Molecular adaptations to diving-induced hypoxia. Diurnal variations in cellular and mitochondrial hypoxia tolerance in the hooded seal (Cystophora cristata)

Abstract

<p>Hypoxia may be defined as a state of O₂ deficiency in which cellular energetic homeostasis is challenged. Prolonged exposure to hypoxia can lead to a bioenergetic collapse as it induces a switch from mitochondrial oxidative metabolism to a mainly glycolytic one, which is not sustainable over time. Recent evidence shows that mitochondrial metabolic response to hypoxia also depends on the circadian clock. Although most species are not able to tolerate hypoxia over time, some live permanently or periodically under hypoxic conditions. Breath-hold diving mammals, like the deep-diving hooded seal (<i>Cystophora cristata</i>), display a set of systemic and cellular/molecular adaptations that convey them enhanced hypoxia tolerance. It has been hypothesised that the hooded seal brain is endowed with alternate metabolic pathways between neurons and astrocytes, in which neurons may periodically rely on glycolysis, with astrocytes metabolising the produced lactate, enabling their brain to tolerate repeated exposure to severely hypoxic conditions. The objectives of this thesis were to investigate 1) mitochondria-clock interactions in this hypoxia-tolerant species and 2) the role of mitochondrial oxidative metabolism in hooded seal neurons and astrocytes. <p>In paper I, we show that hooded seals display diurnal variation in diving behaviour, with longer dives during day than night. Further, by using primary skin fibroblasts we characterised the hooded seal circadian clock: two core clock genes displayed circadian expression which was maintained also under constant conditions. Finally, we show the presence of a clock-mediated change in mitochondrial metabolic efficiency, possibly in coordination with daily changes in diving efforts. In paper II, we found that adult hooded seal astrocytes have higher mitochondrial density than seal neurons, which is opposite to the situation in mice. In addition, via high resolution respirometry of primary astrocytic and neuronal cultures, we show that mitochondria of hooded seal astrocytes have a similar or possibly higher oxidative capacity than neurons. Finally, in paper III, we present three cell lines immortalised from hooded seal primary astrocytes that may serve as <i>in vitro</i> models in future studies into brain function and hypoxia tolerance in diving mammals. High resolution respirometry showed that these astrocytic cell lines have the capacity to oxidise both glucose and lactate for mitochondrial energy production. <p>Overall, the results presented in this thesis work highlight the importance of regulation of mitochondrial oxidative metabolism in hypoxia tolerance physiology and add new knowledge on some of the various hypoxia-induced adaptations in a remarkable species such as the hooded seal.