Visible light influences a range of physiological processes, yet how animals respond to it independently of the visual system remains largely unknown. Here, we uncover a previously undescribed light-induced transcriptional pathway that modulates behavioral plasticity in Caenorhabditis elegans, a roundworm without eyes. We demonstrate that ambient visible light or controlled-intensity visible-spectrum LED activates an effector gene cyp-14A5 in non-neuronal tissues through the bZIP transcription factors ZIP-2 and CEBP-2. Light induction of cyp-14A5 is more prominent at shorter wavelengths but is independent of the known blue light receptors LITE-1 and GUR-3 in C. elegans. This bZIP-dependent genetic pathway in non-neuronal tissues enhances behavioral adaptability and olfactory memory, suggesting a body-brain communication axis. Furthermore, we use the light-responsive cyp-14A5 promoter to drive ectopic gene expression, causing synthetic light-induced sleep and rapid aging phenotypes in C. elegans. These findings advance our understanding of light-responsive mechanisms outside the visual system and offer a new genetic tool for visible light-inducible gene expression in non-neuronal tissues.

Although biosensors for specific cellular ions are widely available, real-time monitoring of overall ionic strength in living organisms remains challenging. Here, we present a genetically encoded nuclear translocation ionic sensor (GENTIS) that enables direct visualization of ionic stress in vivo. Using this sensor alongside longitudinal tracking via an automated microfluidic platform, we find that Caenorhabditis elegans larvae experience highly synchronized, rhythmic elevations in intestinal ionic strength during the molt, a stage during which developmentally timed sleep occurs. Cytosolic proton accumulation through inhibition of vacuolar-type adenosine triphosphatases (V-ATPases) triggers GENTIS nuclear translocation and evokes behavioral quiescence, characterized by reduced feeding, locomotion, and activation of sleep-active neurons. Apical membrane V-ATPases naturally undergo disassembly during molting and stress, conditions that cause proton accumulation and sleep. Notably, this proton-linked sleep is suppressed by proton buffering with ammonium. Together, these findings establish GENTIS as a powerful tool for tracking ionic strength dynamics in vivo and reveal that proton ionic rhythms contribute to the regulation of sleep.

Abstract Suspended animation, a state of profound metabolic, behavioral and developmental quiescence, is a remarkable yet poorly understood stress resilience strategy in animals. Here, we describe a previously uncharacterized form of suspended animation inducible by high-population density in isosmotic liquids in C. elegans throughout larval development and adulthood. Transcriptomic, metabolomic, and live-cell activity reporter imaging analyses reveal striking molecular and cellular landscape changes caused by such liquid-induced suspended animation (LISA), including remodeling of gene expression programs, energy metabolites, lysosomal and mitochondrial morphology. Genetic screens identify mutants with altered stress responses and survival against LISA. While key endo-lysosomal regulators promote survival during LISA, organelle remodeling and a neuronal axis via downstream neuropeptide and cAMP/PKA signaling orchestrate behavioral awakening from LISA. Our findings define a facile paradigm for reversible SA, providing a powerful model system to uncover key molecular and cellular mechanisms governing an extreme case of reversible life arrest and dormancy.

Abstract The inability to selectively trigger therapeutic hypothermia independent of environmental cooling has hindered causal analysis of its broader physiological benefits. Here, we employed P57, a natural product that pharmacologically induces therapeutic hypothermia circumventing external cold stress, to identify the neuronal substrates underlying hypothermia-induced antitumor effects. Integrating functional ultrasound imaging, activity-dependent neuronal labeling, and single-nucleus RNA sequencing, we identify a previously unrecognized population of Foxp2 ⁺ neurons in the medial preoptic area (MPA) that are selectively activated by P57. Inhibition of MPA Foxp2 + neurons abolishes P57-induced hypothermia, whereas the paraventricular hypothalamus (PVH) serves as a critical downstream node. Importantly, targeted activation of MPA Foxp2 ⁺ neurons is sufficient to induce sustained hypothermia, suppress systemic metabolism, and inhibit tumor growth, providing causal evidence that neuronally driven reductions in core body temperature can exert antitumor effects. Together, these findings establish MPA Foxp2 ⁺ neurons as a controllable node for therapeutic hypothermia induction and demonstrate that neuromodulation of defined neuronal populations can achieve therapeutic benefit by directly controlling physiological states.

