Ph. D. Thesis Abstracts

No Oxygen? No Problem! Epigenetic mechanisms of anoxia tolerance in a champion anaerobe, the red-eared slider turtle (Trachemys scripta elegans)

Abstract:

Red-eared sliders (Trachemys scripta elegans) are champion anaerobes that can survive approximately three months of absolute anoxia at 3C and recover with minimal cellular injury. Although various physiological and biochemical adaptations are involved in anoxia tolerance, metabolic rate depression (MRD) is considered to be the most useful response. T.s. elegans can reduce their metabolic rate to 10% of normoxic values by reducing all energy expensive cellular processes including gene expression. However, adaptations of alternate transcriptional regulatory processes are mostly unknown. In the thesis, epigenetic regulation of anoxia tolerance was investigated by exploring the dynamic changes in DNA methylation/demethylation, histone acetylation/deacetylation, and histone lysine methylation during short-term (5 h) anoxia and long-term (20 h) anoxia in several tissues of red-eared sliders. DNA methylation significantly increased in the liver and white skeletal muscle. An increase in DNA methylation could indicate a potential decrease in global gene expression in response to oxygen deprivation in red-eared sliders. Correspondingly, a genomic mark of active transcription, DNA demethylation, decreased in the liver and white skeletal muscle. Establishing a unique balance between global and localized DNA methylation could be an important component of anoxia tolerance. Histone lysine methylation was also anoxia responsive in the liver of red-eared sliders, and suggested a target-specific regulation that could potentially aid in the selective upregulation of genes that are necessary for anoxia survival, while suppressing others. Histone acetylation and deacetylation, implicated in MRD of other stress-tolerant animals, illustrated a strong suppression in the liver of red-eared sliders. A strong suppression in histone H3 acetylation may also indicate an overall decrease in gene expression. Overall, this thesis may enhance our understanding of alternate modes of transcriptional regulation during anoxia tolerance and report several epigenetic mechanisms that are involved the hypometabolic response in T.s. elegans.

Molecular responses to whole-body dehydration in a sequenced vertebrate, Xenopus laevis: Regulation of antioxidants and metabolism by the Sirtuin protein deacetylases

Abstract:

Whole-body dehydration in the African clawed frog, Xenopus laevis, increases hematocrit and blood viscosity, which restrains oxygen delivery. This causes the resting heart rate, differences in arterio-venous blood oxygen contents, and whole-animal lactate to increase.
I hypothesized that dehydration involves changes in cellular signaling through alterations of protein posttranslational acetylation, which can increase antioxidants and regulate metabolism. Seven Sirtuin (Sirt) protein deacetylases were profiled at the mRNA level with RT-qPCR in 6 tissues (liver, muscle, heart, kidney, brain, and lung) of X. laevis under control versus dehydration conditions. At least some sirt transcripts increased in all tissues except for kidney and brain. Similarly, global Sirt activity assays found that Sirt deacetylase activity increased in liver, muscle, heart, and lung. Western blots revealed the relative levels of Ac-SOD2. Results showed that acetylated SOD2 decreased with whole-body dehydration in the lung, heart, and kidney, suggesting that Sirt3 deacetylase activity is triggered by dehydration to activate antioxidant activity in these tissues.
Sirt/PGC-1α/FoxO-mediated upregulation of antioxidants was investigated in lung and brain of X. laevis. Results showed upregulations of these three controllers of antioxidants in lung (but not brain) during dehydration, as evidenced by analyses at the mRNA, protein, and phospho-protein levels. Results suggested that dehydration-induced antioxidant upregulation in X. laevis was mediated by Sirts, in addition to PGC-1a and the FoxO1/3 transcription factors in a tissue-specific manner. Antioxidant capacity assays showed that lung sustained a decrease in antioxidant capacity during dehydration, which suggests that the Sirt/PGC-1α/FoxO response may be a compensatory one to restore antioxidants levels.
In the liver, muscle, and heart, PGC-1α and Hif-1α were assessed for their roles in activating ureagenesis, angiogenesis, and remodelling of the metabolism. MEF2-mediated PGC-1α upregulation occurred in the liver, but not the muscle or heart, whereas Hif-1α increased in all 3 tissues with dehydration. Relative mRNA levels of genes related to glucose metabolism, angiogenesis, ureagenesis and β-oxidation were found to be differentially regulated in response to dehydration. Together, the results suggest that PGC-1α and Hif-1α are modulating gene expression during dehydration to suppress β-oxidation in favour of glycolysis, while ureagenesis and angiogenesis are promoted in liver.

Regulation of gene expression over cycles of torpor-arousal in thirteen-lined ground squirrels

Abstract:

Mammalian hibernators undergo profound behavioural, physiological and biochemical changes to cope with hypothermia, ischemia-reperfusion, and finite fuel reserves during days or weeks of continuous torpor. Against a backdrop of global suppression of energy-expensive processes such as transcription and translation, selected genes/proteins are strategically up-regulated to meet challenges associated with hibernation. Hence, hibernation involves substantial transcriptional and post-transcriptional regulatory mechanisms and provides a model to determine how a set of common genes/proteins can be differentially regulated to enhance stress tolerance beyond that which is possible for nonhibernators. The present research analyzed epigenetic factors, signal transduction pathways, transcription factors, and RNA binding proteins that regulate gene/protein expression programs that define the hibernating phenotype. Epigenetic factors alter gene expression programs by influencing the accessibility of DNA promoter regions to the transcriptional machinery. While DNA methylation was not differentially regulated comparing summer and winter animals, posttranslational modifications on histone proteins were responsive to torpor-arousal, possibly providing a mechanism to dynamically alter chromatin structure. Unique posttranslational modifications on H3 and H2B were identified by mass spectrometry; these have never been found in other organisms. Signal transduction pathways such as mitogen-activated protein kinases convert information received at the cell surface to regulatory targets within cells that promote changes in gene expression. Results showed that MAPK regulation is crucial during arousal from torpor in muscle and heart. Important cytoprotective features needed for hibernation are antioxidant defenses; regulation of antioxidant genes is under primary control of transcription factors, such as Nrf2. Data presented elucidates the regulation of Nrf2 transcription factors by post-translational modifications (e.g. serine phosphorylation, lysine acetylation) and protein-protein interactions with a negative regulator (KEAP1) during hibernation. Finally, a role for RNA binding proteins including TIA-1, TIAR, and PABP-1 is described. Data showed the localization of RNA-binding proteins to subnuclear structures which may represent highly organized storage centers and/or enhance mRNA stability. Taken together, the thesis identifies novel regulatory mechanisms that aid suppression of transcriptional and translational rates, while also coordinating complex pathways that selectively enhance cytoprotective pathways aimed at mitigating stresses associated with torpor-arousal.

Regulation of skeletal muscle carbohydrate metabolism during mammalian hibernation

Abstract:

Thirteen-lined (Ictidomys tridecemlineatus) and Richardson’s (Urocitellus richardsonii) ground squirrels survive harsh winter conditions by entering hibernation, spending the majority of their time in a state of torpor, where metabolic functions are both strongly suppressed and reprioritized to ensure long term survival. This thesis analyzed biochemical controls on carbohydrate metabolism during hibernation by characterizing the regulation of crucial enzymes of the glycolytic and gluconeogenic pathways: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), and fructose-1,6-bisphosphatase (FBPase). Important signal transduction enzymes regulating carbohydrate metabolism were also evaluated: protein phosphatase 2A (PP2A) and glycogen synthase kinase 3 (GSK3). The state of glycolysis in ground squirrel skeletal muscle was assessed by characterizing the bifunctional enzyme GAPDH and the terminal enzyme PK. Results showed that muscle GAPDH and PK activities were substantially suppressed during torpor. PK suppression was linked to reversible serine/threonine phosphorylation. GAPDH regulation was more complex with activity potentially mediated by one or more posttranslational modifications including acetylation, methylation and phosphorylation, as identified through mass spectrometry and Western blot analyses. The gluconeogenic state of muscle was assessed by characterizing FBPase as well as GAPDH operation in its gluconeogenic direction. In both cases, results indicated significant reductions in gluconeogenic function during torpor. Suppressed FBPase activity (i.e. decreased Vmax, increased Km F1,6P2) was linked with an increase in FBPase phosphorylation and allosteric controls by AMP and F2,6P2. Analysis of PP2A catalytic subunit showed that elevated phosphorylation at tyrosine307 accompanied a significant increase in Km peptide, indicating reduced activity of PP2A during torpor. This was corroborated by computational analysis of tyrosine307 phosphorylation effects on substrate binding. Skeletal muscle GSK3 activity also decreased during torpor associated with enhanced GSK3 phosphorylation at serine9. However, the principal substrate of GSK3, glycogen synthase, showed increased phosphorylation suggesting that a different protein kinase was responsible for its control during torpor. Taken together these studies suggest that skeletal muscle glycolysis and gluconeogenesis are suppressed during ground squirrel torpor via posttranslational modification and regulation of key enzymes. Reversible controls over glycolytic and gluconeogenic enzymes would allow for the quick reactivation of muscle metabolism to support shivering thermogenesis and a return to normal euthermic function during arousal.

Front line antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica: An in-depth analysis of mechanisms of enzyme regulation

Abstract:

The wood frog, Rana sylvatica, is one of few species that can survive whole-body freezing during overwintering. Frogs endure freezing of up to 70% of their total body water, and demonstrate a complete lack of respiration, heart beat and brain activity. Freezing imposes multiple stresses including anoxia/ischemia, cellular dehydration when water is lost to extracellular ice masses, wide temperature changes, and potential physical damage by ice. One crucial adaptation for freezing survival is well-developed antioxidant defenses to protect tissues from abiotic stress while frozen and deal with rapid changes in the generation of reactive oxygen species associated with anoxia and reoxygenation over freeze/thaw cycles.

This thesis explores the properties and regulation of key antioxidant enzymes, purified via novel schemes, from frog muscle – both Cu/Zn- and Mn-dependent isoforms of superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT). The studies show that changes in activity, stability, and substrate affinity of antioxidant enzymes during the frozen state may be significant preparatory mechanisms employed by R. sylvatica to support the transition from frozen to thawed states and deal effectively with oxidative stress accompanying reperfusion. Moreover, reversible protein phosphorylation plays a central role in regulating the activity of these enzymes to suit physiological needs throughout freeze-thaw cycles. For example, CuZnSOD from muscle of frozen frogs showed a significantly higher Vmax compared to the control enzyme. Muscle MnSOD from frozen frogs showed a significantly lower Km for O2-, higher phosphorylation, and increased enzyme stability compared to control MnSOD. GR from frog muscle showed a significantly lower Km for GSSG in the face of physiological levels of glucose encountered during freezing, as well as the potential for phosphorylation via endogenous kinases. CAT from muscle of frozen frogs showed a significantly lower Km for H2O2 and a higher level of phosphorylation; furthermore, stimulation of endogenous kinases decreased Km H2O2 similar to what occurred in muscle of frozen animals. This thesis provides compelling evidence for regulation of antioxidant enzymes via reversible protein phosphorylation and augmentation of key antioxidant enzymes during freezing of the frog, likely in preparation to endure oxidative stress encountered during reperfusion over winter freeze-thaw cycles.

Molecular adaptations of mammalian hibernation: Roles of metabolic signaling regulation in the torpor-arousal cycle

Abstract:

For many small mammals, survival over the winter months is a serious challenge because of low environmental temperatures and limited food availability. The solution for thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is hibernation, a metabolically re-programmed state that is characterized by seasonal heterothermy and entry into long periods of torpor. Although many studies have defined the physiological responses of hibernation, including drastic reductions in heart rate, respiration, and body temperature, the regulation of such phenotypic plasticity has yet to be fully characterized at the molecular level. As part of hibernation, metabolic rate is suppressed during torpor to achieve major energy savings through coordinated suppression of non-essential ATP-costly processes. The present thesis examined the role of cell signaling cascades in the regulation of energy dependent cellular processes over the torpor-arousal cycles of hibernation.

The insulin signaling pathway, which functions as the regulator of many pro-growth processes such as protein synthesis, was shown to be regulated during torpor. Significant inhibition of this pathway was most evident in skeletal muscle but not in cardiac muscle during torpor. This inhibition was characterized by reduced phosphorylation of mammalian target of rapamycin kinase (mTOR), which led to subsequent inhibition of various proteins involved in ribosome assembly that are required for protein translation. The cell cycle, another energy dependent metabolic process, was also strongly inhibited during torpor. Cell cycle arrest was evident in liver (proliferative capable) but not in skeletal muscle (terminally differentiated), through mechanisms similar to those observed in G1 and G1/S arrest. The observed cell cycle arrest was characterized by down-regulation of proteins cyclin D and cyclin E which function as positive regulators of cell cycle progression, and up-regulation of the cell cycle inhibitors (CKIs) p15INK4b and p21CIP1. Up-regulation of CKIs during torpor was linked to members of the Smad family of transcription factors, which were activated during torpor, including increased nuclear inclusion and DNA binding activity of Smad 3. Overall, the data presented in this thesis document molecular mechanisms that function to reduce cell-growth and proliferation during torpor, via inhibition of protein synthesis and cell cycle progression.

Roles of Akt signaling and its downstream pathways in wood frog freeze tolerance

Abstract:

Wood frogs, Rana sylvatica, are one of only a few vertebrate species that survive prolonged whole body freezing during the winter. Multiple adaptations of physiology and biochemistry that support freeze tolerance have been identified including accumulation of extreme levels of glucose as a cryoprotectant and entry into a hypometabolic state that reduces the energy needs of the animal while frozen. To date, the stress responsive signal transduction networks that trigger and regulate these adaptations have received little attention. The current thesis addressed this subject by exploring responses and regulation of a major intracellular signaling pathway (the Akt pathway) that is centrally involved in mediating cellular growth and proliferation responses, typically responding to extracellular insulin signals. Analysis of four organs (liver, kidney, heart and skeletal muscle) showed activation of the Akt pathway in liver but signs of inhibition occurred in other tissues in response to freezing. Activation of Akt-dependent anti-apoptosis mechanisms in liver was also indicated to support cell survival in the frozen, anoxic state. However, analysis of multiple protein components of the cell cycle and TORC1-dependent protein synthesis showed strong suppression of these in all tissues, although with lesser inhibition in liver. This demonstrates the importance of suppressing energy-expensive cell processes under stress conditions. The data show that, during whole body freezing of wood frogs, (1) ATP expensive cellular events such as the cell cycle and protein synthesis were suppressed; (2) liver remains more metabolically active than other tissues tested; and (3) freeze responsive Akt activation in liver does not universally activate all of its downstream pathways but rather selectively triggers specific targets, particularly those important to glucose production as a cryoprotectant and to cell preservation. The thesis also investigated freeze-responsive antioxidant defenses in wood frog liver and muscle with a focus on mechanisms regulated by the Nrf2 transcription factor and showed that Nrf2 is glucose-responsive. Furthermore, glucose appears capable of differentially affecting gene expression and posttranslational modifications of proteins in liver. Overall, this thesis showed the central importance of the Akt pathway in freeze tolerance and demonstrated tissue- and environment-specific responses by the pathway and its downstream processes.

Cell cycle regulation by post-translational and post-transcriptional mechanisms in an anaerobic extremist – The anoxic tolerant turtle, Trachemys scripta elegans

Abstract:

As a model for vertebrate long-term survival in oxygen restricted environments, the red-eared slider turtle (T. s. elegans) can adapt at the biochemical level to deal with hibernation occurring in oxygen-free (anoxic) cold water (<10°C). In this thesis I hypothesized that the mechanisms which suppress ATP-expensive cell cycle activity, would contribute to establishing an hypometabolic state. To explore this possibility, this thesis studied the post-transcriptional and post-translational mechanisms of cell cycle arrest during anoxic stress in the freshwater turtle.

Results indicated a general regulation of critical cell cycle components, in addition to the possible regulation by signaling cascades (Akt/GSK-3β and ATR/Chk2) that are known to regulate G1/G0 phases of the cell cycle. Importantly, there is extensive regulation of Cyclin D1 protein by (1) Akt/GSK-3β signaling, (2) post-translational modification, (3) an AU-rich region, and (4) microRNA-induced translational suppression. This study also identified a phase-specific cell cycle arrest mechanism involving the Rb/E2F DNA-binding complex in both anoxic liver and kidney tissues. A novel DNA-binding complex ELISA technique was able to identify that both kidney and liver establish an Rb/E2F1 mediated G1 arrest complex by 5 and 20 h anoxia, repectively. By 20 h anoxia, kidney tissue established a reversible state of G0, characterized by the prescence of a p130/E2F4 DNA-bound complex.

Overall, results from this thesis indicate that both kidney and liver enter into a G1 arrest during anoxia. By contrast, the cell cycle in white skeletal muscle was found to be minimally regulated during anoxia and this finding is likely a reflection of its overall post-mitotic nature. Interestingly, kidney established a state of G1 arrest within 5 h anoxia and subsequently transitioned to a sustainable G0 arrest by 20 h anoxia. However, it appears that liver G1 arrest was not established until 20 h anoxia. Future studies will need to explore the regulation of the cell cycle in liver after longer periods of anaerobiosis to determine whether hepatocytes are also able to transition into G0 arrest in a manner similar to kidney tissue.

A study of the metabolic adaptations of marine gastropod molluscs to environmental anoxia stress

Abstract:

Catalytic and regulatory properties of alanopine dehydrogenase (ADH) and pyruvate kinase (PK) from tissues of anoxia tolerant marine gastropods were studied. Particular attention was given to those properties of the enzymes which could help explain their potential role(s) in anaerobic energy metabolism. The physical and kinetic properties of the terminal glycolytic dehydrogenase, ADH, purified to homogeneity from foot muscle of the common periwinkle, Littorina littorea, were examined. The kinetic properties of ADH favor enzyme function in cytoplasmic redox balance during the recovery period following long-term environmental anoxia. Tissue specific isozymes of ADH were found in another marine gastropod, the channelled whelk,Busycotypus canaliculatum. Three isozymic forms, specific for muscle, gill/kidney and hepatopancreas were identified. The three tissue specific isozymes of ADH were purified to homogeneity from foot muscle, gill and hepatopancreas and their kinetic and physical properties were studied. Muscle ADH showed properties which appear to gear this isozyme for alanopine synthesis as an end product of glycolysis. The hepatopancreas isozyme appears suited for a role in alanopine oxidation in vivo. The properties of gill ADH are intermediate between those of the other two forms. Tissue specific forms of PK were also found in B. canaliculatum. Three isozymic forms, specific for red muscle, white muscle and soft tissues, were identified. Furthermore, each PK isozyme was modified in animals subjected to 21 h of anoxic stress such that several physical and kinetic characteristics were altered. Aerobic and anoxic forms of red muscle PK (RPK-AER and RPK-ANX) were purified to homogeneity from radular retractor tissue of B canaliculatum and the physical and kinetic properties of the enzyme were extensively studied. The differences in kinetic properties between RPK-AER and RPK-ANX indicates that red muscle PK activity is probably greatly depressed in vivo during long-term anoxic stress. The anoxia-dependent, in vivo, covalent incorporation of injected (’32)P orthophosphate into RPK-ANX demonstrated that the enzyme is a phosphoprotein. Evidence for the reversibility of this phosphorylation was provided by the kinetic similarities between purified RPK-AER and homogenous alkaline phosphatase treated RPK-ANX.

Metabolic biochemistry of freeze tolerance in vertebrates

Abstract:

A unique group of vertebrate animals has developed complex metabolic adaptations that enable them to survive freezing. This thesis investigates: i) cryoprotectant synthesis in freeze tolerant frogs, ii) freeze tolerance in a newly identified freeze tolerant vertebrate, the garter snake, and iii) metabolic responses elicited by other stresses (anoxia and dehydration) in the garter snake and two frog species. Investigation of cryoprotectant synthesis in spring frogs,Pseudacris crucifer, revealed large amounts of glucose produced during freezing; approximately 0.1 molar. Changes in the levels of glycolytic intermediates indicated that an activation of glycogen phosphorylase and phosphofructokinase (PFK) directed glycolytic flux to cryoprotectant synthesis. Tissue glucose distribution was much lower than in fall animals. These results suggested seasonal variation in glucose transport mechanisms. A similar investigation of cryoprotectant synthesis in spring Hyla versicolorshowed a maintenance of regulatory enzyme controls at glycogen phosphorylase and PFK directing glycogen carbon to cryoprotectant. Only glucose was synthesized as cryoprotectant; quantities of glycerol (the major cryoprotectant of winter H. versicolor) showed no increase. The amount of cryoprotectant produced was directed correlated to glycogen content in the liver. Investigation of freeze tolerance in garter snakes revealed that these snakes were only partially freeze tolerant. Survival of brief freezing exposures (5-10 h at -2.5°C; 30-50% ice) was possible. Two amino acids, glutamate and taurine, were implicated as possible cryoprotective agents. Comparison of the metabolic responses (adenylate levels, anaerobic end products, glycolytic flux) to freezing in garter snakes were similar to those elicited by anoxia. Dehydration timecourses were investigated in two freeze tolerant frog species, Rana sylvatica andPseudacris crucifer. Even though whole body water contents dropped by 50-60 %, individual tissues exhibited little or no change in water content. There were many similarities between metabolic responses to dehydration and those to freezing. The most remarkable similarity between freezing and dehydration was the accumulation of glucose, presumably acting as a cellular protectant; quantities in liver rose to 127 and 220 micromole per gram in R. sylvatica and P. crucifer, respectively.