Hanane

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.