Mitochondrial Contact Site Research Articles

Structural insights into crista junction formation by the Mic60-Mic19 complex.

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function.

ORP5/8 and MIB/MICOS link ER-mitochondria and intra-mitochondrial contacts for non-vesicular transport of phosphatidylserine.

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes.

The A’-helix of CYP11A1 remodels mitochondrial cristae

BackgroundCYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the abundance of CYP11A1 characterizes the extent of steroidogenic cell differentiation. Besides, the mitochondria of fully differentiated steroidogenic cells are specialized with tubulovesicular cristae. The participation of CYP11A1 in the change of mitochondrial structure and the differentiation of steroid-producing cells, however, has not been investigated.MethodsWe engineered nonsteroidogenic monkey kidney COS1 cells to express CYP11A1 upon doxycycline induction and examined the mitochondrial structure of these cells. We also mapped the CYP11A1 domains that confer structural changes of mitochondria. We searched for CYP11A1-interacting proteins and investigated the role of this interacting protein in shaping mitochondrial structure. Finally, we examined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system.ResultsWe found that CYP11A1 overexpression led to the formation of tubulovesicular cristae in mitochondria. We also identified the A’-helix located at amino acid #57–68 to be sufficient for membrane insertion and crista remodeling. We identified heat shock protein 60 (Hsp60) as the CYP11A1-interacting protein and showed that Hsp60 is required for CYP11A1 accumulation and crista remodeling. Finally, we found that the small MIC10 subcomplex of the mitochondrial contact site and cristae organizing system was reduced when CYP11A1 was overexpressed.ConclusionsCYP11A1 participates in the formation of tubulovesicular cristae in the mitochondria of steroidogenic cells. Its A’-helix is sufficient for the formation of tubulovesicular cristae and for protein integration into the membrane. CYP11A1 interacts with Hsp60, which is required for CYP11A1 accumulation. The accumulation of CYP11A1 leads to the reduction of MIC10 complex and changes mitochondrial structure.

ER-mitochondria contact sites; a multifaceted factory for Ca2+ signaling and lipid transport.

Membrane contact sites (MCS) between organelles of eukaryotic cells provide structural integrity and promote organelle homeostasis by facilitating intracellular signaling, exchange of ions, metabolites and lipids and membrane dynamics. Cataloguing MCS revolutionized our understanding of the structural organization of a eukaryotic cell, but the functional role of MSCs and their role in complex diseases, such as cancer, are only gradually emerging. In particular, the endoplasmic reticulum (ER)-mitochondria contacts (EMCS) are key effectors of non-vesicular lipid trafficking, thereby regulating the lipid composition of cellular membranes and organelles, their physiological functions and lipid-mediated signaling pathways both in physiological and diseased conditions. In this short review, we discuss key aspects of the functional complexity of EMCS in mammalian cells, with particular emphasis on their role as central hubs for lipid transport between these organelles and how perturbations of these pathways may favor key traits of cancer cells.

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.

Transfer of H2O2 from Mitochondria to the endoplasmic reticulum via Aquaporin-11

Some aquaporins (AQPs) can transport H2O2 across membranes, allowing redox signals to proceed in and between cells. Unlike other peroxiporins, human AQP11 is an endoplasmic reticulum (ER)-resident that can conduit H2O2 to the cytosol. Here, we show that silencing Ero1α, an ER flavoenzyme that generates abundant H2O2 during oxidative folding, causes a paradoxical increase in luminal H2O2 levels. The simultaneous AQP11 downregulation prevents this increase, implying that H2O2 reaches the ER from an external source(s). Pharmacological inhibition of the electron transport chain reveals that Ero1α downregulation activates superoxide production by complex III. In the intermembrane space, superoxide dismutase 1 generates H2O2 that enters the ER channeled by AQP11. Meanwhile, the number of ER-mitochondria contact sites increases as well, irrespective of AQP11 expression. Taken together, our findings identify a novel interorganellar redox response that is activated upon Ero1α downregulation and transfers H2O2 from mitochondria to the ER via AQP11.

