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Mitochondria shape cytosolic Ca2+ (cCa2+) transients. Ca2+ entry into the mitochondria is driven by the highly negative mitochondrial membrane potential and through a highly selective channel, the Mitochondrial Calcium Uniporter (MCU). Mitochondrial Ca2+ (mCa2+) is utilized by the matrix dehydrogenases for maintaining cellular bioenergetics. The TCA cycle-derived NADH and FADH2 are mCa2+ dependent thus, feed into the electron transport chain (ETC) to generate ATP. Either loss of mCa2+ or metabolite uptake by the mitochondria results in a bioenergetic crisis and mitochondrial dysfunction. Reciprocally, sudden elevation of cCa2+ under conditions of stroke or ischemia/reperfusion injury (I/R) drives excessive mCa2+ overload that in turn leads to the opening of a large channel, the mitochondrial permeability transition pore (PTP) that triggers necrotic cell death. Thus, Ca2+ and metabolite equilibrium is essential to maintain a healthy mitochondrial pool. Our laboratory has previously showed that loss of mCa2+ uptake leads to decreased ATP generation and cell survival through autophagy. Although metabolite scarcity also results in similar reduction in ATP generation, the molecular mechanisms by which metabolites control mitochondrial ion homeostasis remain elusive. Deprivation of glucose or supplementation of mitochondrial pyruvate carrier (MPC) transport blocker UK5099 and or carnitine-dependent fatty acid blocker etomoxir triggered an increase in the expression of MICU1, a regulator of the mitochondrial calcium uniporter (MCU) but not the MCU core subunit. Consistently, either RNAi-mediated deletion of MPC isoforms or dominant negative human mutant MPC1 R97W showed significant induction of MICU1 protein abundance and inhibition of MCU-mediated mCa2+ uptake. Moreover, TCA cycle substrate-dependent MICU1 expression is under the control of EGR1 transcriptional regulation. Reciprocally, the MICU1 dependent inhibition of mCa2+ uptake exhibited lower NADH production and oxygen consumption and ATP production. The reduction of mitochondrial pyruvate by MPC knockdown is linked to higher production of mitochondrial ROS and elevated autophagy markers. These studies reveal an unexpected regulation of MCU-mediated mCa2+ flux machinery involving major TCA cycle substrate availability and possibly MICU1 to control cellular switch between glycolysis and oxidative phosphorylation. While mCa2+ is required for energy generation, sustained elevation of mCa2+ results in mitochondrial swelling and necrotic death. Hence, it was thought that preventing mCa2+ overload can be protective under conditions of elevated cCa2+. Contrary to this, mice knocked-out for MCU, that demonstrated no mCa2+ uptake and hence no mitochondrial swelling, however failed protect cells from I/R- mediated cell death. MCU-/- animals showed a similar infarct size comparable to that of control animals, suggesting that prevention of MCU-mediated mCa2+ overload alone is not sufficient to protect cells from Ca2+ -induced necrosis. The absence of mCa2+ entry revealed an elevation in the upstream cCa2+ transients in hepatocytes from MCUDHEP. Ultra-structural analysis of liver sections from MCU-/- (MCUDHEP) and MCUfl/fl animals revealed stark contrast in the shape of mitochondria: MCUfl/fl liver sections showed long and filamentous mitochondria (spaghetti-like) while MCUDHEP mitochondria were short and circular (donut-like). Furthermore, challenging MCUfl/fl and MCUDHEP hepatocytes with ionomycin caused a marked increase in cCa2+ and a simultaneous change in mitochondrial shape (from spaghetti to donut), a phenomenon we termed mitochondrial shape transition (MiST) that was independent of mitochondrial swelling. The cCa2+-mediated MiST is induced by an evolutionarily conserved mitochondrial surface EF-hand domain containing Miro1. Glutamate and Ca2+ -stress driven cCa2+ mobilization cause MiST in neurons that is suppressed by expression of Miro1 EF1 mutants. Miro1-dependent MiST is essential for autophagosome formation that is attenuated in cells harboring Miro1 EF1 mutants. Remarkably, loss of cCa2+ sensitization by Miro1 prevented MiST and mitigated autophagy. These results demonstrate that an interplay of ions and metabolites function in concert to regulate mitochondrial shape that in turn dictates the diverse mitochondrial processes from ATP generation to determining mechanisms of cell death. / Biomedical Sciences