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species. The ER-transmembrane protein that supports ER proteostasis and guards the cytosol for antioxidant defense remains unidentified. Here, we combine AlphaFold2 and functional screens in C. elegans to discover a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting ERGU-1 upregulates H2O2 and NRF2/SKN-1-dependent gene expression. ERGU-1 deficiency also impairs organismal reproduction and behavioral responses to H2O2. Both C. elegans ERGU-1 and human homolog TMEM161B localize to ER membranes, forming reticular networks. Human and Drosophila homologs of ERGU-1 rescue C. elegans mutant phenotypes, demonstrating ancient and conserved functions. In addition, purified ERGU-1 and TMEM161B exhibit redox-modulated oligomeric states. Together, our results reveal an ER-membrane-specific machinery, suggesting a conserved mechanism for maintaining ER redox homeostasis and proteostasis in animal cells.

BLTP1 (Bridge-Like Lipid Transfer Protein Family Member 1), previously known as Tweek, Kiaa1109, FSA, or 4932438A13Rik, is a nonvesicular lipid transport protein linked to Alkuraya-Kučinskas syndrome (AKS), an autosomal recessive disorder with severe brain malformations and arthrogryposis in humans. Although BLTP1 is known to be involved in the transfer of phospholipids between organellar membranes at membrane contact sites, its specific role during mammalian development remains poorly defined. Here we characterized the development of the neuromuscular junction (NMJ) in Bltp1 knock-out (Bltp1-/- ) mice of either sex. These mutant mice die perinatally, exhibiting reduced growth of intramuscular motor nerves and a reduction in the size of the NMJ, compared with littermate controls. Electrophysiological analysis of the diaphragm muscle in Bltp1 -/- embryos reveals defects in synaptic transmission at the NMJ. Notably, the frequency of spontaneous neurotransmitter release is markedly increased, whereas evoked neurotransmitter release and quantal content are reduced. In addition, neuromuscular synapses in Bltp1 -/- mice fail to respond to a repetitive stimulation. Remarkably, the impairment of intramuscular nerve growth in Bltp1-/- embryos is restored by supplementing pregnant dams with lecithin, a mixture of unsaturated phospholipids naturally present in foods, although lecithin supplement does not improve the overall survival of these mutant mice. Together, these results demonstrate that BLTP1 plays important roles during development for proper intramuscular nerve growth and neuromuscular synaptic function and suggest that AKS patients may benefit from phospholipid supplementation such as lecithin.

When embryos experience different environments than their parents, plasticity can enable the development of alternate phenotypes that confer higher fitness in the new conditions. Temperature-induced plasticity could be especially critical for species that inhabit areas with considerable thermal variation. We studied transcriptional variation in embryos of mangrove rivulus (Kryptolebias marmoratus)-a self-fertilizing hermaphroditic, eurythermal fish that resides in notoriously spatiotemporally variable mangrove forests-exposed to different thermal regimes during development. To study transcriptional plasticity, we first improved the genome assembly to chromosome length scaffolds (N50 of 28.17 Megabases). Whole transcriptome sequencing revealed that both temperature and developmental timing modulated embryonic gene expression. We found few differences in gene expression between embryos incubated in cold and warm conditions and assessed before the temperature-sensitive period of development, indicating high resistance to stochastic changes in gene expression early in development. Replicate embryos exposed to cold temperatures and sampled after the temperature-sensitive period showed less variation in gene expression than those sampled before, suggesting canalization of the plastic response. DNA replication/repair, organelle, and gas transport pathways were upregulated while nervous system development, cell signaling, and cell adhesion were downregulated in cold-exposed compared to warm-exposed embryos sampled after the temperature-sensitive period. These plastic shifts in gene expression could have major implications for reorganizing the phenotype (e.g., apoptosis, mitosis) in response to environmental changes occurring within a generation.

