M. Sc. Thesis Abstracts

Antioxidant defense in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

Abstract:

Dramatic changes in blood flow and oxygen consumption during torpor-arousal cycles could increase the risk of oxidative stress to tissues of mammalian hibernators. Changes to antioxidant defense systems during hibernation were assessed in thirteen-lined ground squirrels, Spermophilus tridecemlineatus, focusing on heme oxygenase 1 (HO1), superoxide dismutase (SOD) and the thioredoxin (Trx) system. Compared with euthermic control squirrels, inducible HO1 was elevated at both the transcriptional and translational level in multiple tissues of hibernating squirrels. The Nrf2/ARE signal transduction pathway commonly regulates HO1 and protein levels of both the transcription factor Nrf2 and its cofactor MafG were elevated in concert with HO1 implicating this pathway in HO1 expression in hibernation. Both cytosolic Cu/Zn SOD and mitochondrial Mn SOD increased significantly in squirrel muscle during hibernation, probably contributing to antioxidant defense during arousal from torpor when oxygen consumption by muscle increases rapidly to support shivering thermogenesis. Proteins of the thioredoxin system (Trx1, Trx2, TrxR2) also rose in multiple tissues during hibernation, perhaps to remedy a glutathione redox imbalance during hibernation. Amino acid sequencing of HO1 and SOD showed strong conservation of sequence compared with nonhibernating mammals but selected substitutions were found that could aid enzyme function at low body temperatures. The data support the idea that hibernators experience oxidative stress over torpor-arousal cycles and in response activate antioxidant defenses to minimize damage. A better understanding of antioxidant defense systems in hibernation may guide the use of antioxidants in medical treatments for ischemia/reperfusion damage and technology for cold preservation of organs.

Regulation of protein and phospholipid metabolism in the 13-lined ground squirrel, Spermophilus tridecemlineatus and the wood frog, Rana sylvatica.

Abstract:

Mammalian hibernation and ectotherm freeze tolerance are unique forms hypometabolism employed for animal winter survival. The present studies explore selected molecular mechanisms associated with hypometabolism in two model animals, the thirteen-lined ground squirrel, Spermophilus tridecemlineatus, and the wood frog, Rana sylvatica. Conservation of fuel reserves and of macromolecular integrity during hypometabolism depend on the coordinated suppression of pathways of macromolecular synthesis and degradation. Investigations of the regulation of protein degradation, protein synthesis, andphospholipid degradation pathways were undertaken comparing control and stressed conditions in skeletal muscle and liver from both animals. Several eukaryotic initiation factors were analyzed using western blotting and revealed animal specific changes in the regulation of protein synthesis. The activity of the proteasome, responsible for protein degradation, was assayed fluorometrically and was correlated with measurements of oxidatively damaged proteins (protein carbonyls) and immunoblot analysis of levels of ubiquitin-tagged damaged proteins. Proteasome activity was strongly suppressed in frozen wood frogs via changes in both the total amount and the phosphorylation state of the proteasome. However, proteasome activity remained constant in the hibernator model. Ubiquitination increased in the hibernator model, but decreased in the frog system highlighting a difference in the ubiquitin system upstream of the proteasome. Assays of the activity of cytoplasmic phospholipase A₂ (cPLA₂) provided estimates of the role ofarachidonic acid signaling in hibernation and freezing survival. Phospholipase activity decreased significantly during hibernation, but was enhanced during freezing. Control of the proteasome and cPLA₂ have implications for the repair mechanisms that deal with oxidative damage to cellular macromolecules.

Protein chaperones and winter cold hardiness in insects: heat shock proteins and glucose regulated proteins in freeze-tolerant and freeze-avoiding species.

