By buffering cytosolic calcium, mitochondria can shape the magnitude and duration of intracellular calcium transients, which in turn govern key physiological events. Although controlled uptake of calcium into the matrix influences the rate of ATP production, excess calcium within the matrix triggers non-specific permeabilization of the mitochondrial inner membrane, resulting in cell death. Despite its importance in cellular physiology, the molecular identity of the mitochondrial calcium uniporter remained a mystery for nearly five decades. Recently, an approach inspired by comparative genomics was used to identify two proteins required for high-capacity mitochondrial calcium uptake. These include MICU1, an EF-hand protein that may function as a regulatory component by sensing calcium, and MCU, the channel-forming subunit of the uniporter. In this work, I explore two distinct areas within the growing field of molecular mitochondrial calcium biology. First, I discuss the identification of a new protein, MICU1-paralog EFHA1, and present data that implicates it in mitochondrial calcium uptake. Subsequently, I describe efforts to establish an in vitro system to characterize the channel activity of MCU, including my contribution to the development of a liposome-based assay for calcium transport and preliminary work aimed at reconstituting MCU transport activity in proteoliposomes.

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme in the mitochondrial electron transfer chain. It catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of protons across the mitochondrial inner membrane, maintaining the proton motive force used for ATP synthesis. Complex I is the least understood of the respiratory enzymes; although the mechanisms of NADH oxidation and intramolecular electron transfer are gradually becoming appreciated, the mechanisms of quinone binding and reduction and proton translocation remain unknown. Complex I dysfunction has been implicated in a wide range of pathologies including mitochondrial diseases such as Leigh's disease, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's. The work described in the first part of this thesis is aimed at elucidating the structure of either a subcomplex of mitochondrial complex I, or of the intact enzyme itself. A comprehensive investigation revealed that hydrophilic subcomplexes of complex I from bovine heart mitochondria are not suitable for use as models of the intact enzyme. Attempts to prepare intact complex I of sufficient quality for structural work were successful; however, results from a large set of crystallization trials were disappointing. The second part of this thesis describes three studies of the function and mechanism of complex I from bovine heart mitochondria. First, the flavin mononucleotide, the site of NADH oxidation, was identified as the site of the 'inhibitor-insensitive' NADH:ubiquinone oxidoreduction reaction. The formation of semiquinones initiates redox cycling reactions with oxygen, producing vast amounts of reactive oxygen species; further studies revealed that other oxidants, such as paraquat, also react at the flavin site and initiate redox cycling reactions. Second, kinetic studies showed that the reaction between NADH and positively charged oxidants such as HAR (hexaammineruthenium (III)) proceeds by an unusual ternary reaction mechanism at the flavin site of complex I. Finally, double electron-electron resonance spectroscopy was used to show unambiguously that iron sulphur cluster 4Fe[TY]1 gives rise to electron paramagnetic resonance signal N4; the data provide an alternating potential energy profile for electron transfer along the cluster chain between the flavin and the quinone-binding site.

INTRODUCTION Phthalates, a chemical class of plasticizers, are ubiquitous in the environment and recognized as endocrine disrupting compounds (EDCs). Recent data suggest that oxidative stress is a potential mediator of poor male reproductive health associated with phthalate exposure. Mitochondria are implicated in the production of excess oxidative stress and sperm mitochondrial copy number (MtCopy) and deletions (MtDeletion) have been linked with male infertility. However, little is known about the relationship of these mitochondrial biomarkers in sperm with phthalate exposure and oxidative stress. OBJECTIVES To examine associations of urinary phthalate metabolites and isoprostane concentrations on sperm MtCopy and MtDeletions in male partners undergoing assisted reproductive technologies (ART). METHODS A total of (n=97) sperm samples were collected from male partners undergoing ART at Baystate Medical Center, in Springfield, MA from 2014 to 2016 as part of the Sperm Environmental Epigenetics and Development Study (SEEDS). Seventeen urinary phthalate metabolites (n=103) were analyzed by the Centers for Disease Control using tandem mass spectrometry. 15-F2t-Isoprostane (n=101) was measured using a competitive enzyme-linked immonsorbent assay in urine of male individuals. A triplex Taqman probe-based qPCR method was developed for relative quantification of genomic DNA, MtCopy and MtDeletions. Multivariable linear or logistic regression was employed to examine associations with age, BMI, batch and current smoking status with each outcome to determine confounders used for adjustment. RESULTS Quartiles of MtCopy and MtDeletion were positively associated with the odds of male infertility (p for trend < .0001 and 0.007, respectively). Urinary metabolite concentrations of MCNP displayed a positive association with MtCopy (β=1.56; p =0.03). Urinary MEHP concentrations were positively associated with MtDeletion in only infertile individuals (n=30) (β = 0.075; p = 0.006). Urinary isoprostane concentration was not associated with MtCopy or MtDeletion, but was associated with seven phthalate metabolite concentrations (MEOHP, MEHHP, MBzP, MHBP, MiBP, and MHiBP). CONCLUSIONS To our knowledge, this is the first study to investigate the relationship between sperm MtCopy and MtDeletion with oxidative stress and phthalates. These results suggest that certain phthalate metabolites may be associated with a known biomarker of systemic oxidative stress. Sperm mitochondrial function as measured by MtCopy and MtDeletion may be considered biomarkers of male infertility, although no relationship was shown between mitochondrial outcomes and oxidative stress. Future research is investigating these relationships with developmental outcomes including embryo quality.

Cellular senescence represents status, when the cells cease to divide and remain in permanent cell cycle arrest. Senescence is considered to be an active response of the cell to various extrinsic and intrinsic types of stress such as certain oncogene activation, exposing to several cytokines or drugs and damaged and/or uncapped telomeres. Senescent cells are characterised by extensive modification of gene expression, flattened and enlargement of cellular body. Hypothetically, altered gene expression may lead also to increase of certain surface proteins expression. Such protein can be L1 cell adhesive molecule (L1CAM), which is expressed heterogeneously within the population. This Thesis describes current knowledge of cellular senescence, the mechanism, which may result in establishment of senescence phenotype, and also the characteristic markers of senescence. Thesis also puts together the heterogeneity of L1CAM expression in A375 senescent cells with oxygen consumption rate and extracellular acidification rate performed by Seahorse XFe24 metabolic analyser. Therefore, ells were sorted according to their levels of expressing L1CAM onto low and high L1CAM expressing subpopulations. Obtained data show potential correlation between the rate of L1CAM expression in A375 cells and the metabolic rate. Key...

Patients with obesity develop a metabolic heart disease (MHD) of unclear mechanisms and limited therapeutic options. MHD is characterized by left-ventricular hypertrophy and impaired ventricular relaxation and is associated with cardiac lipid accumulation, oxidative stress and impaired energetics. Mitochondria play a critical role in cardiac metabolism and mitochondrial dysfunction results in a pathologic decrease in ATP production and increase in reactive oxygen species (ROS) generation. I hypothesized that nutrient excess and cardiac lipid accumulation impair mitochondrial function and cause cardiac remodeling through mitochondrial oxidative stress. Mice overexpressing fatty acid transport protein 1 (FATP1) in cardiomyocytes have increased uptake and use of cardiac lipids and develop MHD. I observed that FATP1 mice have increased cardiac diacylglycerols and PKC activation and decreased mitochondrial biogenesis, size, and oxygen consumption with unchanged ATP synthesis and ROS production. Overexpression of the antioxidant enzyme catalase in mitochondria (mCAT) does not attenuate MHD in FATP1 mice. This suggests that FATP1-driven cardiac lipid accumulation leads to compensated downregulation of mitochondrial function without oxidant overproduction. Mice fed a high fat, high sucrose (HFHS) diet have myocardial oxidative stress and develop MHD. I observed that cardiac mitochondria of HFHS-fed mice have increased ROS production and decreased ATP synthesis and oxygen consumption. HFHS-fed mCAT mice do not develop mitochondrial dysfunction or cardiac remodeling, suggesting that mitochondrial ROS may mediate HFHS-driven mitochondrial dysfunction and MHD. Mice with partial loss of the mitochondrial transporter ABCB10 exhibit cardiac oxidative stress leading to impaired recovery from ischemic injury. I generated mice with cardiomyocyte-specific ABCB10 deletion and observed that ABCB10 loss decreases mitochondrial oxygen consumption and increases ROS production without altering ATP synthesis or affecting cardiac structure and function. After HFHS feeding, mice with heterozygous loss of cardiomyocyte ABCB10 have exaggerated MHD and increased mortality, suggesting a protective role of ABCB10 in MHD induced by HFHS diet. In summary, cardiac lipid accumulation leads to transcriptional downregulation of mitochondrial respiration, while dietary fat and sugar excess leads to mitochondrial dysfunction and cardiac remodeling driven by mitochondrial oxidative stress and exacerbated by loss of ABCB10. This study suggests that oxidant-driven mitochondrial dysfunction plays a key role in MHD.

The physiological limit to maximum aerobic capacity (VO2max) in vertebrates has been attributed to cardiovascular oxygen delivery, to the ability of the muscle cells to consume oxygen, or to a fine-tuned development of all components of the respiratory system such that no single component can be shown to limit VO2max. The above hypotheses have each been developed using different experiments with different animals. The comparative studies uniting these animals and methods are limited. In order to further our knowledge of the cellular limit to VO2max, skeletal muscle mitochondria were isolated from species representing four vertebrate classes, and endothermic and ectothermic physiology. Mitochondrial VO2 was measured at 15, 25 and 35°C and the results were compared between species and endothermic and ectothermic groups. Mitochondrial enzyme activities were measured at the three treatment temperatures to ascertain which enzyme activity best represents VO2max for all vertebrates. Cytochrome difference spectra were measured to determine the concentrations of mitochondrial cytochromes c+c1 . The results show that mitochondria are unique in all species tested. Each species has its own response to changing temperature and its own mitochondrial enzyme activity profile. In addition, in vitro measurements of mitochondrial VO2 for all species show rates significantly higher than those estimated from whole organism measurements of VO2max, suggesting that mitochondrial oxygen uptake is not a factor limiting organismal V02max. The Q10 for mitochondrial VO2 differed significantly between groups, indicating that differences in VO2max between endotherms and ectotherms cannot be explained solely on the basis of temperature. The activation energy (Ea) of mitochondrial VO2 was significantly higher in endotherms compared to ectotherms. Mitochondrial enzyme activities did not show the same Q10 and Ea differences as the intact mitochondria. Since enzyme activities were measured on mitochondria disrupted with either detergent or sonication, physical properties of the mitochondrial inner membrane are suggested as being responsible for these differences.

Mitochondrial dynamics includes the processes of fusion, fission, and motility. These processes form interdependent adaptive mechanisms that, together with autophagy, maintain mitochondrial function to meet cellular needs. Mitochondrial dynamics control function directly by inducing bioenergetic remodeling or indirectly by promoting turnover of mitochondria via autophagy. Importantly, mitochondrial dysfunction has been implicated in beta-cell failure during type 2 diabetes. This thesis will investigate the role of dynamics and autophagy in regulating mitochondrial and pancreatic beta-cell function during chronic exposure to excess glucose and fatty acids, termed glucolipotoxicity (GLT). It remains ill-defined what role fusion and motility play in determining mitochondrial turnover, as current methodologies to assess turnover lack subcellular resolution. To address this need we developed the use of MitoTimer, a mitochondrial fluorescent probe that undergoes a time-dependent green-to-red transition. Turnover was revealed by the integrated proportions of young (green) and old (red) MitoTimer protein. The results demonstrate that mitochondrial fusion and motility regulate turnover by promoting the distribution of newer protein to subsets of mitochondria in the network. GLT inhibits mitochondrial fusion and networking in pancreatic beta-cells. Since fusion is dependent on motility we tested the hypothesis that GLT impairs fusion by affecting motility. We determined that GLT arrests motility, which may contribute to mitochondrial and beta-cell dysfunction. We show that excess nutrients increase O-linked β-N-acetyl glucosamine (O-GlcNAc) modification of mitochondrial motor adaptor Milton1, which decreases its activity and results in arrest of motility and increased fission. Thus Milton1 O-GlcNAc modification acts as a nutrient-sensor linking fusion, fission, and motility to nutrient supply in the beta-cell. Finally, GLT inhibits autophagic flux with concurrent lysosomal pH increase in beta-cells. To address the hypothesis that impaired lysosomal acidification is a causative event inhibiting autophagic flux and beta-cell function, we developed lysosome-localizing nanoparticles that expand and acidify upon UV photo-activation. Increasing lysosomal acidity with the nanoparticles increased autophagic flux and restored beta-cell function under GLT, establishing lysosomal pH as a key mediator of nutrient-induced beta-cell dysfunction. In summary the work elucidates the interdependence and specific roles of mitochondrial fusion, fission, motility, and autophagy in dictating beta-cell responses to excess nutrient environment. / 2017-06-15T00:00:00Z

