Ph. D. Thesis Abstracts

Frozen but alive: Molecular responses to autophagy, angiogenesis and energy metabolism in the stress-tolerant wood frog, Rana sylvatica

Abstract:

The freeze-tolerant wood frogs (Rana sylvatica) are incredible creatures that can tolerate the freezing of up to ~70% of their total body water during winter. Once frozen, these frogs are considered clinically dead, exhibiting no signs of breathing, heartbeat, muscle movement and nerve conductance; yet, they come back to life, unharmed, after a few hours of thawing. Freezing is associated with ischemia due to the freezing of the blood, with hyperglycemia due to the production of large quantities of glucose for cryoprotection, and with dehydration as water moves from inside the cell to the extracellular space to prevent intracellular freezing. Interestingly, wood frogs can tolerate all these stresses independently of freezing, thereby creating a multifactorial model for studying vertebrate freeze-tolerance. Oxygen availability is very low to non-existing during freezing, anoxia, and dehydration; therefore, wood frogs are hypothesized to reduce their overall metabolic rates to balance energy production with energy expenditure in a process called metabolic rate depression (MRD). Animals that undergo MRD reduce energy expensive or detrimental processes and allocate the limited energy available only to pro-survival responses. This thesis examined the effects of freezing and its associated stress on responses to autophagy, angiogenesis, select group of antioxidant enzymes, and energy metabolism. Molecular responses to autophagy demonstrate a significant reduction in autophagosome formation and lysosomal biogenesis in response to anoxia/reoxygenation and to a lesser degree in response to dehydration/rehydration in liver, whereas these two processes were significantly reduced under all conditions in skeletal muscle. Current results also indicate that angiogenesis is regulated in a temporal and stress-dependent manner, where wood frogs increase the expression of certain pro- and anti-angiogenic factors in anticipation of potential damage to capillaries or injury to tissues. Investigation into the role of ETS1 as a transcriptional activator and repressor demonstrated its potential involvement in promoting the expression of select antioxidant enzymes, while repressing the expression of certain nuclear-encoded mitochondrial proteins. Overall, findings in this thesis demonstrate the complexity of the mechanisms involved in controlling metabolic rate depression in adaptive responses in wood frogs.

The molecular biology of dehydration tolerance: Regulation of gene expression and function in Xenopus laevis

Abstract:

The African clawed frog, Xenopus laevis, has been used as a model organism for cellular and developmental biology for nearly a century. Comparatively unstudied is its natural tolerance to dehydration brought about by seasonal drought evaporating its aquatic habitats. To survive the loss of >30% body water content, these animals employ several tissue-specific adaptations ranging from switching to ureotelism to relying on anaerobic metabolism as oxygen transport decreases with increased blood viscosity. Previous studies have indicated dehydration responsive gene expression and function is regulated with multiple mechanisms. In this thesis I further establish X. laevis as a dehydration tolerance model organism by determining suitable RT-qPCR reference genes in eight tissues. I then investigate regulatory mechanisms capable of large-scale regulation, namely, DNA methylation and histone modifications, microRNA, and reversible protein phosphorylation. Global levels of epigenetic marks showed little response to dehydration apart from increased 5hmC and decreased H3K4me in the liver, suggestive of epigenetic reprogramming. MicroRNAs, which are short RNAs that negatively regulate translation of specific mRNAs, were then examined in the heart. This analysis revealed a trend of downregulation during dehydration, and the enrichment of several important pathways including cardiac muscle contraction and glycolysis and gluconeogenesis. Particularly telling is the near uniform prediction of decreased regulation of all glycolytic enzyme transcripts that may support increased anaerobic glycolysis capacity during dehydration. Next, I analyzed the liver and skeletal muscle phosphoproteomes during dehydration and found a strong and concerted response by the liver and not muscle. Also emerging from the data was the significant upregulation and phosphorylation of a hypoxia inducible PFKFB isozyme in the liver known to support glycolysis in many cancers. Together these results significantly advance our understanding of the molecular biology of dehydration tolerance and provide multiple clear directions for future studies.

Regulation of antioxidant defenses, DNA damage repair, the immune response, and neuroprotection during hibernation in the thirteen-lined ground squirrel

Abstract:

Hibernation is a fascinating survival adaptation that allows animals to transition into a torpid state to survive the winter by coordinating a strong suppression of metabolic rate, conservation of fuel/energy, and reduction of body temperature. This strategy permits thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and other hibernating mammals to endure the harsh winter season when there is little access to food. Many energy-expensive cellular processes are suppressed, including gene transcription and protein synthesis/turnover, but are reactivated rapidly when animals arouse back to euthermia. Both torpor and arousal can have damaging consequences; for example, during arousal, reactive oxygen species flood the cell causing oxidative damage to numerous cellular components. Therefore, hibernation requires many pro-survival mechanisms to mitigate multiple types of damage: e.g. from oxidative damage, DNA damage, and pathogen attack, among others. The research reported in this thesis on damage control processes in hibernators shows that antioxidant enzymes such as PRDXs are upregulated in key tissues but in an isoform-specific and time-specific manner over the torpor-arousal cycle. PRDX2, 3, 4 and 6 were found to be significantly upregulated in specific tissues. Similarly, DNA damage repair is initiated during torpor and is characterized by the binding of repair proteins such as Ku80 and the MRN complex to the site of breaks, but ligation (with XLF) reactions to fully repair DNA do not appear to occur until the arousal period. Pro-inflammatory mechanisms are also used to deal with pathogens; these remain active at basal levels in a tissue-specific manner during torpor, but are up-regulated in the final stages just before arousal or only during arousal depending on the tissue, such as the induction of CCL5, a recruiter of monocytes. A cyto/neuro-protective mitochondrial peptide, s-humanin, was also identified that is induced in a tissue-specific manner, helping to protect key organs such as the brain cortex and adipose tissues. The results show that hibernation is a complex, multi-faceted process that employs specific adaptations of damage prevention/repair pathways to protect squirrel tissues from damage not only during prolonged torpor but over the transitional states to/from torpor and does so expertly while conserving energy until such a time that repair mechanisms may be fully initiated.

Reversible enzyme phosphorylation as a mechanism for metabolic adaptation to dehydration in the skeletal muscle of the African clawed frog, Xenopus laevis

Abstract:

Xenopus laevis, although mainly an aquatic frog, lives in seasonally arid regions of southern Africa where well-developed dehydration tolerance is needed when ponds dry up. Frogs can endure about 40% loss of total body water leading to increased hematocrit and blood viscosity that restrict blood and oxygen delivery to tissues, elevate tissue osmolality, and lead to accumulation of lactate and urea. As one response to dehydration, frogs show restricted blood flow to skeletal muscle to preferentially maintain supply to the brain and internal organs. I hypothesized that dehydration stress triggers modifications to cellular energy production in skeletal muscle and could recruit alternative fuel use. This thesis explores metabolic regulation of enzymes (aldolase, CK, IDH), and energy stress signaling (via AMPK) in skeletal muscle of X. laevis. A particular focus was put on regulation via protein posttranslational phosphorylation to adapt enzyme activity and substrate affinity to changing physiological needs during dehydration. Analysis of kinetic parameters found that aldolase, CK and IDH all showed reduced maximal velocities and altered substrate affinities during dehydration. Downregulation of aldolase suggested a reduction in glycolytic rate during dehydration, moderating the use of glucose, whereas CK regulation modulates phosphocreatine consumption. Substrate affinities of both CK and IDH were dependent on magnesium concentrations. CK was more active at higher Mg2+ concentrations that occur as tissues dehydrate whereas IDH showed increased affinity for Mg2+ that could shift the reaction to favor α-KG production during dehydration. I hypothesized that changes to muscle energetics would stimulate the action of AMPK and its downstream effectors to promote a fuel switching from carbohydrates to include fats during dehydration. However, phosphorylated AMPK (activated) did not increase and the regulation of two key downstream AMPK targets, acetyl-coA carboxylase and Unc-51 like autophagy activating kinase 1, did not indicate recruitment of fatty acid metabolism or autophagy for energy during dehydration in skeletal muscle. Overall, these studies showed that reversible protein phosphorylation has a prominent role in controlling X. laevis skeletal muscle enzyme function and reorganization of metabolic pathways during whole animal dehydration.

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.