Decreasing pdzd8-mediated mito-ER contacts improves organismal fitness and mitigates Aβ42 toxicity.

Mitochondria-ER contact sites (MERCs) orchestrate many important cellular functions including regulating mitochondrial quality control through mitophagy and mediating mitochondrial calcium uptake. Here, we identify and functionally characterize the Drosophila ortholog of the recently identified mammalian MERC protein, Pdzd8. We find that reducing pdzd8-mediated MERCs in neurons slows age-associated decline in locomotor activity and increases lifespan in Drosophila. The protective effects of pdzd8 knockdown in neurons correlate with an increase in mitophagy, suggesting that increased mitochondrial turnover may support healthy aging of neurons. In contrast, increasing MERCs by expressing a constitutive, synthetic ER-mitochondria tether disrupts mitochondrial transport and synapse formation, accelerates age-related decline in locomotion, and reduces lifespan. Although depletion of pdzd8 prolongs the survival of flies fed with mitochondrial toxins, it is also sufficient to rescue locomotor defects of a fly model of Alzheimer's disease expressing Amyloid β42 (Aβ42). Together, our results provide the first in vivo evidence that MERCs mediated by the tethering protein pdzd8 play a critical role in the regulation of mitochondrial quality control and neuronal homeostasis.

Mechanisms of mitochondrial respiratory adaptation.

Mitochondrial energetic adaptations encompass a plethora of conserved processes that maintain cell and organismal fitness and survival in the changing environment by adjusting the respiratory capacity of mitochondria. These mitochondrial responses are governed by general principles of regulatory biology exemplified by changes in gene expression, protein translation, protein complex formation, transmembrane transport, enzymatic activities and metabolite levels. These changes can promote mitochondrial biogenesis and membrane dynamics that in turn support mitochondrial respiration. The main regulatory components of mitochondrial energetic adaptation include: the transcription coactivator peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC1α) and associated transcription factors; mTOR and endoplasmic reticulum stress signalling; TOM70-dependent mitochondrial protein import; the cristae remodelling factors, including mitochondrial contact site and cristae organizing system (MICOS) and OPA1; lipid remodelling; and the assembly and metabolite-dependent regulation of respiratory complexes. These adaptive molecular and structural mechanisms increase respiration to maintain basic processes specific to cell types and tissues. Failure to execute these regulatory responses causes cell damage and inflammation or senescence, compromising cell survival and the ability to adapt to energetically demanding conditions. Thus, mitochondrial adaptive cellular processes are important for physiological responses, including to nutrient availability, temperature and physical activity, and their failure leads to diseases associated with mitochondrial dysfunction such as metabolic and age-associated diseases and cancer.

Balancing energy and protein homeostasis at ER-mitochondria contact sites.

The endoplasmic reticulum (ER) is the largest organelle of the cell and participates in multiple essential functions, including the production of secretory proteins, lipid synthesis, and calcium storage. Sustaining proteostasis requires an intimate coupling with energy production. Mitochondrial respiration evolved to be functionally connected to ER physiology through a physical interface between both organelles known as mitochondria-associated membranes. This quasi-synaptic structure acts as a signaling hub that tunes the function of both organelles in a bidirectional manner and controls proteostasis, cell death pathways, and mitochondrial bioenergetics. Here, we discuss the main signaling mechanisms governing interorganellar communication and their putative role in diseases including cancer and neurodegeneration.

CHCHD2 maintains mitochondrial contact site and cristae organizing system stability and protects against mitochondrial dysfunction in an experimental model of Parkinson's disease.

Background:Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's dementia. Mitochondrial dysfunction is involved in the pathology of PD. Coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2) was identified as associated with autosomal dominant PD. However, the mechanism of CHCHD2 in PD remains unclear.Methods:Short hairpin RNA (ShRNA)-mediated CHCHD2 knockdown or lentivirus-mediated CHCHD2 overexpression was performed to investigate the impact of CHCHD2 on mitochondrial morphology and function in neuronal tumor cell lines represented with human neuroblastoma (SHSY5Y) and HeLa cells. Blue-native polyacrylamide gel electrophoresis (PAGE) and two-dimensional sodium dodecyl sulfate-PAGE analysis were used to illustrate the role of CHCHD2 in mitochondrial contact site and cristae organizing system (MICOS). Co-immunoprecipitation and immunoblotting were used to address the interaction between CHCHD2 and Mic10. Serotype injection of adeno-associated vector-mediated CHCHD2 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration were used to examine the influence of CHCHD2 in vivo.Results:We found that the overexpression of CHCHD2 can protect against methyl-4-phenylpyridinium (MPP+)-induced mitochondrial dysfunction and inhibit the loss of dopaminergic neurons in the MPTP-induced mouse model. Furthermore, we identified that CHCHD2 interacted with Mic10, and overexpression of CHCHD2 can protect against MPP+-induced MICOS impairment, while knockdown of CHCHD2 impaired the stability of MICOS.Conclusion:This study indicated that CHCHD2 could interact with Mic10 and maintain the stability of the MICOS complex, which contributes to protecting mitochondrial function in PD.

Endoplasmic Reticulum-Mitochondria Contacts Modulate Reactive Oxygen Species-Mediated Signaling and Oxidative Stress in Brain Disorders: The Key Role of Sigma-1 Receptor.

Significance: Mitochondria-Associated Membranes (MAMs) are highly dynamic endoplasmic reticulum (ER)-mitochondria contact sites that, due to the transfer of lipids and Ca2+ between these organelles, modulate several physiologic processes, such as ER stress response, mitochondrial bioenergetics and fission/fusion events, autophagy, and inflammation. In addition, these contacts are implicated in the modulation of the cellular redox status since several MAMs-resident proteins are involved in the generation of reactive oxygen species (ROS), which can act as both signaling mediators and deleterious molecules, depending on their intracellular levels. Recent Advances: In the past few years, structural and functional alterations of MAMs have been associated with the pathophysiology of several neurodegenerative diseases that are closely associated with the impairment of several MAMs-associated events, including perturbation of the redox state on the accumulation of high ROS levels. Critical Issues: Inter-organelle contacts must be tightly regulated to preserve cellular functioning by maintaining Ca2+ and protein homeostasis, lipid metabolism, mitochondrial dynamics and energy production, as well as ROS signaling. Simultaneously, these contacts should avoid mitochondrial Ca2+ overload, which might lead to energetic deficits and deleterious ROS accumulation, culminating in oxidative stress-induced activation of apoptotic cell death pathways, which are common features of many neurodegenerative diseases. Future Directions: Given that Sig-1R is an ER resident chaperone that is highly enriched at the MAMs and that controls ER to mitochondria Ca2+ flux, as well as oxidative and ER stress responses, its potential as a therapeutic target for neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Alzheimer, Parkinson, and Huntington diseases should be further explored. Antioxid. Redox Signal. 37, 758-780.

Stbd1-deficient mice display insulin resistance associated with enhanced hepatic ER-mitochondria contact

