Phospholipids can regulate complex I assembly independent of their role in maintaining mitochondrial membrane integrity.

Several phospholipid (PL) molecules are intertwined with some mitochondrial complex I (CI) subunits in the membrane domain of CI, but their function is unclear. We report that when the Drosophila melanogaster ortholog of the intramitochondrial PL transporter, STARD7, is severely disrupted, assembly of the oxidative phosphorylation (OXPHOS) system is impaired, and the biogenesis of several CI subcomplexes is hampered. However, intriguingly, a restrained knockdown of STARD7 impairs the incorporation of NDUFS5 and NDUFA1 into the proximal part of the CI membrane domain without directly affecting the incorporation of subunits in the distal part of the membrane domain, OXPHOS complexes already assembled, or mitochondrial cristae integrity. Importantly, the restrained knockdown of STARD7 appears to induce a modest amount of cardiolipin remodeling, indicating that there could be some alteration in the composition of the mitochondrial phospholipidome. We conclude that PLs can regulate CI biogenesis independent of their role in maintaining mitochondrial membrane integrity.

Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.

The membrane domain of respiratory complex I accumulates during muscle aging in Drosophila melanogaster.

The boot-shaped respiratory complex I (CI) consists of a mitochondrial matrix and membrane domain organized into N-, Q- and P-modules. The N-module is the most distal part of the matrix domain, whereas the Q-module is situated between the N-module and the membrane domain. The proton-pumping P-module is situated in the membrane domain. We explored the effect of aging on the disintegration of CI and its constituent subcomplexes and modules in Drosophila flight muscles. We find that the fully-assembled complex remains largely intact in aged flies. And while the effect of aging on the stability of many Q- and N-module subunits in subcomplexes was stochastic, NDUFS3 was consistently down-regulated in subcomplexes with age. This was associated with an accumulation of many P-module subunits in subcomplexes. The potential significance of these studies is that genetic manipulations aimed at boosting, perhaps, a few CI subunits may suffice to restore the whole CI biosynthesis pathway during muscle aging.

The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury.

Acute Lung Injury (ALI) due to inhaled pathogens causes high mortality. Underlying mechanisms are inadequately understood. Here, by optical imaging of live mouse lungs we show that a key mechanism is the viability of cytosolic Ca2+ buffering by the mitochondrial Ca2+ uniporter (MCU) in the lung's surfactant-secreting, alveolar type 2 cells (AT2). The buffering increased mitochondrial Ca2+ and induced surfactant secretion in wild-type mice, but not in mice with AT2-specific MCU knockout. In the knockout mice, ALI due to intranasal LPS instillation caused severe pulmonary edema and mortality, which were mitigated by surfactant replenishment prior to LPS instillation, indicating surfactant's protective effect against alveolar edema. In wild-type mice, intranasal LPS, or Pseudomonas aeruginosa decreased AT2 MCU. Loss of MCU abrogated buffering. The resulting mortality was reduced by spontaneous recovery of MCU expression, or by MCU replenishment. Enhancement of AT2 mitochondrial buffering, hence endogenous surfactant secretion, through MCU replenishment might be a therapy against ALI.

Mitochondrial respiratory chain protein co-regulation in the human brain.

Mitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimer's disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2-4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.

IDH2-mediated regulation of the biogenesis of the oxidative phosphorylation system.

Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI, but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that genetic disruption of the mitochondrial NADPH-generating enzyme, isocitrate dehydrogenase 2 (IDH2), in Drosophila flight muscles results in elevated ROS levels and impairment of assembly of the oxidative phosphorylation system (OXPHOS). Mechanistically, this begins with an inhibition of biosynthesis of the matrix domain of CI and progresses to involve multiple OXPHOS complexes. Despite activation of multiple compensatory mechanisms, including enhanced coenzyme Q biosynthesis and the mitochondrial unfolded protein response, ferroptotic cell death ensues. Disruption of enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype, thereby implicating hydrogen peroxide as the signaling molecule involved. Thus, IDH2 modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

Dissecting the concordant and disparate roles of NDUFAF3 and NDUFAF4 in mitochondrial complex I biogenesis.

