Genetic Analysis of Neuronal Hypoxia Resistance

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PROJECT SUMMARY Hypoxia (O2 deprivation) plays a central role in diverse human diseases, including ischemic stroke, myocardial infarction, pulmonary hypertension, Cerebral Palsy, COVID-19, and cancer. Metazoans respond to hypoxia by employing the conserved hypoxia response pathway. The pathway senses O2 through a prolyl hydroxylase (PHD) enzyme, which negatively regulates the transcription factor Hypoxia Inducible Factor α (HIFα). When hypoxia ensues, PHD enzymes lack O2 to hydroxylate HIFα, resulting in HIFα stabilization and the transcriptional regulation of multiple target genes that help the organism survive. While the HIFα pathway has been well studied in tissue culture, a full understanding of how it operates in specific tissues (particularly neurons) in vivo to provide tailored responses is needed. This proposal takes advantage of genetics and an intact, isogenic model organism (C. elegans) that can thrive under hypoxia, and whose environment and genetics can be controlled with fidelity and reproducibility. The overall premise of this proposal is that the hypoxia response pathway pathway protects against hypoxic damage by (1) removing mitochondria through mitophagy, which eliminates a source of oxidative stress, and by (2) mobilizing antioxidant metabolism, which detoxifies free radicals during hypoxia and reoxygenation. We hypothesize that HIF-1 promotes this metabolic reprograming by binding an enhancer sequence and activating the expression of the PEP carboxykinase pck-1, a key enzyme for moving metabolites through gluconeogenesis. Aim 1 tests this hypothesis by using CRISPR/Cas9 editing to remove this enhancer, then testing for the effects on HIF-1 binding, pck-1 and global gene expression, metabolism, oxidative stress resistance, neurodegeneration, and hypoxia survival. Preliminary cell biological approaches with a genetically encoded fluorescent reporter for mitophagy suggest that HIF-1 promotes mitophagy. We hypothesize that HIF-1 promotes mitophagy by binding enhancer sequences and activating the expression of the mitophagy receptors fndc-1 and dct-1. Aim 2 tests this hypothesis by using CRISPR/Cas9 editing to remove these enhancers, then testing for the effects on HIF- 1 binding, global gene expression, mitophagy and bulk autophagy, metabolism, oxidative stress resistance, neurodegeneration, and hypoxia survival. Recently, our media sterilizer and automated Petri dish pourer reached the end of its life. This large equipment failure is inhibiting our progress towards both aims, as this fundamental equipment is essential for every experiment we conduct. An administrative supplement is requested to replace this equipment and restore our progress towards understanding how the hypoxia response pathway operates. A better understanding of the pathway will provide therapeutic targets for diseases associated with hypoxia.