Starch binding domain-containing protein 1 (STBD1) is an endoplasmic reticulum (ER)-resident, glycogen-binding protein. In addition to glycogen, STBD1 has been shown to interact with several proteins implicated in glycogen synthesis and degradation, yet its function in glycogen metabolism remains largely unknown. In addition to the bulk of the ER, STBD1 has been reported to localize at regions of physical contact between mitochondria and the ER, known as Mitochondria-ER Contact sites (MERCs). Given the emerging correlation between distortions in the integrity of hepatic MERCs and insulin resistance, our study aimed to delineate the role of STBD1 in vivo by addressing potential abnormalities in glucose metabolism and ER-mitochondria communication associated with insulin resistance in mice with targeted inactivation of Stbd1 (Stbd1KO). We show that Stbd1KO mice at the age of 24 weeks displayed reduced hepatic glycogen content and aberrant control of glucose homeostasis, compatible with insulin resistance. In line with the above, Stbd1-deficient mice presented with increased fasting blood glucose and insulin levels, attenuated activation of insulin signaling in the liver and skeletal muscle and elevated liver sphingomyelin content, in the absence of hepatic steatosis. Furthermore, Stbd1KO mice were found to exhibit enhanced ER-mitochondria association and increased mitochondrial fragmentation in the liver. Nevertheless, the enzymatic activity of hepatic respiratory chain complexes and ER stress levels in the liver were not altered. Our findings identify a novel important role for STBD1 in the control of glucose metabolism, associated with the integrity of hepatic MERCs.

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.

CHCHD10 and SLP2 control the stability of the PHB complex: a key factor for motor neuron viability.

CHCHD10 is an amyotrophic lateral sclerosis/frontotemporal dementia gene that encodes a mitochondrial protein whose precise function is unclear. Here we show that Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing protein 10 interacts with the Stomatin-Like Protein 2 and participates in the stability of the prohibitin complex in the inner mitochondrial membrane. By using patient fibroblasts and mouse models expressing the same CHCHD10 variant (p.Ser59Leu), we show that Stomatin-Like Protein 2 forms aggregates with prohibitins, found in vivo in the hippocampus and as aggresome-like inclusions in spinal motor neurons of Chchd10S59L/+ mice. Affected cells and tissues display instability of the prohibitin complex, which participates at least in part in the activation of the OMA1 cascade with OPA1 processing leading to mitochondrial fragmentation, abnormal mitochondrial cristae morphogenesis and neuronal death found in spinal cord and the hippocampus of Chchd10S59L/+ animals. Destabilization of the prohibitin complex leads to the instability of the mitochondrial contact site and cristae organizing the system complex, probably by the disruption of OPA1-mitofilin interaction. Thus, Stomatin-Like Protein 2/prohibitin aggregates and destabilization of the prohibitin complex are critical in the sequence of events leading to motor neuron death in CHCHD10S59L-related disease.

Yin and Yang of NADPH Oxidases in Myocardial Ischemia-Reperfusion.

Oxidative stress is critically involved in the pathophysiology of myocardial ischemic-reperfusion (I/R) injury. NADPH oxidase (Nox) 2 and 4, major sources of reactive oxygen species (ROS) in cardiomyocytes, are upregulated in response to I/R. Suppression of Nox-derived ROS prevents mitochondrial dysfunction and endoplasmic reticulum (ER) stress, leading to attenuation of myocardial I/R injury. However, minimal levels of ROS by either Nox2 or Nox4 are required for energy metabolism during I/R in the heart, preserving hypoxia-inducible factor-1α (HIF-1α) and peroxisome proliferator-activated receptor-α (PPARα) levels. Furthermore, extreme suppression of Nox activity induces reductive stress, leading to paradoxical increases in ROS levels. Nox4 has distinct roles in organelles such as mitochondria, ER, and ER-mitochondria contact sites (MAMs). Mitochondrial Nox4 exerts a detrimental effect, causing ROS-induced mitochondrial dysfunction during I/R, whereas Nox4 in the ER and MAMs is potentially protective against I/R injury through regulation of autophagy and MAM function, respectively. Although Nox isoforms are potential therapeutic targets for I/R injury, to maximize the effect of intervention, it is likely important to optimize the ROS level and selectively inhibit Nox4 in mitochondria. Here, we discuss the ‘Yin and Yang’ functions of Nox isoforms during myocardial I/R.

Disruption of the MICOS complex leads to an aberrant cristae structure and an unexpected, pronounced lifespan extension in Podospora anserina.