Distinct sub-assemblies (modules) of mitochondrial complex I (CI) are assembled with the assistance of CI Assembly Factors (CIAFs) through mechanisms that are incompletely defined. Here, using genetic analyses in Drosophila, we report that when either of the CIAFs - NDUFAF3 or NDUFAF4 - is disrupted, biogenesis of the Q-, N-, and PP-b-modules of CI is impaired. This is due, at least in part, to the compromised integration of NDUFS3 and NDUFS5 into the Q-, and PP-b-modules, respectively, coupled with a destabilization of another CIAF, TIMMDC1, in assembly intermediates. Notably, forced expression of NDUFAF4 rescues the biogenesis defects in the Q-module and some aspects of the defects in the PP-b-module of CI when NDUFAF3 is disrupted. Altogether, our studies furnish new fundamental insights into the mechanism by which NDUFAF3 and NDUFAF4 regulate CI assembly and raises the possibility that certain point mutations in NDUFAF3 may be rescued by overexpression of NDUFAF4.

Mitochondria in epithelial ovarian carcinoma exhibit abnormal phenotypes and blunted associations with biobehavioral factors.

Malignant tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and mitochondria in malignant tumors remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign tissue and in high-grade epithelial ovarian carcinoma (EOC) in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n = 1435) showed that gene expression of specific mitochondrial proteins in EOC is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in tissue from 51 patients with benign ovarian masses and 128 patients with high-grade EOC revealed that compared to benign tissue, EOCs exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P < 0.001) but similar mitochondrial DNA (mtDNA) levels (- 3.1%), documenting abnormal mitochondrial phenotypes in EOC. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r = - 0.43 to 0.47). In contrast, serous EOCs showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in EOC and warrant further research on the link between biobehavioral factors and mitochondria in cancer.

The Mitochondrial Calcium Uniporter of Pulmonary Type 2 Cells Determines Severity of ARDS.

Acute lung immunity to inhaled pathogens elicits defensive pneumonitis that may convert to the Acute Respiratory Distress Syndrome (ARDS), causing high mortality. Mechanisms underlying the conversion are not understood, but are of intense interest because of the ARDS-induced mortality in the ongoing Covid-19 pandemic. Here, by optical imaging of live lungs we show that key to the lethality is the functional status of mitochondrial Ca2+ buffering across the mitochondrial Ca2+ uniporter (MCU) in the alveolar type 2 cells (AT2), which protect alveolar stability. In mice subjected to ARDS by airway exposure to lipopolysaccharide (LPS), or to Pseudomonas aeruginosa, there was marked loss of MCU expression in AT2. The ability of mice to survive ARDS depended on the extent to which the MCU expression recovered, indicating that the viability of Ca2+ buffering by AT2 mitochondria critically determines ARDS severity. Mitochondrial transfer to enhance AT2 MCU expression might protect against ARDS.

Analyzing the integrity of oxidative phosphorylation complexes in Drosophila flight muscles.

Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities. For complete details on the use and execution of this protocol, please refer to Murari et al. (2020).

An antibody toolbox to track complex I assembly defines AIF's mitochondrial function.

An ability to comprehensively track the assembly intermediates (AIs) of complex I (CI) biogenesis in Drosophila will enable the characterization of the precise mechanism(s) by which various CI regulators modulate CI assembly. Accordingly, we generated 21 novel antibodies to various mitochondrial proteins and used this resource to characterize the mechanism by which apoptosis-inducing factor (AIF) regulates CI biogenesis by tracking the AI profile observed when AIF expression is impaired. We find that when the AIF-Mia40 translocation complex is disrupted, the part of CI that transfers electrons to ubiquinone is synthesized but fails to progress in the CI biosynthetic pathway. This is associated with a reduction in intramitochondrial accumulation of the Mia40 substrate, MIC19. Importantly, knockdown of either MIC19 or MIC60, components of the mitochondrial contact site and cristae organizing system (MICOS), fully recapitulates the AI profile observed when AIF is inhibited. Thus, AIF's effect on CI assembly is principally due to compromised intramitochondrial transport of the MICOS complex.

Circadian regulation of mitochondrial uncoupling and lifespan.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.