Abstract:

Two species of insects with different overwintering strategies were chosen as model organisms to assess the role of chaperone proteins in insect cold hardiness. Epiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly, were used to compare and contrast the two overwintering strategies; both species overwinter in the diapause stage as last instar larvae inside galls on the stems of goldenrod plants. The research reported in this thesis analyzed changes in molecular chaperone levels over the winter season in insects sampled from outdoors as well as in larvae given laboratory exposures to subzero temperatures and hypoxia. Multiple proteins in the heat shock (Hsp10, Hsp40, Hsp60, Hsp70, Hsp110) and glucose-regulated (Grp75, Grp78, Grp94, Grp170) families of chaperones were assessed as well as other chaperones (TCP-1, αA- and αB-crystallins). The heat shock transcription factor (HSF-1) was also analyzed. Despite an overall suppression of transcription and translation during winter diapause, selected chaperone proteins were differentially expressed in the larvae in response to low temperatures, freeze/thaw, or hypoxia exposures. Most of the chaperones were up-regulated directly or indirectly by subzero temperature and/or anoxia exposures to enhance their actions in the stabilization, repair or elimination of misfolded or denatured proteins. These chaperones function incytoplasmic, endoplasmic reticulum and mitochondrial compartments, indicating that multiple endogenous pathways are engaged in maintaining and/or restoring cellular homeostasis in the larvae in response to these insults. Elevated HSF-1 levels indicated that Hsp enhancement in the larvae was due to up-regulation of the genes involved.

Profile of FoxO proteins and MnSOD in two cold-hardy insect species exposed to low temperature.

Abstract:

Expression of FoxO genes and proteins in two insects with different overwintering strategies were studied with Western blotting and RT-PCR. Epiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly were used as model organisms to assess the role of FoxO transcription factors and one of their downstream proteins, manganese superoxide dismutase (MnSOD) in insect cold hardiness. FoxO3a gene and protein levels, as well as MnSOD protein, were analyzed inEpiblema scudderiana over time courses of exposure to 5°C or ‑15°C. Significant changes in total Foxo3a protein, the amount of phosphorylated inactive FoxO3a, FoxO3a mRNA levels, and MnSOD content were identified under different conditions. For example, reduced phospho-FoxO3a (Ser 253) content in larvae exposed to -15°C suggested enhanced transcriptional activation of genes under FoxO3a control at subzero temperatures whereas MnSOD content increased in larvae exposed to 5°C. Sequencing of FoxO3a mRNA from Epiblema scudderiana showed that the transcription factor was highly conserved as compared with mammals. FoxO3a, FoxO1 and MnSOD protein levels were also measured in the freeze tolerant insect Eurosta solidaginis subjected to 5 and ‑15°C exposures as well as a time course of thawing after freezing at -15°C for 1 week. The results implied that FoxO3a and FoxO1 functioned oppositely during cold, freezing and thawing of the larvae. Expression of MnSOD increased with 5°C exposure, remained elevated when larvae were frozen at -15°C, and decreased after thawing. Differential expression of FoxOs and MnSOD in both species suggest that MnSOD may not be transcriptionallycontrolled by FoxO3a or FoxO1 in cold hardy insects.

Involvement of FOXO transcription factors and glycogen synthase kinase 3 in the freeze tolerance capability of the wood frog, Rana sylvatica.  

Abstract: 

Animals cope with the subzero temperatures of winter in different ways. The wood frog, Rana sylvatica, endures whole body freezing and is able to survive weeks completely frozen. Organisms that endure extreme environmental stress on a periodic or seasonal basis have developed ways to strongly suppress their metabolic rate and enter a hypometabolic state to survive. Forkhead box ‘other’ (FOXO) transcription factors have important roles in various cellular processes such as metabolism, cellular proliferation, stress tolerance and lifespan. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frog organs. Active FOXO1 increased in brain during freezing and thawing, possibly due to a need for gluconeogenesis during this stress. The levels of active FOXO3 increased in frog brain, kidney and liver during freezing and thawing and also during anoxia and aerobic recovery after anoxia, which could be due to the need to maintain or enhance antioxidant defenses under these stresses. Glycogen synthase kinase-3 (GSK3) is a protein kinase known to inhibit glycogen synthesis, cell growth and differentiation and protein translation. The amount of active GSK3 increased in the frozen state in brain, heart, kidney, liver and muscle of wood frogs. Furthermore, kinetic analysis of GSK3 showed that the skeletal muscle of frozen frogs appears to have a higher affinity for its substrate when compared to control GSK3. Allosteric effectors of GSK3 were also identified: glucose-6-phosphate activated the enzyme whereas AMP inhibited. The data expand our understanding of metabolic regulation during natural freeze tolerance.

Transcription factor regulation in mammalian hibernation: the thirteen-lined ground squirrel as a model.

Abstract:

The thirteen-lined ground squirrel (Spermophilus tridecemlineatus) is one of a number of mammalian species that hibernate over the winter months. Hibernation is characterized by long periods of deep torpor that are interspersed with periodic arousals. This thesis investigated three transcription factor pathways and their regulation and roles in hibernation using tools including immunoblotting of protein and phospho-protein levels, analysis of nuclear versus cytoplasmic distribution, binding to DNA, PCR to quantify mRNA transcripts, and gene sequencing. The carbohydrate response element-binding protein (ChREBP) regulates genes that are responsible for converting excess dietary carbohydrate (glucose) into triglycerides. During torpor ChREBP protein was decreased in kidney and liver and transcripts of two downstream genes (Fasn, PK-L) were differentially expressed; this was consistent with carbohydrate sparing during torpor. Growth and differentiation processes are also minimized during hibernation and ETS transcription factors are important regulators of these. Although levels of ETS increased in most organs of hibernators, ETS binding activity to DNA was greatly reduced indicating a growth suppressive function during torpor. Upstream effectors of ETS (epidermal growth factor receptor, protein kinases C and D) were also evaluated. To assess transcriptional state during hibernation, the Rb-E2F pathway was investigated. Retinoblastoma protein (Rb) associates with E2F1 and then recruits co-factors to the promoter region of target genes to suppress transcription. Rb and E2F1 proteins were elevated during hibernation and the co-factors HDAC and SUV39H1 showed parallel increases in the organs tested; oppositely, transcription of downstream genes (cMyc, Bcl2) was suppressed. These data confirm a reduced transcriptional state during torpor.

Regulation of glucose-6-phosphate dehydrogenase and hexokinase in anoxia-tolerant mollusks: role of reversible phosphorylation

Abstract:

Metabolic rate depression is key to animal survival without oxygen and requires coordinated suppression of ATP-generating and ATP-consuming cellular functions by stable regulatory mechanisms. This thesis examined the possible role of reversible protein phosphorylation in metabolic suppression in organs of the anoxia-tolerant intertidal marine mollusc, Littorina littorea. Studies focused on glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and hexokinase (HK), an important enzyme at the forefront of carbohydrate metabolism. The data show that hepatopancreas G6PDH is regulated by phosphorylation and entry into anoxia leads to a more dephosphorylated form, whose properties suggest a more active enzyme. This would favour enhanced carbon flow through the PPP to sustain NADPH production for antioxidant defense. Furthermore, the study provides one of the first demonstrations of coordinated regulation of the PPP between active and hypometabolic states and implicates specific protein kinases and phosphatases in G6PDH regulation. Studies of HK showed that it is also regulated by reversible protein phosphorylation, in a tissue-specific manner. In the hepatopancreas, it appears that HK affinity for one of its substrates is enhanced during anoxia whereas in foot muscle, HK activity is suppressed. Differences between control and anoxic HK were also observed in their susceptibility to urea denaturation, response to specific protein kinase and protein phosphatase incubations as well as their elution profiles from an ion-exchange column. Overall, these studies confirm an integral role of reversible protein phosphorylation in the suppression and reorganization of L.littorea metabolism for anoxia survival.