The ε4 variant of human apolipoprotein E ( APOE4 ) is a key genetic risk factor for neurodegeneration in Alzheimer’s disease and elevated all-cause mortality in humans. Understanding the factors and mechanisms that can mitigate the harmful effects of APOE4 has significant implications. In this study, we find that inactivating the VHL-1 (Von Hippel–Lindau) protein can suppress mortality, neural and behavioral pathologies caused by transgenic human APOE4 in Caenorhabditis elegans . The protective effects of VHL-1 deletion are recapitulated by stabilized HIF-1 (hypoxia-inducible factor), a transcription factor degraded by VHL-1. HIF-1 activates a genetic program that safeguards against mitochondrial dysfunction, oxidative stress, proteostasis imbalance, and endolysosomal rupture—critical cellular events linked to neural pathologies and mortality. Furthermore, genetic inhibition of Vhl reduces cerebral vascular injury and synaptic lesions in APOE4 mice, suggesting an evolutionarily conserved mechanism. Thus, we identify the VHL–HIF axis as a potent modulator of APOE4 -induced neural pathologies and propose that targeting this pathway in nonproliferative tissues may curb cellular damage, protect against neurodegeneration, and reduce tissue injuries and mortality.

PALS1-associated tight junction (PATJ) protein is linked to metabolic disease and stroke in human genetic studies. Despite the recognized role of PATJ in cell polarization, its specific functions in metabolic disease and ischemic stroke recovery remain largely unexplored. We explored the functions of PATJ in an in vitro model and in vivo in C. elegans and mice. Using a mouse model of stroke, we found post-ischemic stroke duration-dependent increase of PATJ abundance in endothelial cells. PATJ knock-out (KO) HEK293 cells generated by CRISPR-Cas9 suggest roles for PATJ in cell proliferation, migration, mitochondrial stress response, and interactions with the Yes-associated protein (YAP)-1 signaling pathway. Notably, PATJ deletion altered YAP1 nuclear translocation. PATJ KO cells demonstrated transcriptional reprogramming based on RNA sequencing analysis, and identified dysregulation in genes central to vascular development, stress response, and metabolism, including RUNX1, HEY1, NUPR1, and HK2. Furthermore, we found that mpz-1, the homolog of PATJ, was significantly upregulated under hypoxic conditions in C. elegans. Knockdown of mpz-1 resulted in abnormal neuronal morphology and increased mortality, both of which were exacerbated by hypoxia exposure, indicating a critical protective role of PATJ/MPZ-1 in maintaining neuronal integrity and survival, particularly during oxygen deprivation stress relevant to ischemic stroke. These insights offer a new understanding of PATJ's regulatory functions within cellular and vascular physiology and help lay the groundwork for therapeutic strategies targeting PATJ-mediated pathways for stroke rehabilitation and neurovascular repair.

Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens in C. elegans to discover a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting C. elegans ERGU-1 causes excessive H2O2 and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behavioral responses to H2O2. Both C. elegans and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. Human and Drosophila homologs of ERGU-1 can rescue C. elegans mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. In addition, purified ERGU-1 and human homolog TMEM161B exhibit redox-modulated oligomeric states. Together, our results reveal an ER-membrane-specific protein machinery for peroxide detoxification and suggest a previously unknown and conserved mechanisms for antioxidant defense in animal cells.

The Arctic ground squirrel (AGS, Urocitellus parryii), an extreme hibernator, exhibits remarkable resilience to stressors like hypoxia and hypothermia, making it an ideal model for studying cellular metabolic adaptation. The underlying mechanisms of AGS resilience are largely unknown. Here, we use lipidomic and metabolomic profiling to discover specific downregulation of triglyceride lipids and upregulation of the lipid biosynthetic precursor malonic acid in AGS neural stem cells (NSC) versus murine NSCs. Inhibiting lipid biosynthesis recapitulates hypoxic resilience of squirrel NSCs. Extending this model, we find that acute exposure to hypoxia downregulates key lipid biosynthetic enzymes in C. elegans, while inhibiting lipid biosynthesis reduces mitochondrial fission and facilitates hypoxic survival. Moreover, inhibiting lipid biosynthesis protects against APOE4-induced pathologies and aging trajectories in C. elegans. These findings suggest triglyceride downregulation as a conserved metabolic resilience mechanism, offering insights into protective strategies for neural tissues under hypoxic or ischemic conditions, APOE4-induced pathologies and aging.

