Sergei V. Baranov

Inhibition of mitochondrial protein import by mutant huntingtin

Mitochondrial dysfunction is associated with neuronal loss in Huntingtons disease (HD), a neurodegenerative disease caused by an abnormal polyglutamine expansion in huntingtin (Htt). However, the mechanisms linking mutant Htt and mitochondrial dysfunction in HD remain unknown. We identify an interaction between mutant Htt and the TIM23 mitochondrial protein import complex. Remarkably, recombinant mutant Htt directly inhibited mitochondrial protein import in vitro. Furthermore, mitochondria from brain synaptosomes of presymptomatic HD model mice and from mutant Htt-expressing primary neurons exhibited a protein import defect, suggesting that deficient protein import is an early event in HD. The mutant Htt–induced mitochondrial import defect and subsequent neuronal death were attenuated by overexpression of TIM23 complex subunits, demonstrating that deficient mitochondrial protein import causes mutant Htt-induced neuronal death. Collectively, these findings provide evidence for a direct link between mutant Htt, mitochondrial dysfunction and neuronal pathology, with implications for mitochondrial protein import–based therapies in HD.

Melatonin inhibits the caspase-1/cytochrome c/caspase-3 cell death pathway, inhibits MT1 receptor loss and delays disease progression in a mouse model of amyotrophic lateral sclerosis.

Caspase-mediated cell death contributes to the pathogenesis of motor neuron degeneration in the mutant SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS), along with other factors such as inflammation and oxidative damage. By screening a drug library, we found that melatonin, a pineal hormone, inhibited cytochrome c release in purified mitochondria and prevented cell death in cultured neurons. In this study, we evaluated whether melatonin would slow disease progression in SOD1(G93A) mice. We demonstrate that melatonin significantly delayed disease onset, neurological deterioration and mortality in ALS mice. ALS-associated ventral horn atrophy and motor neuron death were also inhibited by melatonin treatment. Melatonin inhibited Rip2/caspase-1 pathway activation, blocked the release of mitochondrial cytochrome c, and reduced the overexpression and activation of caspase-3. Moreover, for the first time, we determined that disease progression was associated with the loss of both melatonin and the melatonin receptor 1A (MT1) in the spinal cord of ALS mice. These results demonstrate that melatonin is neuroprotective in transgenic ALS mice, and this protective effect is mediated through its effects on the caspase-mediated cell death pathway. Furthermore, our data suggest that melatonin and MT1 receptor loss may play a role in the pathological phenotype observed in ALS. The above observations indicate that melatonin and modulation of Rip2/caspase-1/cytochrome c or MT1 pathways may be promising therapeutic approaches for ALS.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release

Significance This paper describes the finding that mitochondria synthesize and release melatonin and have their selective G protein-coupled receptor (GPCR) in the outer membrane. We further demonstrate that mitochondrial melatonin type 1 receptors respond to melatonin by activating heterotrimeric G proteins located in the intermembrane space and inhibit stress-mediated cytochrome c release. This remarkable insight changes our classical understanding of biological GPCR function by showing that a cellular organelle both synthesizes and has a signaling receptor for a specific ligand. Implicit with our original work is the existence of an automitocrine signaling pathway by which melatonin prevents neurodegeneration associated with mitochondrial cytochrome c release and downstream caspase activation. G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and β-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, “automitocrine,” analogous to “autocrine” when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand–receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

Reactive γ-ketoaldehydes formed via the isoprostane pathway disrupt mitochondrial respiration and calcium homeostasis

Isoketals (IsoKs) are gamma-ketoaldehydes formed via the isoprostane pathway of arachidonic acid peroxidation and are among the most reactive by-products of lipid peroxidation. IsoKs selectively adduct to protein lysine residues and are highly cytotoxic, but the targets and molecular events involved in IsoK-induced cell death are poorly defined. Our previous work established that physiologically relevant aldehydes induce mitochondrial dysfunction (Kristal et al., J. Biol. Chem.271:6033-6038; 1996). We therefore examined whether IsoKs induced mitochondrial dysfunction. Incubation of mitochondria with synthetic IsoKs in the presence or absence of Ca(2+) was associated with alterations in mitochondrial respiration, membrane potential (DeltaPsi), and pyridine nucleotide redox state. IsoKs dose dependently (0.5-4microM) accelerated liver mitochondria swelling induced by low concentrations of Ca(2+) and Zn(2+) or by the prooxidant tert-butylhydroperoxide, and release of cytochrome c, with similar observations in heart/brain mitochondria. The mitochondrial permeability transition (mPT) inhibitor cyclosporine A delayed IsoK-induced mitochondria dysfunction. The actions of IsoKs are consistent with interactions with cytochrome c, a protein rich in lysine residues. Direct reaction of IsoKs with select lysines in cytochrome c was demonstrated using high-resolution mass spectrometry. Overall, these results suggest that IsoKs may, in part, mediate their cytotoxic effects through induction of the mPT and subsequent activation of downstream cell death cascades.

Cardiolipin signaling mechanisms: collapse of asymmetry and oxidation.

SIGNIFICANCE An ancient anionic phospholipid, cardiolipin (CL), ubiquitously present in prokaryotic and eukaryotic membranes, is essential for several structural and functional purposes. RECENT ADVANCES The emerging role of CLs in signaling has become the focus of many studies. CRITICAL ISSUES In this work, we describe two major pathways through which mitochondrial CLs may fulfill the signaling functions via utilization of their (i) asymmetric distribution across membranes and translocations, leading to the surface externalization and (ii) ability to undergo oxidation reactions to yield the signature products recognizable by the executionary machinery of cells. FUTURE DIRECTIONS We present a concept that CLs and their oxidation/hydrolysis products constitute a rich communication language utilized by mitochondria of eukaryotic cells for diversified regulation of cell physiology and metabolism as well as for inter-cellular interactions.

Kinetic Model for Ca2+-induced Permeability Transition in Energized Liver Mitochondria Discriminates between Inhibitor Mechanisms

Cytotoxicity associated with pathophysiological Ca2+ overload (e.g. in stroke) appears mediated by an event termed the mitochondrial permeability transition (mPT). We built and solved a kinetic model of the mPT in populations of isolated rat liver mitochondria that quantitatively describes Ca2+-induced mPT as a two-step sequence of pre-swelling induction followed by Ca2+-driven, positive feedback, autocatalytic propagation. The model was formulated as two differential equations, each directly related to experimental parameters (Ca2+ flux/mitochondrial swelling). These parameters were simultaneously assessed using a spectroscopic approach to monitor multiple mitochondrial properties. The derived kinetic model correctly identifies a correlation between initial Ca2+ concentration and delay interval prior to mPT induction. Within the models framework, Ru-360 (a ruthenium complex) and Mg2+ were shown to compete with the Ca2+-stimulated initiation phase of mPT induction, consistent with known inhibition at the phenomenological level of the Ca2+ uniporter. The model further reveals that Mg2+, but not Ru-360, inhibits Ca2+-induced effects on a downstream stage of mPT induction at a site distinct from the uniporter. The analytical approach was then applied to promethazine, an FDA-approved drug previously shown to inhibit both mPT and ischemia-reperfusion injury. Kinetic analysis revealed that promethazine delayed mPT induction in a manner qualitatively distinct from that of lower concentrations of Mg2+. In summary, we have developed a kinetic model to aid in the quantitative characterization of mPT induction. This model is consistent with/informative about the biochemistry of several mPT inhibitors, and its success suggests that this kinetic approach can aid in the classification of agents or targets that modulate mPT induction.