In the majority of sexual eukaryotes, mitochondria are inherited predominantly from a single, usually the female, parent Like the majority of higher plants and animals, the pathogenic yeast Cryptococcus neoformans has two mating types (sexes), however, these two sexes are morphologically similar. In this study, I examined the distribution of the mating types and how mating types influence the inheritance of mitochondria in C. neoformans. My survey of mating type alleles in 358 isolates collected from four geographic areas in the US showed a biased distribution of mating type alleles with most isolates containing mating type a alleles. To characterize the role of mating type locus on mitochondrial inheritance, I constructed two pairs of congenic strains that differed only at the mitochondrial genome and mating type locus. Mating between these two pairs of strains demonstrated that uniparental inheritance in C. neoformans was controlled by the mating type locus and progeny predominantly inherited mitochondria from the mating type a parent. Specifically, we identified two genes within the mating type locus, SXIIa in mating type a strain and SXI2a in mating type a strain, that control mitochondrial inheritance. Disruption of these two genes resulted in biparental mitochondrial inheritance in sexual crosses. These two genes are the first ones identified capable of controlling uniparental mitochondrial inheritance in any organism. In addition, we determined that the deletion of the SXIIa gene enhanced the spread of mitochondrial introns in sexual crosses. This discovery is consistent with the hypothesis that uniparental lnheritance might have evolved to prevent the spread of selfish cytoplasmic elements. / Thesis / Doctor of Philosophy (PhD)

Beluga whales are migratory over much of their range, congregating in small groups around shallow river estuaries in summer, and overwintering in large groups in areas with reliable open water. This complicates management issues because it is unclear if belugas from the common wintering ground represent one large group with exchange of individuals, or if each summer estuarine concentration should be managed as a separate stock. To examine the genetic structuring, we analyzed variation in mitochondrial DNA (mtDNA) restriction sites among 101 beluga whales from 10 regions across North America, including Greenland. Using 11 restriction enzymes, 9 haplotypes were identified among 71 whales. The remaining 30 whales were tested with only the six restriction enzymes found to identify polymorphisms. We found a marked segregation of divergent haplotypes for both sexes between eastern and western Hudson Bay. Haplotype 1 was found in 19 out of 21 animals on the east coast, while haplotype 5 was found in 18 out of 20 animals on the west coast. Sequence divergence among the 71 belugas was estimated to be 2.03%. Haplotypes fell into two major phylogenetic groups, labelled lineage I and II. Lineage I haplotypes occurred primarily in the St. Lawrence Estuary and the eastern Hudson Bay. Lineage II haplotypes occurred primarily along the western Hudson Bay, Southern Baffin Island, western Greenland, the Canadian high arctic, and the Beaufort Sea. These findings support the hypothesis that belugas exhibit maternally directed philopatry to summering grounds, and are consistent with the hypothesis that after deglaciation, the arctic was recolonized by at least two stocks of belugas divergent in their mtDNA, possibly representing Atlantic and Pacific stocks. / Thesis / Master of Science (MS)

Mitochondrial uncouplers have the capacity of passively shuttling protons from the mitochondrial intermembrane space to the mitochondrial matrix, independent of ATP synthase. This results in the disruption of oxidative phosphorylation and increased rate of metabolism as a counter action from the mitochondria. Therefore, small molecule mitochondrial uncouplers have potential for the treatment of obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), neurodegenerative disorders, amongst others. A one-pot method for the synthesis of 1H-imidazo[4,5-b]pyrazines from [1,2,5]oxadiazolo[3,4-b]pyrazines is herein disclosed. In the presence of Fe, Yb(OTf)3, and the desired electrophile partner, in situ reduction of the oxadiazole fragment followed by cyclization afforded imidazolopyrazines in moderate to good yields. The selection of different orthoesters as electrophiles also allowed functionalization on the 2-position of the imidazole ring. This new method was used to synthesize 1H-imidazo[4,5-b]pyrazines to perform structure-activity relationship studies. Thus, a library of 75 compounds was synthesized and characterized for mitochondrial uncoupling activity. The biological activity of the compounds was demonstrated in oxygen consumption rate assays affording potent mitochondrial uncouplers. The method was further applied to the synthesis of 5-alkoxy-2-(trifluoromethyl)-1H-imidazo[4,5-b]pyrazin-6-amines, with over 50 derivatives synthesized. A structure-activity relationship study was performed using a variety of substituents to fine-tune the scaffold's potency. The installation of a methoxy group at the 5-position of the scaffold resulted in the discovery of compound 4.3.20, which exhibited the best activity with an EC50 of 3.6 ± 0.4 μM in rat L6 myoblasts and a half-life of 4.4 h in mice. Compound 4.3.20 displayed potential as an anti-obesity agent in a mouse model with an effective dose of 50 mg kg-1 without changes in food intake or lean mass. Tissue distribution studies revealed predominance in the liver and both white and brown adipose tissue. In addition, 4.3.20 improved serum markers of insulin sensitivity and hyperlipidemia such as