Chronic social stress disrupts the intracellular redistribution of brain hexokinase 3 induced by shifts in peripheral glucose levels.

Chronic stress has the potential to impair health and may increase the vulnerability for psychiatric disorders. Emerging evidence suggests that specific neurometabolic dysfunctions play a role herein. In mice, chronic social defeat (CSD) stress reduces cerebral glucose uptake despite hyperglycemia. We hypothesized that this metabolic decoupling would be reflected by changes in contact sites between mitochondria and the endoplasmic reticulum, important intracellular nutrient sensors, and signaling hubs. We thus analyzed the proteome of their biochemical counterparts, mitochondria-associated membranes (MAMs) from whole brain tissue obtained from CSD and control mice. This revealed a lack of the glucose-metabolizing enzyme hexokinase 3 (HK3) in MAMs from CSD mice. In controls, HK3 protein abundance in MAMs and also in striatal synaptosomes correlated positively with peripheral blood glucose levels, but this connection was lost in CSD. We conclude that the ability of HK3 to traffic to sites of need, such as MAMs or synapses, is abolished upon CSD and surmise that this contributes to a cellular dysfunction instigated by chronic stress. KEY MESSAGES : Chronic social defeat (CSD) alters brain glucose metabolism CSD depletes hexokinase 3 (HK3) from mitochondria-associated membranes (MAMs) CSD results in loss of positive correlation between blood glucose and HK3 in MAMs and synaptosomes.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

ND3 Cys39 in complex I is exposed during mitochondrial respiration.

Mammalian complex I can adopt catalytically active (A-) or deactive (D-) states. A defining feature of the reversible transition between these two defined states is thought to be exposure of the ND3 subunit Cys39 residue in the D-state and its occlusion in the A-state. As the catalytic A/D transition is important in health and disease, we set out to quantify it by measuring Cys39 exposure using isotopic labeling and mass spectrometry, in parallel with complex I NADH/CoQ oxidoreductase activity. To our surprise, we found significant Cys39 exposure during NADH/CoQ oxidoreductase activity. Furthermore, this activity was unaffected if Cys39 alkylation occurred during complex I-linked respiration. In contrast, alkylation of catalytically inactive complex I irreversibly blocked the reactivation of NADH/CoQ oxidoreductase activity by NADH. Thus, Cys39 of ND3 is exposed in complex I during mitochondrial respiration, with significant implications for our understanding of the A/D transition and the mechanism of complex I.

Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart.

PURPOSE: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide. METHODS AND RESULTS: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group. CONCLUSION: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice.

Convergent evolution of conserved mitochondrial pathways underlies repeated adaptation to extreme environments.

Extreme environments test the limits of life; yet, some organisms thrive in harsh conditions. Extremophile lineages inspire questions about how organisms can tolerate physiochemical stressors and whether the repeated colonization of extreme environments is facilitated by predictable and repeatable evolutionary innovations. We identified the mechanistic basis underlying convergent evolution of tolerance to hydrogen sulfide (H2S)-a toxicant that impairs mitochondrial function-across evolutionarily independent lineages of a fish (Poecilia mexicana, Poeciliidae) from H2S-rich springs. Using comparative biochemical and physiological analyses, we found that mitochondrial function is maintained in the presence of H2S in sulfide spring P. mexicana but not ancestral lineages from nonsulfidic habitats due to convergent adaptations in the primary toxicity target and a major detoxification enzyme. Genome-wide local ancestry analyses indicated that convergent evolution of increased H2S tolerance in different populations is likely caused by a combination of selection on standing genetic variation and de novo mutations. On a macroevolutionary scale, H2S tolerance in 10 independent lineages of sulfide spring fishes across multiple genera of Poeciliidae is correlated with the convergent modification and expression changes in genes associated with H2S toxicity and detoxification. Our results demonstrate that the modification of highly conserved physiological pathways associated with essential mitochondrial processes mediates tolerance to physiochemical stress. In addition, the same pathways, genes, and-in some instances-codons are implicated in H2S adaptation in lineages that span 40 million years of evolution.

Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.

Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.

Species- and tissue-specific differences in ROS metabolism during exposure to hypoxia and hyperoxia plus recovery in marine sculpins.

Animals that inhabit environments that fluctuate in oxygen must not only contend with disruptions to aerobic metabolism, but also the potential effects of reactive oxygen species (ROS). The goal of this study was to compare aspects of ROS metabolism in response to O2 variability (6 h hypoxia or hyperoxia, with subsequent normoxic recovery) in two species of intertidal sculpin fishes (Cottidae, Actinopterygii) that can experience O2 fluctuations in their natural environment and differ in whole-animal hypoxia tolerance. To assess ROS metabolism, we measured the ratio of glutathione to glutathione disulfide as an indicator of tissue redox environment, MitoP/MitoB ratio to assess in vivo mitochondrial ROS generation, thiobarbituric acid reactive substances (TBARS) for lipid peroxidation, and total oxidative scavenging capacity (TOSC) in the liver, brain and gill. In the brain, the more hypoxia-tolerant Oligocottusmaculosus showed large increases in TBARS levels following hypoxia and hyperoxia exposure that were generally not associated with large changes in mitochondrial H2O2 In contrast, the less-tolerant Scorpaenichthysmarmoratus showed no significant changes in TBARS or mitochondrial H2O2 in the brain. More moderate increases were observed in the liver and gill of O. maculosus exposed to hypoxia and hyperoxia with normoxic recovery, whereas S. marmoratus had a greater response to O2 variability in these tissues compared with the brain. Our results show a species- and tissue-specific relationship between hypoxia tolerance and ROS metabolism.