Mortality rate increases with age and can accelerate upon extrinsic or intrinsic damage to individuals. Identifying factors and mechanisms that curb population mortality rate has wide-ranging implications. Here, we show that targeting the VHL-1 (Von Hippel-Lindau) protein suppresses C. elegans mortality caused by distinct factors, including elevated reactive oxygen species, temperature, and APOE4, the genetic variant that confers high risks of neurodegeneration in Alzheimer's diseases and all-cause mortality in humans. These mortality factors are of different physical-chemical nature, yet result in similar cellular dysfunction and damage that are suppressed by deleting VHL-1. Stabilized HIF-1 (hypoxia inducible factor), a transcription factor normally targeted for degradation by VHL-1, recapitulates the protective effects of deleting VHL-1. HIF-1 orchestrates a genetic program that defends against mitochondrial abnormalities, excess oxidative stress, cellular proteostasis dysregulation, and endo-lysosomal rupture, key events that lead to mortality. Genetic Vhl inhibition also alleviates cerebral vascular injury and synaptic lesions in APOE4 mice, supporting an evolutionarily conserved mechanism. Collectively, we identify the VHL-HIF axis as a potent modifier of APOE4 and mortality and propose that targeting VHL-HIF in non-proliferative animal tissues may suppress tissue injuries and mortality by broadly curbing cellular damage.

Insulin-mTOR signaling drives anabolic growth during organismal development, while its late-life dysregulation may detrimentally contribute to aging and limit lifespans. Age-related regulatory mechanisms and functional consequences of insulin-mTOR remain incompletely understood. Here we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin INS-7 is drastically over-produced in early life and shortens lifespan in lpd-3 mutants, a C. elegans model of human Alkuraya-Kučinskas syndrome. LPD-3 forms a bridge-like tunnel megaprotein to facilitate phospholipid trafficking to plasma membranes. Lipidomic profiling reveals increased abundance of hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1 (Homolog of Yeast Longevity). Reducing HYL-1 activity decreases INS-7 levels and rescues the lifespan of lpd-3 mutants through insulin receptor/DAF-2 and mTOR/LET-363. LPD3 antagonizes SINH-1, a key mTORC2 component, and decreases expression with age in wild type animals. We propose that LPD-3 acts as a megaprotein brake for aging and its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. Increased TSP-1 abundance persists even after transient early-life heat stress. Such regulation requires CBP-1, a histone acetyltransferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity.

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml‐3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML‐3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L‐rhamnose, a cell‐wall polysaccharide component. C. elegans rml‐3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress‐resistant dauer stage. rml‐3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L‐rhamnose. We propose that interdomain HGT of an ancient bacterial rml‐3 homolog has enabled L‐rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene. Interdomain horizontal gene transfer (iHGT) between species can sometimes occur in evolution. Here, phylogenetic and experimental analyses reveal a C. elegans gene originating from bacteria that is crucial for exoskeleton resilience against environmental toxins. Phylogenetic and experimental analyses suggest that a key gene conferring nematode exoskeleton resilience against environmental toxins has been acquired from the bacterial kingdom.

Insulin-mTOR signaling drives anabolic growth in organismal development, while its late-life antagonistic pleiotropy affects aging and compromises lifespan across animal phylogeny. Here we identify LPD-3 as a megaprotein that orchestrates the tempo of insulin-mTOR signaling during C. elegans aging. We find that an agonist insulin INS-7 is drastically over-produced and shortens lifespan in lpd-3 mutants, a C. elegans model of human Alkuraya-Kučinskas syndrome. LPD-3 forms a bridge-like tunnel megaprotein to facilitate phospholipid trafficking to plasma membrane. Lipidomic profiling reveals increased abundance of hexaceramide species in lpd-3 mutants, accompanied by up-regulation of hexaceramide biosynthetic enzymes, including HYL-1 (Homolog of Yeast Longevity). Reducing HYL-1 activity decreases INS-7 levels and rescues the shortened lifespan of lpd-3 mutants through insulin receptor/DAF-2 and mTOR/LET-363. LPD-3 antagonizes SINH-1, a key mTORC2 component, and reduces protein abundance with age in wild type animals. We propose that LPD-3 acts as a megaprotein brake for aging and its age-dependent decline restricts lifespan through the sphingolipid-hexaceramide and insulin-mTOR pathways.

Background: Parkin, which when mutated leads to early-onset Parkinson’s disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in Caenorhabditis elegans ( C. elegans) to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. Methods: We generated a transgene encoding mCherry-tagged C. elegans Parkin – Parkinson’s Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. Results: We show that the mCherry::pdr-1 transgene rescues the mitochondrial phenotype of pdr-1 mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator ant-1.1/hANT, or hybrid ubiquitin genes ubq-2/h UBA52 and ubl-1/h RPS27A encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. Conclusions: These data show that the abundant reporter of C. elegans Parkin affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases.