Ph. D. Thesis Abstracts

Enzymatic regulation of hepatic carbohydrate metabolism in freeze-tolerant wood frog, Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a widely researched vertebrate species due to their ability to endure natural freeze tolerance. These frogs can survive months of sub-zero temperatures during winter, even when 65-70% of their total body water is frozen as extracellular ice. However, this freezing results in the cessation of blood circulation, heartbeat, and breathing, leading to limited oxygen supply. Wood frogs must depend on anaerobic glycolysis for energy production during this time. Two of the basic mechanisms underlying freeze tolerance in wood frogs are metabolic rate depression (MRD) and the production of high concentrations of glucose as a cryoprotectant by the liver. This thesis aimed to investigate the regulation of key enzymatic checkpoints in hepatic carbohydrate metabolism in wood frogs. The research revealed the downregulation of pyruvate kinase (PK) during freezing, leading to the inhibition of glycolysis. The study also shed light on the suppression of fructose-1,6-bisphosphate (FBPase) and citrate synthase (CS), which subsequently inhibited flux through gluconeogenesis and TCA, respectively. This suppression is likely to aid in the survival of MRD during severe winters. Moreover, it was found that glycerol-3-phosphate dehydrogenase (G3PDH)—an enzyme linking lipid and carbohydrate metabolism—is upregulated despite the hypometabolic conditions during freezing. This upregulation of G3PDH activity likely plays a vital role in supporting the metabolic survival strategies of wood frogs. Overall, this thesis uncovered an intricate yet synchronized network of enzymes that support MRD and initiate hepatoprotective mechanisms allowing wood frogs to endure prolonged freezing and maintain cellular homeostasis.

Complex yet coordinated: regulation of transcriptional factors and cell signaling pathways to endure anoxia in Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a well-studied vertebrate model of natural freeze tolerance, surviving several months of winter subzero temperatures with 65-70% of total body water frozen as extracellular ice. Freezing halts blood circulation, heartbeat and breathing, restricting oxygen availability throughout the body and requiring a switch to anaerobic glycolysis for energy production, with its much lower ATP yield. To survive, wood frogs suppress their metabolic rate by about 90% to match ATP availability from glycolysis alone. Multiple cellular processes are regulated and suppressed, sustaining only pro-survival pathways until thawing occurs. Episodes of anoxia/reoxygenation also elevate reactive oxygen species (ROS) production that can surpass the antioxidant capacity of cells causing oxidative stress and tissue damage. This thesis examined a network of stress-responsive transcription factors (NRF2, OCT1, OCT4, YAP/TEAD, and RBPJ) and their associated pathways to determine their response and regulation over the anoxia/reoxygenation cycle. Decreased binding of transcriptional complexes to the promoter regions of target genes indicated a global reduction in transcription/translation processes. The data show also “functional switching” of OCT1, OCT4, and MAML while selectively upregulating antioxidants in a stress/organ specific manner. The present studies also shed new light on tissue repair mechanisms by demonstrating upregulation of selected pathway proteins. An increase in AHCY levels in liver also suggests maintenance of redox control, and elevated JMJD2C, TAZ, and MAML in skeletal and cardiac muscles indicates a potential increase in the expression of MyoD for muscle regeneration. Overall, the findings of this thesis document a complex yet coordinated network of transcriptional factors that support metabolic rate depression during freezing, combat oxidative stress, and initiate tissue repair mechanisms to endure prolonged anoxia and maintain cellular homeostasis in frozen wood frogs.

Role of glucose-induced transcription factor signalling and mitochondrial epigenetics in stress tolerant wood frog, Rana sylvatica

Abstract:

The freeze-tolerant wood frogs, Rana sylvatica are one of only a few vertebrate species in the animal kingdom, which are extensively studied to understand vertebrate freeze tolerance. They undergo whole-body freezing during winter and become ice solid with no heartbeat, brain activity and blood flow but amazingly come back to life during spring unharmed without any major changes in their body. Freeze survival is challenging, with wood frogs facing ischemia due to freezing of blood, dehydration via cell volume reductions due to loss of 60-70% of total body water into extracellular space as well as hyperglycemia, producing a huge amount of glucose as a cryoprotectant. Interestingly, wood frogs can also tolerate these stresses independent of freezing. Also, winter survival by wood frogs is associated with a metabolic reorganization to reduce their energy demands to a bare minimum by globally suppressing energy-expensive pathways and selectively regulating genes to prioritize available energy use for pro-survival pathways. This thesis examined the effects of freezing and dehydration-induced hyperglycemic response in selectively inducing transcription factor MondoA in regulating glucose-induced transcription and activating an adaptive transcriptional response to induce stress response via inflammasome activation, mitochondrial dysfunction and mitochondrial epigenetics. The current findings establish MondoA in guiding an adaptive transcriptional response to activate genes regulating glucose homeostasis and circadian rhythm in a tissue-specific manner in the liver during the freeze/thaw cycle. Also, the role of TXNIP (downstream to MondoA) and its PTMs, in activating inflammasome via NLRP-3 in stress-specific way during freezing was shown. Moreover, the higher mitochondrial presence of TXNIP did not correlate to protein expression of its downstream targets in inducing mitochondrial dysfunction in any of the stresses, which were attributed to its low/weak binding to TRX-2. Investigating the role of mitochondrial methylation suggests its tissue-specific regulation in the liver and potential role in maintaining a tight regulation of mitochondrial transcriptional and gene expression response. Altogether, findings from this thesis demonstrate that a highly synchronized and intricate control via multiple levels of regulation is present in activating mechanisms that are involved in maintaining cellular milieu during stress in wood frogs.

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.