Browsing by Subject "Mitochondria"

Nearly all animals live in environments with fluctuations in nutrient availability. The ability to sense and respond to these changes is essential for survival. Nutrition impacts physiology immediately, but can also have long-lasting effects across generations. The nematode Caenorhabditis elegans is particularly well-adapted to thrive in conditions of variable food availability. Here we find that starvation responses in C. elegans are largely independent of the larval stage at which worms experience starvation. Starvation in worms results in shrinkage, delayed growth upon recovery, and ultimately death. In order to adapt to starvation, metabolism is dramatically altered. At a gross level, this can be seen in a reduction of mitochondrial genomes and a more fragmented network of mitochondria.

Insulin-like signaling is a key cell signaling pathway controlling nutrient responses. We interrogate the role of insulin-like signaling in regulation of the acute starvation response. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant, but also serves as a glycolytic input. Further, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

In addition to acute changes in response to the nutrient environment, effects can persist intergenerationally. Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more off- spring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.

SIRT3 is an NAD+-dependent mitochondrial protein deacetylase purported to influence cellular and systemic metabolism through modulation of the mitochondrial acetylproteome. Fuel-stimulated insulin secretion from pancreatic islets involves mitochondrial metabolism and might be susceptible to SIRT3-mediated effects. To investigate this idea, we used CRISPR/Cas9 technology to obtain complete SIRT3 knockout in the INS-1 832/13 insulinoma cell line. In the context of this SIRT3 knockout cell line, we re-expressed wild-type SIRT3, β-Galactosidase, or one of three enzymatically inactive mutant forms of SIRT3 to generate lines representing a wide range of SIRT3 expression and mitochondrial protein deacetylase activity. We performed large-scale acetylproteome profiling by mass spectrometry on the different lines, and observed wide-spread, SIRT3-dependent changes in acetylation of enzymes involved in fatty acid oxidation, the TCA cycle, and the electron transport chain. Remarkably, despite these broad changes, the cell lines had indistinguishable insulin secretion responses to glucose or pyruvate, and exhibited no differences in function or viability in response to metabolic or ER stress-inducing agents. Moreover, metabolomic profiling revealed that, when compared to SIRT3-null cell lines, expression of wild-type SIRT3 does not result in appreciable changes in a host of organic acid, amino acid or fatty acid-derived acylcarnitine metabolites during glucose stimulation.

We also studied mice with global SIRT3 knockout (KO) fed a standard chow (STD) or high-fat/high-sucrose (HFHS) diet. Importantly, we performed these studies in the C57Bl/6J background in which we replaced the mutant allele of nicotinamide nucleotide transhydrogenase (NNT) present in the “J” substrain, with the wild-type allele in order to restore endogenous NNT function. SIRT3 KO and wild-type (WT) mice fed a STD diet exhibited no differences in insulin secretion during oral or IP glucose tolerance tests, and the function of islets isolated from these mice was indistinguishable in islet perifusion studies conducted with a broad array of secretagogues. Only when chronically fed a HFHS diet did SIRT3 KO animals exhibit a modest impairment in insulin secretion, but without an effect on glycemic control. Our broad conclusion is that major changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism. However, under conditions of chronic nutritional stress (feeding of a HFHS diet for 12 weeks), a negative effect on function appears, suggesting that islets are more susceptible to nutrition-induced factors (oxidative stress, local cytokine production, etc.) when SIRT3 is absent. Further studies will be required to identify factors that may interact with SIRT3 deficiency and mitochondrial protein hyperacetylation to increase the risk of -cell dysfunction.

