Mitochondrial Oxidative Phosphorylation in Viral Infections.

Mitochondria have been identified as the "powerhouse" of the cell, generating the cellular energy, ATP, for almost seven decades. Research over time has uncovered a multifaceted role of the mitochondrion in processes such as cellular stress signaling, generating precursor molecules, immune response, and apoptosis to name a few. Dysfunctional mitochondria resulting from a departure in homeostasis results in cellular degeneration. Viruses hijack host cell machinery to facilitate their own replication in the absence of a bonafide replication machinery. Replication being an energy intensive process necessitates regulation of the host cell oxidative phosphorylation occurring at the electron transport chain in the mitochondria to generate energy. Mitochondria, therefore, can be an attractive therapeutic target by limiting energy for viral replication. In this review we focus on the physiology of oxidative phosphorylation and on the limited studies highlighting the regulatory effects viruses induce on the electron transport chain.

Contemporary Antiretroviral Therapy Dysregulates Iron Transport and Augments Mitochondrial Dysfunction in HIV-Infected Human Microglia and Neural-Lineage Cells.

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hµglia) cell line harboring inducible HIV provirus and in SH-SY5Y cells after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20-30%, with the most pronounced effects in latently infected HIV+ hµglia and SH-SY5Y cells. Latently HIV-infected hµglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hµglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron-sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hµglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency.

The MNRR1 activator nitazoxanide abrogates lipopolysaccharide-induced preterm birth in mice.

Intra-amniotic inflammation leading to preterm birth is one of the leading causes of neonatal morbidity and mortality. We recently reported that the mitochondrial levels of MNRR1 (Mitochondrial Nuclear Retrograde, Regulator 1; also called CHCHD2, AAG10, or PARK22), an important bi-organellar regulator of cellular function, are reduced in the context of inflammation and that genetic and pharmacological increases in MNRR1 levels can counter the inflammatory profile. Herein, we show that nitazoxanide, a clinically approved drug, is an activator of MNRR1 and abrogates preterm birth in a well-characterized murine model caused by intra-amniotic lipopolysaccharide (LPS) injection.

MNRR1 is a driver of ovarian cancer progression.

Cancer progression requires the acquisition of mechanisms that support proliferative potential and metastatic capacity. MNRR1 (also CHCHD2, PARK22, AAG10) is a bi-organellar protein that in the mitochondria can bind to Bcl-xL to enhance its anti-apoptotic function, or to respiratory chain complex IV (COX IV) to increase mitochondrial respiration. In the nucleus, it can act as a transcription factor and promote the expression of genes involved in mitochondrial biogenesis, migration, and cellular stress response. Given that MNRR1 can regulate both apoptosis and mitochondrial respiration, as well as migration, we hypothesize that it can modulate metastatic spread. Using ovarian cancer models, we show heterogeneous protein expression levels of MNRR1 across samples tested and cell-dependent control of its stability and binding partners. In addition to its anti-apoptotic and bioenergetic functions, MNRR1 is both necessary and sufficient for a focal adhesion and ECM repertoire that can support spheroid formation. Its ectopic expression is sufficient to induce the adhesive glycoprotein THBS4 and the type 1 collagen, COL1A1. Conversely, its deletion leads to significant downregulation of these genes. Furthermore, loss of MNRR1 leads to delay in tumor growth, curtailed carcinomatosis, and improved survival in a syngeneic ovarian cancer mouse model. These results suggest targeting MNRR1 may improve survival in ovarian cancer patients.

Lysophosphatidylcholine acyltransferase 1 protein is present in maternal blood in the third trimester and is upregulated by antenatal corticosteroids.

OBJECTIVES: Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is involved in the production of fetal lung surfactant. We have shown that LPCAT1 mRNA is present in amniotic fluid and maternal plasma and that its quantity correlates with the amniotic fluid lamellar body count. The purpose of the present study was to assay maternal plasma for the LPCAT1 protein in term and preterm pregnancies; and to measure the impact of antenatal corticosteroids. METHODS: Maternal and newborn plasma samples were obtained from 7 women admitted to the hospital for induction of labor. Maternal plasma was also obtained before administration of corticosteroids and 24 h after the second dose of corticosteroids from 12 women with premature labor and premature rupture of membranes. After sample preparation, LPCAT1 protein levels were determined using sandwich ELISA. RESULTS: We discovered LPCAT1 protein in maternal plasma in measurable quantities after 32 weeks gestation. Further, there was a rise of maternal plasma LPCAT1 in response to the clinical administration of antenatal corticosteroids. CONCLUSIONS: Quantitation of maternal plasma LPCAT1 protein offers promise in the ongoing study of fetal lung maturation.