OBJECTIVE: Diabetes is characterized by pancreatic ß-cell dedifferentiation. Dedifferentiating ß cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the ß-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. METHODS: Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to ß-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. RESULTS: The expression of Cyb5r3 is decreased in FoxO1-deficient ß cells. Mice with ß-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient ß cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient ß cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. CONCLUSIONS: The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate ß-cell failure.

Insights from Drosophila on mitochondrial complex I.

NADH:ubiquinone oxidoreductase, more commonly referred to as mitochondrial complex I (CI), is the largest discrete enzyme of the oxidative phosphorylation system (OXPHOS). It is localized to the mitochondrial inner membrane. CI oxidizes NADH generated from the tricarboxylic acid cycle to NAD+, in a series of redox reactions that culminates in the reduction of ubiquinone, and the transport of protons from the matrix across the inner membrane to the intermembrane space. The resulting proton-motive force is consumed by ATP synthase to generate ATP, or harnessed to transport ions, metabolites and proteins into the mitochondrion. CI is also a major source of reactive oxygen species. Accordingly, impaired CI function has been associated with a host of chronic metabolic and degenerative disorders such as diabetes, cardiomyopathy, Parkinson's disease (PD) and Leigh syndrome. Studies on Drosophila have contributed to our understanding of the multiple roles of CI in bioenergetics and organismal physiology. Here, we explore and discuss some of the studies on Drosophila that have informed our understanding of this complex and conclude with some of the open questions about CI that can be resolved by studies on Drosophila.

Assembly of the complexes of oxidative phosphorylation triggers the remodeling of cardiolipin.

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

Activin signaling mediates muscle-to-adipose communication in a mitochondria dysfunction-associated obesity model.

Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. We analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-ß signaling pathway were induced in the fat body. Strikingly, expression of the TGF-ß family ligand, Activin-ß (Actß), was dramatically increased in the muscles by NF-κB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actß expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling.

Regulation of Mitochondrial Complex I Biogenesis in Drosophila Flight Muscles.

The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. We report the mechanism of CI biogenesis in Drosophila flight muscles and show that it proceeds via the formation of ∼315, ∼550, and ∼815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting an ∼700 kDa transient CI assembly intermediate into the ∼815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.

Stress signaling between organs in metazoa.

Many organisms have developed a robust ability to adapt and survive in the face of environmental perturbations that threaten the integrity of their genome, proteome, or metabolome. Studies in multiple model organisms have shown that, in general, when exposed to stress, cells activate a complex prosurvival signaling network that includes immune and DNA damage response genes, chaperones, antioxidant enzymes, structural proteins, metabolic enzymes, and noncoding RNAs. The manner of activation runs the gamut from transcriptional induction of genes to increased stability of transcripts to posttranslational modification of important biosynthetic proteins within the stressed tissue. Superimposed on these largely autonomous effects are nonautonomous responses in which the stressed tissue secretes peptides and other factors that stimulate tissues in different organs to embark on processes that ultimately help the organism as a whole cope with stress. This review focuses on the mechanisms by which tissues in one organ adapt to environmental challenges by regulating stress responses in tissues of different organs.

Modeling metabolic homeostasis and nutrient sensing in Drosophila: implications for aging and metabolic diseases.

Over the past decade, numerous reports have underscored the similarities between the metabolism of Drosophila and vertebrates, with the identification of evolutionarily conserved enzymes and analogous organs that regulate carbohydrate and lipid metabolism. It is now well established that the major metabolic, energy-sensing and endocrine signaling networks of vertebrate systems are also conserved in flies. Accordingly, studies in Drosophila are beginning to unravel how perturbed energy balance impinges on lifespan and on the ensuing diseases when energy homeostasis goes awry. Here, we highlight several emerging concepts that are at the nexus between obesity, nutrient sensing, metabolic homeostasis and aging. Specifically, we summarize the endocrine mechanisms that regulate carbohydrate and lipid metabolism, and provide an overview of the neuropeptides that regulate feeding behavior. We further describe the various efforts at modeling the effects of high-fat or -sugar diets in Drosophila and the signaling mechanisms involved in integrating organ function. Finally, we draw attention to some of the cardinal discoveries made with these disease models and how these could spur new research questions in vertebrate systems.