UC San Diego

Damage caused by lack of oxygen is a key factor in many serious disease states ranging from stroke and heart attack to asthma and high altitude mountain sickness. Determining the pathways that can ameliorate injuries caused by hypoxia is key to developing treatments and prevention of hypoxic damage. This dissertation examines the role of Notch signaling in potentiating Drosophila survival under chronic low oxygen conditions.

Pathway-level analysis of micro-array data of hypoxia-selected flies, the Notch signaling pathway was significantly up-regulated. Using the UAS-GAL4 system, the temporal and spatial specificity of Notch signaling was determined. The most robust results were found with glia-specific GAL4 drive, hence they were chosen for further characterization. Expression patterns of the GAL-4 glial drivers were confirmed by crossing them to a UAS-GFP transgenic fly line. Immunohistochemical analysis confirmed NICD (Notch Intracellular Domain) over-expression in glia cells. The developmental importance of Eaat1 glia was shown by crossing them to the UAS-reaper line; the results were embryonic lethal. Transcriptional up-regulation of Notch signaling was demonstrated by crossing flies stably over-expressing NICD with a reporter stock.

To address the question of what mechanism Notch utilizes to potentiate survival during hypoxia, several hypotheses were considered. First, Notch signaling may allow Eaat1-expressing glia to form a stem cell niche. This niche, in turn, allows the replacement of damaged/dead neurons injured during hypoxia.

Alternatively, other pathways may mediate the survival of these glial cells, which have been shown to be necessary to the survival of the whole organism. The metabolism of these glia could be altered via pyruvate dehydrogenase (PDH). Akt and NF-&kappaB are also known to interact with the Notch pathway. Perhaps canonical downstream target genes of Notch signaling, such as m-&alpha act as mechanism to allow survival of these crucial glia and thus the whole organism.

To study Notch interacting with other pathways, flies stably over-expressing NICD under the Eaat1 driver were crossed to UAS-RNAi flies targeting genes involved in pathways known to potentiate survival during hypoxia. Both Relish and m-&alpha act downstream of Notch signaling to mediate survival, demonstrating novel means of Notch up-regulation leading to hypoxic survival.