Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy.

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome.

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by approximately 7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.

Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat.

Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinson's disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP(+), or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP(+), or paraquat is independent of complex I inhibition.

Age modifies respiratory complex I and protein homeostasis in a muscle type-specific manner.

Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age-induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I-derived H2 O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial-targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue-specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.

Effect of omega-3 fatty acid oxidation products on the cellular and mitochondrial toxicity of BDE 47.

High levels of the flame retardant 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) have been detected in Pacific salmon sampled near urban areas, raising concern over the safety of salmon consumption. However, salmon fillets also contain the antioxidants eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), whose oxidation products induce cellular antioxidant responses. Because oxidative stress is a mechanism of BDE 47 toxicity, we hypothesized that oxidized EPA and DHA can ameliorate the cellular and mitochondrial toxicity of BDE 47. HepG2 cells were treated with a mixture of oxidized EPA and DHA (oxEPA/oxDHA) at a ratio relevant to salmon consumption (1.5/1 oxEPA/oxDHA) followed by exposure to 100 µM BDE 47. Pretreatment with oxEPA/oxDHA for 12 h prior to BDE 47 exposure prevented BDE 47-mediated depletion of glutathione, and increased expression of antioxidant response genes. oxEPA/oxDHA also reduced the level of reactive oxygen species production by BDE 47. The oxEPA/oxDHA antioxidant responses were associated with partial protection against BDE 47-induced loss of viability and also mitochondrial membrane potential. Mitochondrial electron transport system functional analysis revealed extensive inhibition of State 3 respiration and maximum respiratory capacity by BDE 47 were partially reversed by oxEPA/oxDHA. Our findings indicate that the antioxidant effects of oxEPA/oxDHA protect against short exposures to BDE 47, including a protective role of these compounds on maintaining cellular and mitochondrial function.

Mitochondrial-targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice.

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial-targeted peptide SS-31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg(-1) of SS-31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and (31) P magnetic resonance spectroscopy. Age-related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS-31 treatment, while SS-31 had no observable effect on young muscle. These effects of SS-31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H2 O2 emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS-31 treatment, and eight days of SS-31 treatment led to increased whole-animal endurance capacity. These data demonstrate that SS-31 represents a new strategy for reversing age-related deficits in skeletal muscle with potential for translation into human use.

Altered anesthetic sensitivity of mice lacking Ndufs4, a subunit of mitochondrial complex I.

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABA(A) and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.

Fatal breathing dysfunction in a mouse model of Leigh syndrome.

Leigh syndrome (LS) is a subacute necrotizing encephalomyelopathy with gliosis in several brain regions that usually results in infantile death. Loss of murine Ndufs4, which encodes NADH dehydrogenase (ubiquinone) iron-sulfur protein 4, results in compromised activity of mitochondrial complex I as well as progressive neurodegenerative and behavioral changes that resemble LS. Here, we report the development of breathing abnormalities in a murine model of LS. Magnetic resonance imaging revealed hyperintense bilateral lesions in the dorsal brain stem vestibular nucleus (VN) and cerebellum of severely affected mice. The mutant mice manifested a progressive increase in apnea and had aberrant responses to hypoxia. Electrophysiological recordings within the ventral brain stem pre-Bötzinger respiratory complex were also abnormal. Selective inactivation of Ndufs4 in the VN, one of the principle sites of gliosis, also led to breathing abnormalities and premature death. Conversely, Ndufs4 restoration in the VN corrected breathing deficits and prolonged the life span of knockout mice. These data demonstrate that mitochondrial dysfunction within the VN results in aberrant regulation of respiration and contributes to the lethality of Ndufs4-knockout mice.

Mitochondrion-derived reactive oxygen species lead to enhanced amyloid beta formation.

AIMS: Intracellular amyloid beta (Aß) oligomers and extracellular Aß plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aß production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. RESULTS: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aß. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aß, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aß was partly reduced by an antioxidant, indicating that Aß formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aß levels in vivo. INNOVATION: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aß production in vitro and in vivo. CONCLUSION: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aß itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.

Reduced coupling of oxidative phosphorylation in vivo precedes electron transport chain defects due to mild oxidative stress in mice.

Oxidative stress and mitochondrial function are at the core of many degenerative conditions. However, the interaction between oxidative stress and in vivo mitochondrial function is unclear. We used both pharmacological (2 week paraquat (PQ) treatment of wild type mice) and transgenic (mice lacking Cu, Zn-superoxide dismutase (SOD1(-/-))) models to test the effect of oxidative stress on in vivo mitochondrial function in skeletal muscle. Magnetic resonance and optical spectroscopy were used to measure mitochondrial ATP and oxygen fluxes and cell energetic state. In both models of oxidative stress, coupling of oxidative phosphorylation was significantly lower (lower P/O) at rest in vivo in skeletal muscle and was dose-dependent in the PQ model. Despite this reduction in efficiency, in vivo mitochondrial phosphorylation capacity (ATPmax) was maintained in both models, and ex vivo mitochondrial respiration in permeabilized muscle fibers was unchanged following PQ treatment. In association with the reduced P/O, PQ treatment led to a dose-dependent reduction in PCr/ATP ratio and increased phosphorylation of AMPK. These results indicate that oxidative stress uncouples oxidative phosphorylation in vivo and results in energetic stress in the absence of defects in the mitochondrial electron transport chain.

Synthesis and in vitro evaluation of new benzovesamicol analogues as potential imaging probes for the vesicular acetylcholine transporter.

Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition against binding of [3H]vesamicol to human VAChT. Of the eight enantiomers, (E)-(R,R)-5-AOIBV [(R,R)-3], and (R,R)-5-FPOBV [(R,R)-4] displayed the highest binding affinities for VAChT (Kd=0.45 and 0.77 nM, respectively), which indicated that an elongation of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-(E)-3-iodoallyloxy or 5-fluoropropoxy substituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer (R,R)-4-MAIBV [(R,R)-16], which retains the basic structure of (-)-5-IBVM but possess an additional aminomethyl substituent in the 4-position of the piperidine ring, displayed lower binding affinity (Kd=8.8 nM). Nevertheless, the result suggests that substitution at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected, the corresponding (S,S) enantiomers displayed lower Kd values, they were approximately 10-fold lower in the case of (S,S)-5-FPOBV (Kd=8.4 nM) and (E)-(S,S)-5-AOIBV (Kd=4.3 nM). (R,R)-3, and (R,R)-4 showed the same high affinity for VAChT as (-)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals.