activity, mtDNA, and Cyt c content in PBICs (211%, 243%, and 331% of controls for the respective indicators were found at days 6 to 7; P < 0.0001). No differences in respiratory capacities were noted between survivors and nonsurvivors at any of the time points measured.ConclusionsPBICs from patients with sepsis displayed higher mitochondrial respiratory capacities compared with controls, due to an increased mitochondrial content, as indicated by increased mitochondrial DNA, protein content, and enzyme activity. The results argue against mitochondrial respiratory dysfunction in this type of cells in sepsis.

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.

Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume.

Modulation of K+ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K+ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca2+ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K+ or H+ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoKATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K+ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H2O2-induced protein kinase Cϵ activation. Rather, increased K+ conductance reduced H2O2 generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC‐1β

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post‐neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER‐resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild‐type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue‐specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator‐activated receptor coactivator (PGC)‐1β, and Syvn1 mutants showed upregulation of PGC‐1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS‐102 abolished the negative regulation of PGC‐1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post‐translational regulator of PGC‐1β and a potential therapeutic target in obesity treatment.

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.

Probing the molecular mechanisms of neuronal degeneration: importance of mitochondrial dysfunction and calcineurin activation

Cerebral injury is a critical aspect of the management of patients in intensive care. Pathological conditions induced by cerebral ischemia, hypoxia, head trauma, and seizure activity can result in marked residual impairment of cerebral function. We have investigated the potential mechanisms leading to neuronal cell death in pathological conditions, with the aim of discovering therapeutic targets and methods to minimize neuronal damage resulting from insults directed at the central nervous system (CNS). Over the years, deeper understanding of the mechanisms of neuronal cell death has indeed evolved, enabling clinical critical care management to salvage neurons that are at the brink of degeneration and to support recovery of brain function. However, no substantial breakthrough has been achieved in the quest to develop effective pharmacological neuroprotective therapy directed at tissues of the CNS. The current situation is unacceptable, and preservation of function and protection of the brain from terminal impairment will be a vital medical issue in the twenty-first century. To achieve this goal, it is critical to clarify the key mechanisms leading to neuronal cell death. Here, we discuss the importance of the calcineurin/immunophilin signal transduction pathway and mitochondrial involvement in the detrimental chain of events leading to neuronal degeneration.