Detection of changes in mitochondrial hydrogen sulfide i n vivo in the fish model Poecilia mexicana (Poeciliidae).

In this paper, we outline the use of a mitochondria-targeted ratiometric mass spectrometry probe, MitoA, to detect in vivo changes in mitochondrial hydrogen sulfide (H2S) in Poecilia mexicana (family Poeciliidae). MitoA is introduced via intraperitoneal injection into the animal and is taken up by mitochondria, where it reacts with H2S to form the product MitoN. The MitoN/MitoA ratio can be used to assess relative changes in the amounts of mitochondrial H2S produced over time. We describe the use of MitoA in the fish species P. mexicana to illustrate the steps for adopting the use of MitoA in a new organism, including extraction and purification of MitoA and MitoN from tissues followed by tandem mass spectrometry. In this proof-of-concept study we exposed H2S tolerant P. mexicana to 59 µM free H2S for 5 h, which resulted in increased MitoN/MitoA in brain and gills, but not in liver or muscle, demonstrating increased mitochondrial H2S levels in select tissues following whole-animal H2S exposure. This is the first time that accumulation of H2S has been observed in vivo during whole-animal exposure to free H2S using MitoA. This article has an associated First Person interview with the first author of the paper.

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.

Attenuation of oxidative damage by targeting mitochondrial complex I in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Establishing sustained reoxygenation/reperfusion ensures not only the recovery, but may initiate a reperfusion injury in which oxidative stress plays a major role. This study offers the mechanism and this mechanism-specific therapeutic strategy against excessive release of reactive oxygen species (ROS) associated with reperfusion-driven recovery of mitochondrial metabolism. AIMS AND METHODS: In neonatal mice subjected to cerebral hypoxia-ischaemia (HI) and reperfusion, we examined conformational changes and activity of mitochondrial complex I with and without post-HI administration of S-nitrosating agent, MitoSNO. Assessment of mitochondrial ROS production, oxidative brain damage, neuropathological and neurofunctional outcomes were used to define neuroprotective strength of MitoSNO. A specificity of reperfusion-driven mitochondrial ROS production to conformational changes in complex I was examined in-vitro. RESULTS: HI deactivated complex I, changing its conformation from active form (A) into the catalytically dormant, de-active form (D). Reperfusion rapidly converted the D-form into the A-form and increased ROS generation. Administration of MitoSNO at the onset of reperfusion, decelerated D→A transition of complex I, attenuated oxidative stress, and significantly improved neurological recovery. In cultured neurons, after simulated ischaemia-reperfusion injury, MitoSNO significantly reduced ROS generation and neuronal mortality. In isolated mitochondria subjected to anoxia-reoxygenation, MitoSNO restricted ROS release during D→A transitions. CONCLUSION: Rapid D→A conformation in response to reperfusion reactivates complex I. This is essential not only for metabolic recovery, but also contributes to excessive release of mitochondrial ROS and reperfusion injury. We propose that the initiation of reperfusion should be followed by pharmacologically-controlled gradual reactivation of complex I.

MitoNeoD: A Mitochondria-Targeted Superoxide Probe.

Mitochondrial superoxide (O2⋅-) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅-, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅- probe, MitoNeoD, which can assess O2⋅- changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅--sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅- over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅- from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅- production in health and disease.

Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA.

Hydrogen sulfide (H2S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H2S source and sink, and many of the biological effects of H2S relate to its interactions with mitochondria. However, the significance of mitochondrial H2S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H2S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H2S probe, MitoA, which can be used to assess relative changes in mitochondrial H2S levels in vivo MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo There, the aryl azido group reacts with H2S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H2S in vivo Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H2S levels in vivo As a proof of principle we used MitoA to show that H2S levels increase in vivo during myocardial ischemia.

Design, synthesis, and characterization of peptide-based rab geranylgeranyl transferase inhibitors.

Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors.

A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications.

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.

Use of cerny epoxides for the accelerated synthesis of glycosaminoglycans.

[reaction: see text]. 1,6:2,3-Dianhydrohexopyranoses (Cerny epoxides) are versatile intermediates for the synthesis of glycosaminoglycans. Complex heparan and chondroitin sulfate disaccharide synthons can be assembled from a single common precursor in a short sequence of steps.