Ph. D. Thesis Abstracts

Regulation of citric acid cycle enzymes and related pathways in the skeletal muscle of hibernating Richardson’s ground squirrels, Urocitellus Richardsonii

Abstract:

Richardson’s ground squirrels (Urocitellus richardsonii) are small rodents inhabiting western Canada that spend a large portion of their life in hibernation. Hibernation is accompanied by a profound drop in body temperature to a minimum of 2-3 °C and a notable shift from carbohydrate to lipid consumption that involves large-scale rearrangements of central metabolic processes. This thesis investigated the regulation of key enzymatic checkpoints in the citric acid cycle (CAC) as well as enzymes that shuttle substrates into the CAC in skeletal muscle of ground squirrels during hibernation. Initial work investigated regulation of the pyruvate dehydrogenase complex (PDC) that bridges glycolysis and the CAC. Muscle PDC showed few changes in properties in terms of activity and inhibitory phosphorylation of the enzyme. This was in stark contrast to liver where strong suppression of PDC activity occurred during hibernation correlated with increased inhibitory phosphorylation on serine-300. This then led to investigation of two crucial irreversible regulatory steps of the CAC in the muscle: citrate synthase (CS) and the α-ketoglutarate dehydrogenase complex (KGDC). CS activity decreased significantly during hibernation. This correlated with decreased lysine succinylation of CS that reflected increased SIRT5 levels, the enzyme responsible for desuccinylase activity in mitochondria. KGDC also showed decreased affinity for coenzyme A in hibernating squirrels and marked differences in posttranslational modifications including increased tyrosine phosphorylation on all three enzyme subunits and increased serine phosphorylation on E2 subunit. Stimulating the action of endogenous protein kinases demonstrated decreased affinity for coenzyme A. Finally, regulation of muscle glutamate dehydrogenase (GDH) was analyzed to ascertain how GDH regulation mediated the flow of α-ketoglutarate into the CAC from amino acid catabolism. Most GDH kinetic parameters were unaffected between hibernating and euthermic states, except that glutamate affinity was substantially lower at 8 °C (a physiologically relevant temperature) for the enzyme from hibernating squirrels. GDH from hibernating animals also exhibited significantly higher ADP-ribosylation, suggesting a regulatory mechanism for modulating GDH. Taken together these findings suggest that enzymatic regulation in Richardson’s ground squirrel muscle is actively mediated by a variety of posttranslational mechanisms of the CAC and related enzymes to coordinate metabolic suppression during hibernation.

Molecular adaptations of mammalian hypoxia tolerance: Regulation of oxidative damage, neuroprotection, and microRNA

Abstract:

Prolonged exposure to limited oxygen can be lethal. Investigating the biological consequences of oxygen-deprivation in a hypoxia tolerant mammalian model can provide us with novel insights that could be applied to alleviate the ischemic insults experienced during stroke, or to better tolerate the hypoxia of high-altitude. Naked mole-rats (Heterocephalus glaber) represent nature’s solution to the problem of both acute and chronic oxygen limitation among mammals, solutions that have developed over evolutionary time. In this thesis I investigate their unique adaptations. The data I collected paints a picture of intricate signalling mechanisms in place to facilitate metabolic reorganization and protection during hypoxia. I determine that naked mole-rats are not as vulnerable to hypoxia-induced oxidative damage, as compared to hypoxia intolerant animals, and that brains appear to be the most resilient. The cell-survival proteins I profile implicate the induction of mechanisms responsible for conserving energy and maintaining neural integrity under low oxygen levels. Next, I perform the first microRNA-sequencing analysis in naked mole-rats, focusing on the hypoxic brain. Hypoxia-induced microRNAs suppress ATP-expensive processes, activate central signalling pathways, and coordinate a shift to non-fructose based anaerobic glycolysis. I then examine global metabolic reorganization and characterize a microRNA-mediated, AMPK-driven shift to carbohydrate metabolism in hypoxic skeletal muscles that may support tissue-specific prioritization of energy for more essential organs. Taken together, these findings advance our understanding of mammalian hypoxia tolerance and highlight the molecular mechanisms and complex layered regulatory controls required to endure frequent hypoxia exposures, as well as provide directions for future studies.