The French-Canadian variant of Leigh syndrome (LSFC) is a distinct and particularly severe presentation of Leigh syndrome characterized by the onset of unpredictable acidotic crises leading to death of 80% of them before the age of five. This autosomal recessive disorder is caused by mutations in LRPPRC, encoding an mRNA binding protein of the same name with a high affinity for mitochondrial transcripts. As a result of the mutations, levels of LRPPRC are decreased in all tissues and cause a severe deficiency of complex IV of the respiratory chain, with a deeper involvement of brain and liver. To gain better knowledge on the pathophysiology of this disease, and of the impact of the OXPHOS defect on the liver, our research consortium developed a mouse model of the disease harboring a liver specific inactivation of Lrpprc (H-Lrpprc). The goal of this thesis is to investigate the in vivo consequences of hepatic Lrpprc inactivation and to test potential therapy for mitochondrial diseases. The characterization of this model and the analysis of the mitochondrial phenotype are presented in Chapter 2 (Cuillerier et al, Human Molecular Genetics, 2017). Despite this severe phenotype, H-Lrpprc mice show no signs of overt liver failure and maintain energy levels, suggesting mechanisms are in place to sustain residual complex IV function. The underlying compensatory mechanisms granting these mice a remarkable resilience were explored and are presented in Chapter 4 (Cuillerier et al, Communications Biology, 2021). Along this project, we developed a protocol, and the optimized conditions of this method are described in Chapter 3 (Cuillerier and Burelle, JoVE, 2019). Although great progress has been made, there are currently no effective or curative treatments for LSFC and mitochondrial diseases. Recently, extensive pre-clinical and clinical studies supported the emergence and safety of mesenchymal stem cells therapy in the treatment of various diseases. Following transplantation, MSCs promote repair through various mechanisms including secretion of cytokines/exosomes, and transfer of mitochondria directly to target cells with impaired mitochondria offering a possibility to replace mutant dysfunctional organelles, which is relevant in the context of genetic mitochondrial diseases. Based on this, the objective of the last chapter of this thesis is to test the therapeutic potential of MSCs for genetic mitochondrial disorders using MSC-based approaches and LSFC as a disease model. Unfortunately, we encountered several obstacles along the way, including the departure of our main collaborator and stem cell expert, and delays in experimental procedures due to the COVID-19 pandemic. Consequently, this study was not completed at the moment of submission of this thesis, and is therefore presented as a pilot study in the form of a manuscript in Chapter 5. Overall, these projects unveiled alterations of mitochondrial functions that go beyond OXPHOS, a complex network of compensatory mechanisms in place to palliate these defects, and finally, encouraging preliminary results suggest MSC therapy could be beneficial for the treatment of mitochondrial diseases.

<p> The mitochondrial inner membrane is highly selective with regard to permeability to solutes and the movement of a large number of large or charged molecules across it therefore requires specific transport processes provided by specific membrane proteins. </p> <p> In order to study the spatial arrangement of one such protein the adenine nucleotide translocator protein which transports ADP and ATP across the mitochondrial inner membrane, a number of chemical labelling studies of the mitochondrial inner membrane were carried out. </p> <p> Mitochondrial inner membrane preparations of normal (mitoplasts) and inverted (submitochondrial particles) config~ration with respect to mitochondria have been isolated and the external phosphatidylethanolamine and proteins modified by 3H isethionyl acetimidate. An upper limit of 40-46% of the total PE in mitoplasts was found to be located in the external monolayer. </p> <p> Differences in protein labelling patterns of isethionyl acetimidate modified mitochondria and SMP was observed. JAI was found to penetrate the outer membrane but not the inner membrane of intact mitochondria. </p> <p> A tritiated photoreactive phospholipid, 1 palmitoyl-2-(mdiazirinophenoxynonanoyl) phosphatidylcholine (DAP-PC) was incorporated into mitoplasts and submitochondrial particles symmetrically into both monolayers by sonication and asymmetrically using phospholipid exchange protein isolated from beef heart. Photolysis yielded the translocator as a major crosslinked product in both types of particles and with both methods of incorporation. </p> <p> It was shown that the adenine nucleotide translocator can be asymmetrically labelled by modification of membrane.particles of opposite orientation by water soluble and membrane soluble probes. </p> / Thesis / Master of Science (MSc)

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