Dipyrone Inhibits Neuronal Cell Death and Diminishes Hypoxic/Ischemic Brain Injury

BACKGROUND:Dipyrone is an analgesic and antipyretic drug usually prescribed for patients with inflammatory conditions. We recently identified dipyrone as an antiapoptotic agent by screening a library of 1040 compounds for their ability to inhibit cytochrome c release from isolated mitochondria. OBJECTIVE:We investigated the potential neuroprotective properties of dipyrone in cerebral ischemia. METHODS:We evaluated the protective effects of dipyrone in experimental models of neuronal hypoxia/ischemia, including an oxygen/glucose deprivation model in primary cerebrocortical neurons and a focal cerebral ischemia model in mice. RESULTS:Dipyrone reduced hypoxia/ischemia injury in both cellular and animal models. Dipyrone inhibited the release of cytochrome c and other mitochondrial apoptogenic factors from mitochondria into the cytoplasm, and attenuated subsequent caspase-9 and caspase-3 activation both in vitro and in vivo. Moreover, dipyrone prevented ischemia-induced changes in Bcl-2 and tBid, and ameliorated oxygen/glucose deprivation-mediated loss of mitochondrial membrane potential. Dipyrone also inhibited ischemia-induced reactive microgliosis. In the cellular models evaluated, dipyrone did not inhibit oxygen/glucose deprivation-induced cyclooxygenase-2 activation. CONCLUSION:Dipyrone is remarkably neuroprotective in cerebral ischemia, and its cyclooxygenase-independent protective properties are, at least in part, due to the inhibition of mitochondrial cell death cascades.

Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization

Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes, and cancer. There is growing recognition of the importance of mitochondria both as targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semiautomated methods to disrupt tissue using kidney and muscle mitochondria preparations as exemplars. These methods use the Barocycler, the PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of overhomogenization. Mitochondria isolated using the PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of the PCT Shredder and the Barocycler to be used to purify mitochondrial preparations.

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q9; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.

Calcium controls the assembly of the photosynthetic water-oxidizing complex: a cadmium(II) inorganic mutant of the Mn4Ca core

Perturbation of the catalytic inorganic core (Mn4Ca1OxCly) of the photosystem II-water-oxidizing complex (PSII-WOC) isolated from spinach is examined by substitution of Ca2+ with cadmium(II) during core assembly. Cd2+ inhibits the yield of reconstitution of O2-evolution activity, called photoactivation, starting from the free inorganic cofactors and the cofactor-depleted apo-WOC-PSII complex. Ca2+ affinity increases following photooxidation of the first Mn2+ to Mn3+ bound to the ‘high-affinity’ site. Ca2+ binding occurs in the dark and is the slowest overall step of photoactivation (IM1→IM1* step). Cd2+ competitively blocks the binding of Ca2+ to its functional site with 10- to 30-fold higher affinity, but does not influence the binding of Mn2+ to its high-affinity site. By contrast, even 10-fold higher concentrations of Cd2+ have no effect on O2-evolution activity in intact PSII-WOC. Paradoxically, Cd2+ both inhibits photoactivation yield, while accelerating the rate of photoassembly of active centres 10-fold relative to Ca2+. Cd2+ increases the kinetic stability of the photooxidized Mn3+ assembly intermediate(s) by twofold (mean lifetime for dark decay). The rate data provide evidence that Cd2+ binding following photooxidation of the first Mn3+, IM1→IM1*, causes three outcomes: (i) a longer intermediate lifetime that slows IM1 decay to IM0 by charge recombination, (ii) 10-fold higher probability of attaining the degrees of freedom (either or both cofactor and protein d.f.) needed to bind and photooxidize the remaining 3 Mn2+ that form the functional cluster, and (iii) increased lability of Cd2+ following Mn4 cluster assembly results in (re)exchange of Cd2+ by Ca2+ which restores active O2-evolving centres. Prior EPR spectroscopic data provide evidence for an oxo-bridged assembly intermediate, Mn3+(μ-O2−)Ca2+, for IM1*. We postulate an analogous inhibited intermediate with Cd2+ replacing Ca2+.