Roles of inflammatory signaling and microRNA in the adipose stress response of hibernating Ictidomys tridecemlineatus

Abstract:

Hibernating ground squirrels have an interesting ability to avoid organ dysfunction despite months of obesity, starvation, and low body temperature. However, pro-inflammatory signaling and conserved miRNA expression patterns have yet to be investigated in white and brown adipose tissues (WAT, BAT), organs with roles in fat storage and heat production, respectively. The inflammasome was activated in BAT during torpor and arousal relative to the control, as evidenced by increased inflammasome priming, elevated protein levels of NLRP3, AIM2, cleaved gasdermin D and IL-18, as well as increased caspase-1 activity. By contrast, caspase-1 activity, the ultimate indicator of inflammasome activation, was decreased during torpor and arousal in WAT relative to the euthermic control. Pro-inflammatory cytokines, matrix metalloproteinases (MMPs), and their inhibitors were also investigated to determine if cytokines and tissue remodeling proteins could be important in the stress response in hibernator adipose tissue. An increase in IL-1α during torpor in BAT furthered the idea that BAT may use pro-inflammatory pathways as part of the response to cell stress. By contrast, the only change in WAT was a decrease in the total protein levels of MMP2, suggesting tissue remodeling may not be important in the maintenance of WAT homeostasis. Finally, conserved BAT and WAT miRNAs were analyzed. There was an association between the BAT miRNA expression profile and condition (control or torpor), but no association between the two variables in WAT. Consistently, fewer miRNAs were differentially expressed in WAT than BAT, with more being downregulated than upregulated. As expected, microRNAs were predicted to inhibit energy expensive pathways during torpor in both tissues, suggesting an important role for non-coding RNAs in the regulation of metabolic rate suppression. Unexpectedly, KEGG pathway analysis suggested miRNAs were less likely to target pathways involved in damage sensing and wound repair in BAT, and DNA damage repair in WAT. Together, the data in this thesis suggest an upregulation of stress sensing and response in BAT in torpid and arousing ground squirrels through the regulation of inflammasomes, inflammatory signaling, and miRNA expression. By contrast, DNA repair may be increased in WAT but generally, pro-inflammatory pathways were suppressed.

Regulation and modification of peripheral circadian molecular clocks in 13-lined ground squirrels during hibernation

Abstract:

During winter, hibernators are able to conserve energy during times of limited resources through the virtual cessation of energetically expensive processes that are thought to be intrinsic to the cell in homeostasis. During prolonged hibernation, these mammals, such as the 13-lined ground squirrel (Ictidomys tridecemlineatus), shut down the bulk of transcription and translation in order to preserve resources yet still require the expression of subsets of genes to assist with the challenges encountered during hibernation. Hibernators provide a unique opportunity for examining the dynamics of circadian clock activation in a system that requires the selection of groups of transcripts against a backdrop of suppressed cellular activity. This research shows that peripheral circadian clocks are regulated and have adapted to function in a tissue-specific manner that is congruent with the tissues functions during hibernation.

In addition, substantial transcriptional and post-transcriptional machineries are required to endure deep torpor and low body temperature, including increased regulation over genomic activity by epigenetic enzymes. Both RNA adenosine and protein arginine methylation act to regulate activity within the circadian clock via epigenetic mechanisms and provide novel opportunities to uncover information about the post-translational modifications used during hibernation. RNA N6-methyladenosine (m6A) dynamics were maintained during hibernation and levels of m6A were increased on mRNA transcripts during torpor in liver. Responses by protein arginine methyltransferase (PRMT) enzymes were tissue-specific and within liver and white adipose, revealed responses that characterized metabolic reprogramming, whereas skeletal muscle PRMT activity was centered around transcriptional regulation. This research suggests that dynamic epigenetic modifications provide a mechanism for maintaining translation of selected groups of necessary transcripts during hibernation, including core circadian clock genes, against a backdrop of stunted transcript processing. These data also provide evidence that the circadian clock is an important and integral regulator of peripheral tissues within the mammalian hibernation phenotype.

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.