Lipopolysaccharide induces placental mitochondrial dysfunction in murine and human systems by reducing MNRR1 levels via a TLR4-independent pathway.

Mitochondria play a key role in placental growth and development, and mitochondrial dysfunction is associated with inflammation in pregnancy pathologies. However, the mechanisms whereby placental mitochondria sense inflammatory signals are unknown. Mitochondrial nuclear retrograde regulator 1 (MNRR1) is a bi-organellar protein responsible for mitochondrial function, including optimal induction of cellular stress-responsive signaling pathways. Here, in a lipopolysaccharide-induced model of systemic placental inflammation, we show that MNRR1 levels are reduced both in mouse placental tissues in vivo and in human trophoblastic cell lines in vitro. MNRR1 reduction is associated with mitochondrial dysfunction, enhanced oxidative stress, and activation of pro-inflammatory signaling. Mechanistically, we uncover a non-conventional pathway independent of Toll-like receptor 4 (TLR4) that results in ATM kinase-dependent threonine phosphorylation that stabilizes mitochondrial protease YME1L1, which targets MNRR1. Enhancing MNRR1 levels abrogates the bioenergetic defect and induces an anti-inflammatory phenotype. We therefore propose MNRR1 as an anti-inflammatory therapeutic in placental inflammation.

Intraperitoneal Triamcinolone Reduces Postoperative Adhesions, Possibly through Alteration of Mitochondrial Function.

Adhesions frequently occur postoperatively, causing morbidity. In this noninterventional observational cohort study, we enrolled patients who presented for repeat abdominal surgery, after a history of previous abdominal myomectomy, from March 1998 to June 20210 at St. Vincent's Catholic Medical Centers. The primary outcome of this pilot study was to compare adhesion rates, extent, and severity in patients who were treated with intraperitoneal triamcinolone acetonide during the initial abdominal myomectomy (n = 31) with those who did not receive any antiadhesion interventions (n = 21), as documented on retrospective chart review. Adhesions were blindly scored using a standard scoring system. About 32% of patients were found to have adhesions in the triamcinolone group compared to 71% in the untreated group (p < 0.01). Compared to controls, adhesions were significantly less in number (0.71 vs. 2.09, p < 0.005), severity (0.54 vs. 1.38, p < 0.004), and extent (0.45 vs. 1.28, p < 0.003). To understand the molecular mechanisms, human fibroblasts were incubated in hypoxic conditions and treated with triamcinolone or vehicle. In vitro studies showed that triamcinolone directly prevents the surge of reactive oxygen species triggered by 2% hypoxia and prevents the increase in TGF-ß1 that leads to the irreversible conversion of fibroblasts to an adhesion phenotype. Triamcinolone prevents the increase in reactive oxygen species through alterations in mitochondrial function that are HIF-1α-independent. Controlling mitochondrial function may thus allow for adhesion-free surgery and reduced postoperative complications.

Molecular mechanisms regulating lysophosphatidylcholine acyltransferase 1 (LPCAT1) in human pregnancy.

INTRODUCTION: Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) is necessary for surfactant production in fetal lungs. Mechanisms responsible for its regulation during gestation remain to be elucidated. Our goal is to evaluate molecular mechanisms regulating LPCAT1 expression during gestation and after glucocorticoid administration. METHODS: Placentas throughout gestation were assayed for LPCAT1 protein levels. A placental cell line, HTR-8/SVneo (HTR), was used as a model to test the effects of placental oxygen tension found during pregnancy as well as the effects of dexamethasone used therapeutically in the clinic. RESULTS: LPCAT1 protein levels are maximal in late third trimester placental samples and are expressed strongly on the basal plate. LPCAT1 was maximally upregulated at 4% O2 (P < 0.01), corresponding to oxygen tension found in placenta at term. Mitochondrial nuclear retrograde regulator 1 (MNRR1), a bi-organellar (mitochondria and nucleus) regulator, transcriptionally activates LPCAT1. Antenatal corticosteroids (ACS) upregulate LPCAT1, at least in part, by an MNRR1-dependent pathway. HTR cells treated with 25 nM dexamethasone for 24 h exhibited a 2-fold increase in LPCAT1 levels compared to controls. In MNRR1 knockout cells, the response to ACS is significantly blunted. DISCUSSION: LPCAT1 appears to be induced by MNRR1. Hypoxia and corticosteroids increase LPCAT1 expression through an MNRR1 dependent pathway. LPCAT1 protein levels can be measured in maternal plasma and rise throughout gestation and in response to ACS.

Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) rescues the cellular phenotype of MELAS by inducing homeostatic mechanisms.

MNRR1 (CHCHD2) is a bi-organellar regulator of mitochondrial function that directly activates cytochrome c oxidase in the mitochondria and functions in the nucleus as a transcriptional activator for hundreds of genes. Since MNRR1 depletion contains features of a mitochondrial disease phenotype, we evaluated the effects of forced expression of MNRR1 on the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) syndrome. MELAS is a multisystem encephalomyopathy disorder that can result from a heteroplasmic mutation in the mitochondrial DNA (mtDNA; m.3243A > G) at heteroplasmy levels of ∼50 to 90%. Since cybrid cell lines with 73% m.3243A > G heteroplasmy (DW7) display a significant reduction in MNRR1 levels compared to the wild type (0% heteroplasmy) (CL9), we evaluated the effects of MNRR1 levels on mitochondrial functioning. Overexpression of MNRR1 in DW7 cells induces the mitochondrial unfolded protein response (UPRmt), autophagy, and mitochondrial biogenesis, thereby rescuing the mitochondrial phenotype. It does so primarily as a transcription activator, revealing this function to be a potential therapeutic target. The role of MNRR1 in stimulating UPRmt, which is blunted in MELAS cells, was surprising and further investigation uncovered that under conditions of stress the import of MNRR1 into the mitochondria was blocked, allowing the protein to accumulate in the nucleus to enhance its transcription function. In the mammalian system, ATF5, has been identified as a mediator of UPRmt MNRR1 knockout cells display an ∼40% reduction in the protein levels of ATF5, suggesting that MNRR1 plays an important role upstream of this known mediator of UPRmt.

Decreased membrane cholesterol in liver mitochondria of the point mutation mouse model of juvenile Niemann-Pick C1, Npc1nmf164.

It has long been known that there is decreased mitochondrial function in several tissues of Niemann-Pick C1 model mice and cultured cells. These defects contribute to the accumulation of Reactive Oxygen Species (ROS) and tissue damage. It is also well established that there is increased unesterified cholesterol, stored in late endosomes/lysosomes, in many tissues in mutant humans, mouse models, and mutant cultured cells. Using a mouse model with an NPC1 point mutation that is more typical of the most common form of the disease, and highly purified liver mitochondria, we find markedly decreased mitochondrial membrane cholesterol. This is compared to previous reports of increased mitochondrial membrane cholesterol. We also find that, although in wild-type or heterozygous mitochondria cytochrome c oxidase (COX) activity decreases with age as expected, surprisingly, COX activity in homozygous mutant mice improves with age. COX activity is less than half of wild-type amounts in young mutant mice but later reaches wild-type levels while total liver cholesterol is decreasing. Mutant mice also contain a decreased number of mitochondria that are morphologically abnormal. We suggest that the decreased mitochondrial membrane cholesterol is causative for the mitochondrial energy defects. In addition, we find that the mitochondrial stress regulator protein MNRR1 can stimulate NPC1 synthesis and is deficient in mutant mouse livers. Furthermore, the age curve of MNRR1 deficiency paralleled levels of total cholesterol. The role of such altered mitochondria in initiating the abnormal autophagy and neuroinflammation found in NPC1 mouse models is discussed.

Tris-heteroleptic ruthenium(II) polypyridyl complexes: Synthesis, structural characterization, photophysical, electrochemistry and biological properties.

Three water-soluble tris-heteroleptic ruthenium(II) polypyridyl complexes [Ru(bpy)(phen)(bpg)]2+ (1), [Ru(bpy)(dppz)(bpg)]2+ (2), and [Ru(phen)(dppz)(bpg)]2+ (3) (where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c] phenazine, bpg = 4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10] phenanthroline-6,13-dione) have been synthesized and characterized. Molecular structures of complexes 1 and 3 are confirmed by single crystal X-ray structure determination. Interaction of complexes 1-3 with DNA is explored by various spectroscopic techniques. The complexes 1-3 show solvent dependent photophysical properties. Complexes 2 and 3 show extensive "molecular light switch" effect for DNA. The complexes 1-3 are low toxic towards HeLa (human cervical cancer) and HL-60 (human promyelocytic leukemia) cell lines. Further, the cellular uptake of complexes 2 and 3 by cells shows that complexes mainly localised on the nucleus of the cells.