Regulation of NF-κB and p53 in the liver and skeletal muscle of the freeze tolerant wood frog, Rana sylvatica

Abstract:   

The wood frog, Rana sylvatica, is the primary model animal used for studying vertebrate freeze tolerance.  During Canadian winters, wood frogs can endure the freezing of about 70% of their total body water and then thaw and resume life in the spring.  Frogs have multiple ways to protect themselves against potential freezing injuries including adaptive changes to intermediary metabolism and gene expression.  One way that wood frogs deal with freezing stress is via upregulation of several freeze-responsive genes.  Previous studies provided excellent presumptive evidence for the involvement of the NF-κB and p53 transcription factors in freeze tolerance.  The studies in this thesis used Western blotting to quantify levels of NF-κB subunits p50 and p65, its inhibitor, p-IκB, and downstream targets (ferritin heavy chain, manganese superoxide dismutase) as well as protein levels of p53, post-translationally modified p53, and some p53 downstream genes in the muscle and liver of control versus frozen wood frogs. Nuclear distributions of NF-κB and p53 were also assessed. RT-PCR was used to quantify transcript levels of select targets of NF-κB and p53.  Significant increases in the expression levels of NF-κBand its downstream targets as well as in levels of p53, post-translationally modified p53, and its downstream targets were observed during freezing.  These findings suggest the activation of NF-κB antioxidant defenses in the wood frog during freezing in anticipation of reperfusion during thawing and the activation of p53 in the wood frog which would lead to cell cycle arrest in the frozen state.

Regulation of the Tb-E2F pathway in the freeze tolerant wood frog, Rana sylvatica

Abstract:   

The North American wood frog, Rana sylvatica, is a primary model animal used in the study of freeze tolerance in vertebrates. During Canadian winters, wood frogs endure the freezing of about 65-70% of their total body water. Anoxia, ischemia, oxidative stress and many other consequences are a result of freeze/thaw cycles. A variety of adaptations are known that protect the frogs against potential freezing injuries as well as regulate their intermediary metabolism and gene expression to support survival. Selected transcription factors have critical roles to play in freezing survival by regulating the expression of genes that control the adaptations needed to handle freezing stress. In my present study, the retinoblastoma (Rb) protein coupled with the E2F transcription factor family were demonstrated to have roles in controlling the cell cycle in wood frog liver and skeletal muscle during freezing and associated stresses (anoxia, dehydration). Western blotting was used to quantify total Rb, phosphorylation or acetylation at different sites on Rb, E2F members and selected downstream targets under E2F control. Other central regulators of the cell cycle were also quantified including Cyclins, Cyclin dependent kinases (Cdks), and checkpoint proteins. Nuclear distributions of Rb-E2F and Cdk:Cyclins were also assessed. RT-PCR was used to quantify mRNA transcript levels of Cyclin D1 which decreased significant during freezing as well as c-Myc, a downstream target of E2F. The data indicate that the cell cycle is under regulation during freezing through E2F upregulation and Rb phosphorylation via Cdk:Cyclin activity.

Phosphorylation of glycolytic enzymes in hibernation.

Abstract:

Glycerol-3-phosphate dehydrogenase (G3PDH) and lactate dehydrogenase (LDH) were examined for differential phosphorylation, accompanying kinetics and stability inSpermophilus richardsonii liver and skeletal muscle. Hibernator G3PDH had a higher phosphate content and differential kinetics in both tissues which could be manipulated by kinase and phosphatase incubations in the G3P utilizing direction. Arrhenius plots and activation energies (Ea) showed that the hibernator form had a lower Ea in both tissues, especially in the liver.Euthermic LDH had a higher phosphate content and differential kinetics which could be manipulated by kinase and phosphatase incubations in both directions. Euthermic forms had lower Ea values in both tissues. G3PDH and LDH had differential urea I50 values with the hibernator more susceptible to urea denaturation in both tissues. Urea I50 values could be manipulated in LDH by kinase and phosphatase incubations in the liver but not in muscle.