insulin, glucose, triglycerides, cholesterol, and HOMA-IR. Taken together, compound 4.3.20 and related mitochondrial uncouplers show promise for further development in the treatment of obesity and other diseases. / Doctor of Philosophy / The mitochondria, which is an organelle within our cells, is where all the nutrients ingested in the form of food are metabolized, and either used for energy or stored as fat if they are not used. The latter is the main cause of obesity, carrying with it a myriad other comorbidities, such as high blood pressure, heart disease, diabetes, certain types of cancer. Obesity has become a great concern with an incidence of 42% in the US. Mitochondrial uncouplers are molecules that target the mitochondria with a mechanism of action of converting some of the energy ingested in the form of nutrients to be lost as heat instead of being stored as fat. The potential result is a regulated form of weight-loss. Herein, we developed a method for the synthesis of a novel mitochondrial uncoupler scaffold and disclose the mitochondrial uncoupler activity of over 150 molecules. In particular, compound 4.3.20 was tested in an obesity mouse model and was shown to induce fat loss with mice fed a high fat diet. Our investigations support potential use of mitochondrial uncouplers as a mechanism for the treatment and prevention of obesity and other metabolic diseases.

Small molecule mitochondrial uncouplers are compounds that dissipate the proton motive force independent of ATP synthase that results in increased energy expenditure. Mild mitochondrial uncoupling has therapeutic potential in treating obesity, diabetes, neurological diseases, non-alcoholic steatohepatitis (NASH), and aging. Our group has previously reported the discovery of a small molecule mitochondrial uncoupler BAM15, which was efficacious in an obesity mouse model. Herein, we describe the design and synthesis of two scaffolds as well as their characterization as mitochondrial uncouplers through a series of in vitro and in vivo assays. Compounds that pass as bona fide mitochondrial uncouplers are administered in mice to determine pharmacokinetic properties and promising compounds are then tested in a mouse model of obesity. The first series of mitochondrial uncouplers are anilinopyrazines. By changing the substitution pattern and electronics on the aniline rings, our investigations reveal the importance of the proximity of aniline rings on the pyrazine core, with the 2,3-positions being crucial for uncoupling activity. We found that mitochondrial uncouplers 2.5g and 2.5l elicited a maximum oxygen consumption rate (OCR) of 260% and 343% with an EC50 of 2.5 and 5.9 µM, respectively. Utilizing the knowledge gained from the anilinopyrazine series, we designed a second novel chemical scaffold based on a related BAM15 analog 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol. The new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives have a pyridine instead of pyrazine core that is decorated with aniline substituents. We found that derivatives with electron withdrawing groups (EWG) substitutions in the 2,5-position on the aniline ring exhibited the greatest uncoupling activity compared to other structural isomers. Strong EWGs CF3/OCF3/SO2CF3 were well tolerated and demonstrated the highest uncoupling activity compared to other EWGs. Our studies indicated that placement of the hydroxyl group in the 2-position of the pyridine moiety was crucial for uncoupling activity. Several of the most promising compounds tested in vitro were examined in vivo and found to have good oral bioavailability in mice with ranges in Cmax of 10-90 µM and t1/2 of 0.9 to >24 hours. We found that analogs that have F/OCF3/SO2CF3 groups on the 4-position exhibited the longest t1/2 compared to other structural isomers, suggesting that this position is a site of metabolic lability. Among the 51 derivatives tested, SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR and an EC50 of 17.1 µM in L6 myoblast cells. SHM20519115 was found to have good oral bioavailability with a Cmax of 57 µM and a t1/2 of 4.4 hours. Additionally, SHM20519115 had significant distribution in adipose tissue where it can promote mitochondrial uncoupling. In a mouse model of obesity, SHM20519115 prevented fat mass gain by 59% compared to the western diet (WD) control group. Importantly, weight loss did not alter lean mass or food intake. Further characterization demonstrated that SHM20519115 prevented glucose and insulin intolerance in mice. Taken together, our investigations support the utility of mitochondrial uncouplers for the treatment of obesity and other metabolic disorders. / Doctor of Philosophy / Obesity is commonly considered a modern-day epidemic with more than 40% of adult Americans being classified as obese. The higher prevalence of obesity over the course of the last century has been attributed to a more sedentary lifestyle and high calorie diet. Obesity has been shown to negatively impact every organ system and increases the risk for heart disease, cancer, neurological diseases, non-alcoholic steatohepatitis (NASH), and diabetes. Moreover, obesity has further burdened the healthcare system with an estimated expenditure of $190 billion a year in the US. Although diet and exercise has shown excellent results in weight loss, long-term compliance with these regiments is extremely low. Current non-invasive treatments provide varying efficacies and a myriad of side-effects. Invasive procedures, which is restricted to those who are classified as 'morbidly obese' with a BMI > 40, have shown excellent results in facilitating weight loss but come with high cost and risks to patients. This excludes individuals in the BMI range of 30-40 unless they are qualified with additional comorbidities. In recent years, mitochondrial uncouplers have reemerged as a potential therapeutic treatment for obesity. This dissertation discusses the structure-activity relationship study of anilinopyrazines and 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives as mitochondrial uncouplers. Building on previous work on BAM15, we investigated uncoupling activity of anilinopyrazines. We discovered that although anilinopyrazines were previously found to be inactive, modifications to the aniline rings could result in uncoupling activity. We found that strong electron withdrawing groups placed in the meta and para positions were most favorable. We also determined that the 2,3-disubstitution on the aniline rings was crucial for uncoupling activity. From this study, we discovered 2.5g and 2.5l that elicited a maximum oxygen consumption rate (OCR) of 260 and 343% with EC50 of 2.5 and 5.9 µM, respectively. Furthermore, we recently reported a new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives and identified SHM20519115 as a mitochondrial uncoupler. Our studies determined that SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR with an EC50 of 17.1µM in L6 myoblasts cells. In a mouse model of obesity, SHM20519115 was found to be efficacious at a 130 mg/kg dose. Pharmacokinetic studies SHM20519115 showed greater overall distribution in adipose tissue in mice. Additionally, when examined in a mouse obesity prevention model, SHM20519115 successfully prevented 59% fat mass gain compared to the western diet (WD) control group. Finally, we found that SHM20519115 prevents glucose and insulin intolerance in mice. Taken together, these results support a role for mitochondrial uncouplers in the treatment of obesity.

The gram-negative endotoxin, lipopolysaccharide (LPS), has been extensively researched in high doses (10-200ng/ml) and is well-documented in the literature for its ability to result in devastating effects such as multi-organ failure, sepsis, and septic shock. In high doses, LPS signals through Toll-like-receptor 4 (TLR4) and triggers a cascade of events culminating in the release of pro- and anti-inflammatory cytokines and the activation of NF-κB. In contrast, super-low doses of LPS (1-100pg/ml) are able to trigger the persistent release of pro-inflammatory mediators while evading the compensatory activation of NF-κB. This mild yet persistent induction of inflammation may lie at the heart of numerous inflammatory diseases and disorders and warrants studies such as this to elucidate the novel mechanisms. In this study, we explored the novel mechanisms utilized by super-low dose LPS in cellular stress and low-grade inflammation. In the first study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and necroptosis were examined. We show that in the presence of super-low dose LPS (50pg/ml), the key regulators of mitochondrial fission and fusion, Drp1 and Mfn1 respectively, are inversely regulated. An increase in mitochondrial fragmentation and cell death which was not dependent on caspase activation was observed. In addition, super-low dose LPS was able to activate RIP3, a kinase responsible for inducing the inflammatory cell death, necroptosis. These mechanisms were regulated in an Interleukin-1 receptor-associated kinase 1 (IRAK-1) dependent manner. In the second study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and endosome/lysosome fusion were examined. In the presence of low-dose LPS (50pg/ml), endosomal-lysosomal fusion is inhibited and a loss of endosomal acidification required for the successful clearance of cellular debris and resolution of inflammation was observed. Additionally, super-low dose LPS induced the accumulation of p62 indicative of the suppression of autophagy. Tollip and Interleukin-1 receptor-associated kinase 3 (IRAK-M) appear to be critical regulators in this process. Collectively, these studies show that low-dose endotoxemia is capable of causing persistent cellular stress, not observed in the presence of high-dose LPS (10-200ng/ml), and that it promotes necroptotic cell death while suppressing mechanisms necessary for the resolution of inflammation such as endosome-lysosome fusion. This research reveals novel mechanisms utilized by low-dose endotoxemia which could aid future efforts to develop prevention and treatment for various debilitating inflammatory diseases. / Ph. D.

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.