My thesis work has focused on characterizing mechanisms by which human fungal pathogens regulate their adaptive cellular responses in order to survive and cause disease in the human host. Unlike most microbial fungi found in the environment, Cryptococcus neoformans has become a successful human pathogen due to two intrinsic abilities: 1) to survive and grow at human body temperature and 2) to employ virulence factors to combat host immune defenses. Over the past two decades, the fungal pathogenesis field has made enormous progress in identifying and characterizing C. neoformans proteins responsible for these adaptive cellular responses with a particular focus on enzymes, like those involved in cell cycle progression or those responsible for synthesizing components of the fungal cell surface. Although we know a substantial amount about the functions of these enzymes and their implications on fungal pathogenesis, the mechanisms by which these enzymes are regulated are less clear. I have attempted to address this gap in knowledge by focusing my thesis work on the identification and characterization of C. neoformans non-enzymatic proteins that regulate enzymes important for adaptive cellular responses. I have identified and characterized the C. neoformans arrestin proteins as regulators of enzyme ubiquitination, and likely enzyme function, in response to specific extracellular stressors (Chapters 2 & 3). I have also characterized a Cryptococcus-specific protein, Mar1, as an important modulator of host-fungal interactions due to its regulation of cell surface remodeling through maintenance of mitochondrial metabolic activity and homeostasis in response to cellular stress (Chapters 4 & 5). Furthermore, I also performed a comprehensive comparative analysis of different RNA enrichment methods for RNA sequencing applications and long non-coding RNA identification in C. neoformans, which can help researchers select appropriate tools for studying adaptive cellular responses from the RNA level (Chapter 6). These studies collectively have demonstrated that non-enzymatic proteins are important “cellular coordinators” in human fungal pathogens; they regulate the activity of many different enzymes in response to distinct extracellular signals, and as a result are required for both fungal growth and virulence factor employment in response to host-relevant stressors.

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Heart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.

Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.

Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.

Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.

Mitochondria are the main cellular producers of ATP, and play key roles in cellular signaling and apoptosis. Mitochondria also contain their own genomes (mtDNA), which encode 13 subunits of the electron transport chain (ETC), 22 tRNAs, and 2 rRNAs, making mtDNA integrity critical to both mitochondrial and organismal health. Mitochondria are dynamic organelles that fuse and divide to maintain mitochondrial shape, number, and size. However, mitochondrial fission and fusion also play a major role in the mitochondrial stress response. For example, mildly damaged mitochondria can fuse with healthy mitochondria allowing contents to mix, resulting in the generation of healthy mitochondria, which is known as functional complementation. Alternatively, when mitochondria become damaged beyond repair, they are targeted for autophagosomal degradation, or mitophagy. The overall importance of fission, fusion, mitophagy, and mtDNA is demonstrated by the fact that deficiencies in these processes and mtDNA content cause human disease. Interestingly, the age of onset, and severity of clinical manifestations of mitochondrial disease vary from patient to patient, even in individuals harboring identical mutations. These observations suggest a role for the environment in the development and progression of certain mitochondrial diseases; however, the relationship remains poorly understood.

To investigate the role of environmental toxicants in the development, progression, and exacerbation of mitochondrial disease I have taken two approaches using the in vivo model organism Caenorhabditis elegans. First, ten known and suspected mitochondrial toxicants (2,4-dinitrophenol (DNP), acetaldehyde, acrolein, aflatoxin B1 (AfB1), arsenite, cadmium, cisplatin, doxycycline, paraquat, rotenone) were screened for exacerbation of larval growth delay in wild-type, fission-, fusion-, and mitophagy-deficient nematodes using the COPAS Biosort. Second, a C. elegans model of mtDNA depletion was developed using chronic low-dose ethidium bromide exposure. Five toxicants (AfB1, arsenite, paraquat, rotenone, ultraviolet C radiation (UVC)) were tested for exacerbation of mitochondrial function (assessed via changes in steady-state ATP levels) in nematodes with reduced mtDNA content. Mitochondrial health was then further assessed for some of the identified gene-environment interactions. Mitochondrial respiration was measured using the Seahorse XFe24 Extracellular Flux Analyzer, while steady-state ATP levels were assessed using transgenic luciferase expression nematodes and traditional extraction protocols. Gene expression, mtDNA, and nuclear DNA copy number were assessed using real-time PCR, while enzyme activity was assessed using microplate reader-based assays.

Results from the fission, fusion, and mitophagy toxicant screen revealed that fusion-deficient nematodes were sensitive to a variety of toxicants (DNP, AfB1, arsenite, cisplatin, paraquat, rotenone), while pink-1 mitophagy-deficient nematodes were sensitive to rotenone, and fission- and pdr-1 mitophagy-deficient nematodes were only mildly sensitive to paraquat, and rotenone, respectively. As mitochondrial disease is rare, but chronic arsenite exposure is widespread, we further investigated the mechanisms underlying arsenite sensitivity in fission- and fusion-deficient nematodes. Although not sensitive in the larval growth assay, fission-deficient nematodes were sensitive to arsenite later in life in both reproduction and lethality assays. Seahorse and ATP analysis revealed that arsenite disrupts mitochondrial function in fusion-deficient nematodes at multiple life stages (L4, 8- and 12-days of age), while enhancing mitochondrial function in 8-day old wild-type nematodes, and has minimal effect on mitochondrial function in fission-deficient nematodes. Lastly, arsenite inhibited both pyruvate and isocitrate dehydrogenase activity in fusion-deficient nematodes, suggesting a disruption of pyruvate metabolism and Krebs cycle activity underlie the observed mitochondrial dysfunction. These results suggest that deficiencies in mitochondrial fusion may sensitive individuals to arsenite toxicity.

Lastly, I have found that reducing mtDNA content 35-55% only mildly sensitized nematodes to certain secondary toxicant exposures, including UVC and arsenite. Alternatively, reduced mtDNA content did not sensitize nematodes to acute or chronic paraquat or AfB1 exposure, and provided resistance to rotenone. However, we also found that EtBr can induce cytochrome P450s (CYPs), which play a major role in rotenone metabolism; thus, it is likely that induction of CYPs and not reduced mtDNA content is responsible for rotenone resistance. These results suggest that individuals with reduced mtDNA content may be sensitive to certain toxicant exposures, but also highlight the robust mechanism that exist to maintain the integrity of mitochondria and mtDNA.

Collectively, these results suggest individuals suffering from mitochondrial disease caused by mutations in mitochondrial fission, fusion, or mitophagy genes, or by depletion of mtDNA, may be especially sensitive to certain environmental toxicant exposures, including arsenic. Arsenic’s pervasive contamination of drinking water results in chronic exposure for over 100 million people worldwide; thus, dramatically increasing the probability of exposure for individuals suffering from mitochondrial disease, and warrants further investigation in the human populous.

Fungi of the basidiomycete genus Cryptococcus cause disease in an estimated quarter of a million people, annually. Cryptococcus neoformans and Cryptococcus deneoformans are the two most prevalent disease causing species within the Cryptococcus clade, with isolates of these species exhibiting considerable variation in their pathogenicity, ranging from benign to highly virulent. A wide variety of traits, such as thermal tolerance, melanin production, and an extracellular capsule contribute to virulence, yet our understanding of the genetic architecture of such traits is limited. In the studies reported here, I describe the first genome-wide analyses of recombination in C. neoformans and C. deneoformans and provide the first high-resolution genetic mapping studies of virulence traits in these important fungal pathogens.

In studying recombination, I considered both the nuclear and mitochondrial genomes, and estimated recombination rates for both opposite- and same-sex matings. With respect to recombination of the nuclear genome, I found that progeny from opposite-sex mating have more crossovers on average than those from same-sex mating. These analyses also suggest differences in recombination rate between C. neoformans and C. deneoformans. Similarly, analyses of mitochondrial inheritance and recombination point to differences between offspring from opposite- and same-sex matings, though with much lower overall rates of recombination as compared to the nuclear genome.

To dissect the genetic architecture of complex virulence traits, I employed quantitative trait locus (QTL) mapping. A unique aspect of these QTL studies was the application of functional data analysis methods that exploit time-series data and multiple experimental conditions. I mapped QTL for thermal tolerance, melanization, capsule size, salt tolerance, and antifungal drug susceptibility in C. deneoformans. For several QTL, I was able to identify candidate causal variants that underlie these loci. Two major effect QTL for amphotericin B resistance map to SSK1 and SSK2; regulators of the high osmolarity glycerol (HOG) pathway that governs responses to osmotic stress. Epistatic interactions between SSK1 and SSK2 were also shown to govern fludioxonil sensitivity. A third major effect, pleiotropic QTL was mapped to the gene, RIC8, a regulator of cAMP-PKA signaling. RIC8 variation is predicted to contribute to differences in thermal tolerance, melanin production, and capsule size.

In combination, the studies reported here advance our understanding of the mechanisms that generate and maintain variation in Cryptococcus and implicate genetic variants in key stress-responsive signaling pathways as a major contributor to phenotypic variation between lineages of Cryptococcus.

Cellular senescence is a fundamental cell fate playing significant and complex roles during tumorigenesis and natural aging process. However, the molecular determinants distinguishing senescence from other temporary and permanent cell-cycle arrest states such as quiescence and post-mitotic state and the specified mechanisms underlying cell-fate decisions towards senescence versus cell death in response to cellular stress stimuli remain less understood. In our studies, we aimed to employ multi-omics approaches to deepen our understanding of cellular senescence, in particular, regarding the specific molecular determinants distinguishing cellular senescence from other non-dividing cell fates.

Notably, one of the most prominent features of cellular senescence differing from other non-dividing cell fates is the increased expression of senescence-associated beta-galactosidase. Because 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG) is known as the substrate catalyzed by beta-galactosidase for producing a green fluorescent product, we applied this compound to the cells undergoing G1 cell-cycle arrest (a mixture of senescent and quiescent cells). Employing fluorescence-activated cell sorting, we separated and collected senescent and quiescent cell populations based on green fluorescence intensity. As cellular senescence is more than just the non-dividing cell fate, we therefore systematically compared the gene expression between senescence and quiescence to provide insights into the specific features underlying senescence programming beyond cell cycle arrest. Following this strategy for the comparative gene expression analysis, we identified and characterized several genes critically involved in the program of cellular senescence, and one of the major findings was to identify IMMP2L, a nuclear-encoded mitochondrial intermembrane peptidase, can act as a molecular switch for determining the cell fates of healthy living, cell death, and senescence.

Inhibiting IMMP2L signaling through either the suicidal protease inhibitor SERPINB4 or transcriptional downregulation was sufficient to initiate cellular senescence by reprogramming the mitochondria functionality. Employing proteomics, we identified at least two mitochondrial target proteins processed by IMMP2L, including metabolic enzyme GPD2 and cell death regulator/electron transport chain complex I component AIF. Functional study suggests that, in healthy cells, the IMMP2L-GPD2 axis catalyzes redox reactions to produce phospholipid precursor Glycerol 3-phosphate; while under oxidative stress, IMMP2L cleaves AIF into its truncated pro-apoptotic form leading to cell death initiation to remove cells with irreparable damage. For cells programmed to senesce, the IMMP2L-GPD2 axis is switched off to block phospholipid biosynthesis leading to reduced availability of membrane building blocks for cell growth together with the disruption of mitochondrial localization of certain phospholipid-binding kinases, such as protein kinase C-δ (PKC-δ) and its downstream signaling. These alterations in mitochondria-associated metabolism and signaling network promote entry into a senescent state featuring high levels of reactive oxygen species (ROS). Simultaneously, blockage of pro-apoptotic AIF generation, which is due to the loss of IMMP2L, ensures the viability of senescent cells under ROS-mediated oxidative stress. Taken together, we have mechanistically uncovered IMMP2L-mediated signaling as a key regulatory pathway in the control of fates of healthy, apoptotic, and senescent cells.

In the physiological conditions, we observed that IMMP2L is downregulated in the muscle tissues and the blood samples of geriatric groups compared to that from young cohorts. Besides, centenarians display better genomic integrity at the IMMP2L locus when compared with the general population. Taken together, it suggests IMMP2L could also be an important player associated with the natural aging process.