Mitochondria are dynamic eukaryotic organelles involved in a variety of essential cellular processes including the generation of adenosine triphosphate (ATP) and reactive oxygen speciesas well as in the control of apoptosis and autophagy. Impairments of mitochondrial functions lead to aging and disease. Previous workwith the ascomycete Podospora anserina demonstrated that mitochondrial morphotype as well as mitochondrial ultrastructure change during aging. The latter goes along with an age-dependent reorganization of the inner mitochondrial membrane leading to a change from lamellar cristae to vesicular structures. Particularlyfrom studies with yeast, it is known that besides the F1 Fo -ATP-synthase and the phospholipid cardiolipin also the "mitochondrial contact site and cristae organizing system"(MICOS) complex, existing of the Mic60- and Mic10-subcomplex, is essential for proper cristae formation. In the present study, we aimed to understand the mechanistic basis of age-related changes in the mitochondrial ultrastructure. We observed that MICOS subunits are coregulated at the posttranscriptional level. This regulation partially depends on the mitochondrial iAAA-protease PaIAP. Most surprisingly, we made the counterintuitive observation that, despite the loss of lamellar cristae and of mitochondrial impairments, the ablation of MICOS subunits (except for PaMIC12) leads to a pronounced lifespan extension. Moreover, simultaneous ablation of subunits of both MICOS subcomplexes synergistically increases lifespan, providing formal genetic evidence that both subcomplexes affect lifespan by different and at least partially independent pathways. At the molecular level, we found that ablation of Mic10-subcomplex components leads to a mitohormesis-induced lifespan extension, while lifespan extension of Mic60-subcomplex mutants seems to be controlled by pathways involved in the control of phospholipid homeostasis. Overall, our data demonstrate that both MICOS subcomplexes have different functions and play distinct roles in the aging process of P. anserina.

Prime Real Estate: Metals, Cofactors and MICOS.

Metals are key elements for the survival and normal development of humans but can also be toxic to cells when mishandled. In fact, even mild disruption of metal homeostasis causes a wide array of disorders. Many of the metals essential to normal physiology are required in mitochondria for enzymatic activities and for the formation of essential cofactors. Copper is required as a cofactor in the terminal electron transport chain complex cytochrome c oxidase, iron is required for the for the formation of iron-sulfur (Fe-S) clusters and heme, manganese is required for the prevention of oxidative stress production, and these are only a few examples of the critical roles that mitochondrial metals play. Even though the targets of these metals are known, we are still identifying transporters, investigating the roles of known transporters, and defining regulators of the transport process. Mitochondria are dynamic organelles whose content, structure and localization within the cell vary in different tissues and organisms. Our knowledge of the impact that alterations in mitochondrial physiology have on metal content and utilization in these organelles is very limited. The rates of fission and fusion, the ultrastructure of the organelle, and rates of mitophagy can all affect metal homeostasis and cofactor assembly. This review will focus of the emerging areas of overlap between metal homeostasis, cofactor assembly and the mitochondrial contact site and cristae organizing system (MICOS) that mediates multiple aspects of mitochondrial physiology. Importantly the MICOS complexes may allow for localization and organization of complexes not only involved in cristae formation and contact between the inner and outer mitochondrial membranes but also acts as hub for metal-related proteins to work in concert in cofactor assembly and homeostasis.

The role of IR‐induced swelling in OPA1‐MICOS interaction in cardiac mitochondria

