Cellular and transcriptional profiling of hypoxic cells in the brain
von Umer Javed Butt
Datum der mündl. Prüfung:2019-12-09
Erschienen:2021-07-08
Betreuer:Prof. Dr. Dr. Hannelore Ehrenreich
Gutachter:Prof. Dr. Dörthe Katschinski
Gutachter:Prof. Dr. Tiago Fleming Outeiro
Gutachter:Prof. Dr. Klaus-Armin Nave
Gutachter:Prof. Dr. Susann Boretius
Gutachter:Dr. Hauke Werner
Dateien
Name:PhD Thesis Umer Javed Butt.pdf
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Zusammenfassung
Englisch
Hypoxia is a critical regulator of the hypoxic response driving neurodevelop-ment, angiogenesis, hematopoiesis, and tissue regeneration. In the central nervous system, oxygen is a fundamental constituent of cellular homeostasis and is tightly controlled down to the single cells to maintain physiological func-tions. Under challenging conditions, slight alterations are detected and adaptive mechanisms are activated to adjust the homeostatic balance. In the brain, neu-ronal metabolic and network activity demands tightly controlled oxygen supply and any disruption results in detrimental outcomes. We therefore hypothesized that high neuronal activity would result in transient reduction of oxygen leading to physiological, ‘functional’ hypoxia followed by initiation of an adaptive gene response. A novel transgenic and robust approach, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recom-binase driven by CAG promoter was developed to report and label hypoxic cells upon breeding with tdTomato reporter mice. CAG-CreERT2-ODD::R26R-tdTomato transgenic mice allowed us to fate map hypoxic cells in the behaving brain under normoxia and hypoxia. We demonstrate an increase in the number of hypoxic neurons in the hippocampus upon complex motor cognitive chal-lenge. Light-sheet microscopy whole-brain spatial mapping revealed a global distribution of hypoxic cells in the brain. Hypoxic cells were particularly abundant in the hippocampus and increased under inspiratory hypoxia. Hypoxic cell type characterization by immuno-labelling revealed that the major population of tdTomato+ cell were neurons followed by small subset of astrocytes, endothelial cells, oligodendrocytes and pericytes. Unexpectedly, the microglial population was devoid of tdTomato+ fluorescence in the brain. However, single-cell tran-scriptome analysis indicated comparable expression of ODD/tdTomato mRNA across cell-types in the hippocampus. These findings point to variable hypoxia tolerance and response of different cells in the brain. Furthermore, association of high neuronal activity with cognitive challenge was confirmed in new neuron-specific CaMKIIα-Cre-ERT2-ODD::R26R-tdTomato transgenic mouse model. Hippocampal hypoxic neuron quantification results indicate an increase upon exposure to a challenging cognitive and motor task. Light-sheet microscopy analysis provided the spatial and temporal distribution of hypoxic neurons in the whole-brain. Taken together, these data provide evidence of the existence of physiological hypoxia in the brain under normoxia and its increase upon com-plex motor learning tasks.
Keywords: Hypoxia; Brain; cognition