Expanding high-impact mentorship and research in the Bracht Laboratory

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Abstract of Supplement: The proposed supplemental funding will be used to both expand mentoring of a diverse student research group in the Bracht laboratory at American University, and expand the research proposed in the parent grant (1R15GM146207). The parent grant abstract is reproduced below; the supplemental research is within the scope of this parent grant. Abstract of parent award: A critical and well-studied cellular stress response pathway, the Unfolded Protein Response (UPR), protects organisms against several stressors including heat, hypoxia, starvation, and toxins. Helping to repair cellular damage, the UPR can also trigger apoptosis if the stress is ongoing, severe, and unrecoverable. Therefore, proper regulation of this pathway is essential, particularly since its malfunction contributes to human pathologies including autoimmune disorders, cancer, and neurodegenerative diseases. The Bracht lab recently published the genome of a nematode, Halicephalobus mephisto, isolated from the deep terrestrial subsurface of South Africa, over a kilometer underground. This organism has adapted to a stressful environment: hot, hypoxic, and rich in methane. Therefore the organism displays a naturally evolved resilience to stresses that would normally cause lethality; we also found that its UPR pathway is a site where adaptation has occurred. We have confirmed that RNA Interference (RNAi) by feeding can be used to modulate gene expression in this organism, setting the stage for a molecular investigation of stress resilience. New supplemental aims: Aim 1. Single-cell RNA-seq of ARMET RNAi in H. mephisto. In the parent grant, we described a gene, Arginine-Rich, Mutated in Early-stage Tumors / Mesencephalic Astrocyte derived Neurotrophic Factor (ARMET/MANF), that is highly expressed when the nematodes are grown at high temperatures. Surprisingly, our RNAseq data have uncovered an exciting and previously unknown link between the UPR pathway and insulin signaling. We propose to perform single-cell sequencing to identify which tissues are implicated in this process. Aim 2. Analysis of a more efficient mitochondrial proton pump in H. mephisto. In this aim we extend the research into H. mephisto adaptation into hypoxia, an environmental stress described in the parent grant. We found that the mitochondrial genome of H. mephisto exhibits an altered proton pump in the cytochrome c oxidase complex. We show that this pump is more effective than that encoded by C. elegans, a standard laboratory nematode, non-hypoxia adapted worm. We propose to perform extensive metabolic analysis of H. mephisto to confirm and extend these findings to whole- organism physiology.