The Conserved VPS-50 Protein Functions in Dense-Core Vesicle Maturation and Acidification and Controls Animal Behavior

The modification of behavior in response to experience is crucial for animals to adapt to environmental changes. Although factors such as neuropeptides and hormones are known to function in the switch between alternative behavioral states, the mechanisms by which these factors transduce, store, retrieve, and integrate environmental signals to regulate behavior are poorly understood. The rate of locomotion of the nematode Caenorhabditis elegans depends on both current and past food availability. Specifically, C. elegans slows its locomotion when it encounters food, and animals in a food-deprived state slow even more than animals in a well-fed state. The slowing responses of well-fed and food-deprived animals in the presence of food represent distinct behavioral states, as they are controlled by different sets of genes, neurotransmitters, and neurons. Here we describe an evolutionarily conserved C. elegans protein, VPS-50, that is required for animals to assume the well-fed behavioral state. Both VPS-50 and its murine homolog mVPS50 are expressed in neurons, are associated with synaptic and dense-core vesicles, and control vesicle acidification and hence synaptic function, likely through regulation of the assembly of the V-ATPase complex. We propose that dense-core vesicle acidification controlled by the evolutionarily conserved protein VPS-50/mVPS50 affects behavioral state by modulating neuropeptide levels and presynaptic neuronal function in both C. elegans and mammals.National Institutes of Health (U.S.) (Grant GM024663

Regulation of C. elegans behavior and physiology by the hypoxia-response pathway

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged student-submitted from PDF version of thesis. Vita.Includes bibliographical references.The transcriptional response controlling adaptation to internal and environmental hypoxia is broadly conserved in animals. The key mediator of this response is the transcription factor HIF (hypoxia-inducible factor), which is active only in hypoxia due to the function of its negative regulators, the prolyl hydroxylase EGLN and the E3 ubiquitin ligase complex recognition subunit pVHL. HIF drives transcription of hundreds of targets that promote hypoxia adaptation. Recent work has also described important and broad roles for HIF outside of the traditional hypoxia response, including functions in immunity, oxidative and other stress responses, and behavior; how HIF targets drive these aspects of animal physiology is poorly understood. In this dissertation, I describe genetic analyses of the nematode C. elegans that have provided insight into the function of HIF targets in regulating animal physiology and behavior. The EGLN family was defined by the C. elegans homolog, EGL-9. Prior to the identification of EGL-9 as a HIF hydroxylase, our laboratory discovered the egl-9 gene from studies of egg-laying behavior. egl-9 loss-of-function mutants, in which HIF is constitutively active, are egg-laying defective; the mechanism regulating egg laying downstream of HIF has been unknown. From a screen for suppressors of the egl-9(lf) egg-laying defect, we identified the gene cyp-36A1, which encodes a cytochrome P450 enzyme and is likely a direct transcriptional target of HIF. In addition to modulating egg-laying behavior downstream of HIF, CYP-36A1 controls expression of more than a third of HIF-upregulated genes and regulates multiple stress responses. A screen for suppressors of cyp-36A1(lf) identified the nuclear hormone receptor NHR-46. We propose that CYP-36A1 functions as a hormone biosynthetic enzyme for the ligand of this receptor, thus mediating gene expression changes that alter stress physiology and behavior. We also found site-of-action and genetic evidence for at least one additional pathway acting downstream of EGL-9 and HIF-1 to regulate egg-laying behavior. These studies have identified novel HIF effectors that broadly affect physiology and behavior in C. elegans, and reveal new avenues for future work on regulation of HIF-controlled biology.by Corinne L. Pender.Ph. D

Hypoxia-inducible factor cell non-autonomously regulates C. elegans stress responses and behavior via a nuclear receptor

The HIF (hypoxia-inducible factor) transcription factor is the master regulator of the metazoan response to chronic hypoxia. In addition to promoting adaptations to low oxygen, HIF drives cytoprotective mechanisms in response to stresses and modulates neural circuit function. How most HIF targets act in the control of the diverse aspects of HIF-regulated biology remains unknown. We discovered that a HIF target, the C. elegans gene cyp-36A1, is required for numerous HIF-dependent processes, including modulation of gene expression, stress resistance, and behavior. cyp-36A1encodes a cytochrome P450 enzyme that we show controls expression of more than a third of HIF-induced genes. CYP-36A1 acts cell non-autonomously by regulating the activity of the nuclear hormone receptor NHR-46, suggesting that CYP-36A1 functions as a biosynthetic enzyme for a hormone ligand of this receptor. We propose that regulation of HIF effectors through activation of cytochrome P450 enzyme/nuclear receptor signaling pathways could similarly occur in humans.National Institutes of Health (Grant GM024663)National Institutes of Health (Grant T32GM007287

Cytochrome P450 Drives a HIF-Regulated Behavioral Response to Reoxygenation by C. elegans

Oxygen deprivation followed by reoxygenation causes pathological responses in many disorders, including ischemic stroke, heart attacks, and reperfusion injury. Key aspects of ischemia-reperfusion can be modeled by a Caenorhabditis elegans behavior, the O2-ON response, which is suppressed by hypoxic preconditioning or inactivation of the O[subscript 2]-sensing HIF (hypoxia-inducible factor) hydroxylase EGL-9. From a genetic screen, we found that the cytochrome P450 oxygenase CYP-13A12 acts in response to the EGL-9–HIF-1 pathway to facilitate the O2-ON response. CYP-13A12 promotes oxidation of polyunsaturated fatty acids into eicosanoids, signaling molecules that can strongly affect inflammatory pain and ischemia-reperfusion injury responses in mammals. We propose that roles of the EGL-9–HIF-1 pathway and cytochrome P450 in controlling responses to reoxygenation after anoxia are evolutionarily conserved.National Institutes of Health (U.S.) (Grant GM24663)National Science Foundation (U.S.). Graduate Research Fellowship ProgramMassachusetts Institute of Technology. Undergraduate Research Opportunities ProgramHelen Hay Whitney Foundation (Postdoctoral Fellowship

The Hyaluronidase, TMEM2, Promotes ER Homeostasis and Longevity Independent of the UPRER

Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPRER, which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPRER pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPRER activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1

Lysosomal recycling of amino acids affects ER quality control

Recent work has highlighted the fact that lysosomes are a critical signaling hub of metabolic processes, providing fundamental building blocks crucial for anabolic functions. How lysosomal functions affect other cellular compartments is not fully understood. Here, we find that lysosomal recycling of the amino acids lysine and arginine is essential for proper ER quality control through the UPRER. Specifically, loss of the lysine and arginine amino acid transporter LAAT-1 results in increased sensitivity to proteotoxic stress in the ER and decreased animal physiology. We find that these LAAT-1-dependent effects are linked to glycine metabolism and transport and that the loss of function of the glycine transporter SKAT-1 also increases sensitivity to ER stress. Direct lysine and arginine supplementation, or glycine supplementation alone, can ameliorate increased ER stress sensitivity found in laat-1 mutants. These data implicate a crucial role in recycling lysine, arginine, and glycine in communication between the lysosome and ER