The cellular stress proteins CHCHD10 and MNRR1 (CHCHD2): Partners in mitochondrial and nuclear function and dysfunction.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) and CHCHD2 (MNRR1) are homologous proteins with 58% sequence identity and belong to the twin CX9C family of proteins that mediate cellular stress responses. Despite the identification of several neurodegeneration-associated mutations in the CHCHD10 gene, few studies have assessed its physiological role. Here, we investigated CHCHD10's function as a regulator of oxidative phosphorylation in the mitochondria and the nucleus. We show that CHCHD10 copurifies with cytochrome c oxidase (COX) and up-regulates COX activity by serving as a scaffolding protein required for MNRR1 phosphorylation, mediated by ARG (ABL proto-oncogene 2, nonreceptor tyrosine kinase (ABL2)). The CHCHD10 gene was maximally transcribed in cultured cells at 8% oxygen, unlike MNRR1, which was maximally expressed at 4%, suggesting a fine-tuned oxygen-sensing system that adapts to the varying oxygen concentrations in the human body under physiological conditions. We show that nuclear CHCHD10 protein down-regulates the expression of genes harboring the oxygen-responsive element (ORE) in their promoters by interacting with and augmenting the activity of the largely uncharacterized transcriptional repressor CXXC finger protein 5 (CXXC5). We further show that two genetic CHCHD10 disease variants, G66V and P80L, in the mitochondria exhibit faulty interactions with MNRR1 and COX, reducing respiration and increasing reactive oxygen species (ROS), and in the nucleus abrogating transcriptional repression of ORE-containing genes. Our results reveal that CHCHD10 positively regulates mitochondrial respiration and contributes to transcriptional repression of ORE-containing genes in the nucleus, and that genetic CHCHD10 variants are impaired in these activities.

MNRR1, a Biorganellar Regulator of Mitochondria.

The central role of energy metabolism in cellular activities is becoming widely recognized. However, there are many gaps in our knowledge of the mechanisms by which mitochondria evaluate their status and call upon the nucleus to make adjustments. Recently, a protein family consisting of twin CX9C proteins has been shown to play a role in human pathophysiology. We focus here on two family members, the isoforms CHCHD2 (renamed MNRR1) and CHCHD10. The better studied isoform, MNRR1, has the unusual property of functioning in both the mitochondria and the nucleus and of having a different function in each. In the mitochondria, it functions by binding to cytochrome c oxidase (COX), which stimulates respiration. Its binding to COX is promoted by tyrosine-99 phosphorylation, carried out by ABL2 kinase (ARG). In the nucleus, MNRR1 binds to a novel promoter element in COX4I2 and itself, increasing transcription at 4% oxygen. We discuss mutations in both MNRR1 and CHCHD10 found in a number of chronic, mostly neurodegenerative, diseases. Finally, we propose a model of a graded response to hypoxic and oxidative stresses, mediated under different oxygen tensions by CHCHD10, MNRR1, and HIF1, which operate at intermediate and very low oxygen concentrations, respectively.

Abl2 kinase phosphorylates Bi-organellar regulator MNRR1 in mitochondria, stimulating respiration.

We previously showed that MNRR1 (Mitochondrial Nuclear Retrograde Regulator 1, also CHCHD2) functions in two subcellular compartments, displaying a different function in each. In the mitochondria it is a stress regulator of respiration that binds to cytochrome c oxidase (COX) whereas in the nucleus it is a transactivator of COX4I2 and other hypoxia-stimulated genes. We now show that binding of MNRR1 to COX is promoted by phosphorylation at tyrosine-99 and that this interaction stimulates respiration. We show that phosphorylation of MNRR1 takes place in mitochondria and is mediated by Abl2 kinase (ARG). A family with Charcot-Marie-Tooth disease type 1A with an exaggerated phenotype harbors a Q112H mutation in MNRR1, located in a domain that is necessary for transcriptional activation by MNRR1. Furthermore, the mutation causes the protein to function suboptimally in the mitochondria in response to cellular stress. The Q112H mutation hinders the ability of the protein to interact with Abl kinase, leading to defective tyrosine phosphorylation and a resultant defect in respiration.