Mitochondria are membrane‐bound organelles composed of two membranes, the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM), with the intermembrane space (IMS) localized between them. The IMM is divided into two subcompartments, the inner boundary membrane (IBM) that lies parallel to the OMM, and the cristae membrane (CM), which are IMM invaginations folded into cristae. The IBM and CM are connected at terminals forming circular‐like openings known as cristae junctions (CJs). The structural integrity of the mitochondrial CJs is maintained by the mitochondrial contact site and cristae organizing system (MICOS), a specific protein complex containing several structural and regulatory proteins. The MICOS proteins control the IMM architecture through direct membrane shaping, formation of contact sites, and biogenesis of proteins and lipids. In addition, the optic atrophy 1 (OPA1), a mitochondrial fusion protein, localized also in the CM, has been shown to participate in IMM remodeling which mediates the fusion of the IMM. The structural organization of the IMM is regulated by changes in the matrix volume of mitochondria; excessive matrix swelling in response to energetic and oxidative stress induced by pathological stimuli such as cardiac ischemia‐reperfusion (IR) impairs the integrity of CJs and thereby, alters mitochondrial function. The main goal of this study is to investigate the effects of cardiac IR‐induced swelling on OPA1 and MICOS proteins. Hearts were isolated from male Sprague Dawley rats and perfused with Krebs‐Henseleit solution (KHS) using the Langendorff‐mode technique at a constant flow rate (10‐12 ml/min). The animals were randomly assigned to the following groups: i) perfusion (no ischemia) for 55 min (C‐55 group), ii) ischemia (I group) for 25 min, iii) ischemia in the presence of sanglifehrin A (SfA), an inhibitor of the permeability transition pore (IS), iv) perfusion (no ischemia) for 95 min (C‐95), v) ischemia for 25‐min followed by 40‐min reperfusion (IR group), and vi) IR in the presence of SfA (IRS). SfA (0.5 µM) was present 10 min before ischemia (IS group) and throughout the entire period of reperfusion (IRS group). LDH activity was determined in the coronary effluent as a marker of cell death. At the end of reperfusion, mitochondria were isolated from the hearts by differential centrifugation for analysis of oxygen consumption rates, mitochondrial swelling, and protein levels of long and short forms of OPA1, and MICOS proteins. Our results show that ischemia diminished post‐ischemic recovery of the hearts as evidenced by impaired cardiac contractility, increased LDH activity in the coronary effluent, and reduced mitochondrial respiration. Ischemia alone and IR differently affected the expression of both, OPA1 and mitofilin (Mic‐60, a core MICOS protein) as well as other MICOS components. In conclusion, our data suggest that OPA1 and MICOS proteins are affected differently by ischemia alone and IR.

Lifespan Extension of Podospora anserina Mic60-Subcomplex Mutants Depends on Cardiolipin Remodeling.

Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the ‘mitochondrial contact site and cristae organizing system’ (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.

MicroRNA-200c coordinates HNF1 homeobox B and apolipoprotein O functions to modulate lipid homeostasis in alcoholic fatty liver disease

Hepatic steatosis is an initial manifestation of alcoholic liver disease. An imbalance of hepatic lipid processes including fatty acid uptake, esterification, oxidation, and triglyceride secretion leads to alcoholic fatty liver (AFL). However, the precise molecular mechanisms underlying the pathogenesis of AFL remain elusive. Here, we show that mice deficient in microRNAs (miRs)-141 and -200c display resistance to the development of AFL. We found that miR-200c directly targets HNF1 homeobox B (Hnf1b), a transcriptional activator for microsomal triglyceride transfer protein (Mttp), as well as apolipoprotein O (ApoO), an integral component of the mitochondrial contact site and cristae organizing system complex. We show that expression of these miRs is significantly induced by chronic ethanol exposure, which is accompanied by reduced HNF1B and APOO levels. Furthermore, miR-141/200c deficiency normalizes ethanol-mediated impairment of triglyceride secretion, which can be attributed to the restored levels of HNF1B and MTTP, as well as phosphatidylcholine abundance. Moreover, we demonstrate that miR-141/200c deficiency restores ethanol-mediated inhibition of APOO expression and mitochondrial dysfunction, improving mitochondrial antioxidant defense capacity and fatty acid oxidation. Taken together, these results suggest that miR-200c contributes to the modulation of lipid homeostasis in AFL disease by cooperatively regulating Hnf1b and ApoO functions.