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The dopamine transporter (DAT) is a transmembrane protein that regulates dopamine (DA) neurotransmission by binding to and moving DA from the synaptic cleft back into the neurons. Besides moving DA and other endogenous monoamines, DAT is also a neuronal carrier for exogenous compounds such as the psychostimulant amphetamine (Amph), and several studies have shown that Amph-induced behaviors require a functional DAT. Here, we demonstrate that exposure to Amph during early development causes behavioral, functional, and epigenetic modifications at the Caenorhabditis elegans DAT gene homolog, dat-1, in C. elegans offspring. Specifically, we show that, while embryos exposed to Amph generate adults that produce offspring with no obvious behavioral alterations, both adults and offspring exhibit an increased behavioral response when challenged with Amph. Our functional studies suggest that a decrease in DAT-1 expression underlies the increased behavioral response to Amph seen in offspring. Moreover, our epigenetic data suggest that histone methylation is a mechanism utilized by Amph to maintain changes in DAT-1 expression in offspring. Taken together, our data reveal that Amph, by altering the epigenetic landscape of DAT, propagates long-lasting functional and behavioral changes in offspring., (© 2024 International Society for Neurochemistry.)

(1) Background: Vitamin B 12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B 12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B 12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B 12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7 , which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid β, we found that vitamin B 12 deficiency did not affect the gene and protein expressions of amyloid β but increased the formation of dityrosine crosslinking in the amyloid β protein. (3) Conclusions: Vitamin B 12 -deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B 12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid β formation, which might promote its stabilization and toxic oligomerization.

In addition to performing digestion and nutrient absorption, the intestine serves as one of the first barriers to the external environment, crucial for protecting the host from environmental toxins, pathogenic invaders, and other stress inducers. The gene regulatory network (GRN) governing embryonic development of the endoderm and subsequent differentiation and maintenance of the intestine has been well-documented in C. elegans. A key regulatory input that initiates activation of the embryonic GRN for endoderm and mesoderm in this animal is the maternally provided SKN-1 transcription factor, an ortholog of the vertebrate Nrf1 and 2, which, like C. elegans SKN-1, perform conserved regulatory roles in mediating a variety of stress responses across metazoan phylogeny. Other key regulatory factors in early gut development also participate in stress response as well as in innate immunity and aging and longevity. In this review, we discuss the intersection between genetic nodes that mediate endoderm/intestine differentiation and regulation of stress and homeostasis. We also consider how direct signaling from the intestine to the germline, in some cases involving SKN-1, facilitates heritable epigenetic changes, allowing transmission of adaptive stress responses across multiple generations. These connections between regulation of endoderm/intestine development and stress response mechanisms suggest that varying selective pressure exerted on the stress response pathways may influence the architecture of the endoderm GRN, thereby leading to genetic and epigenetic variation in early embryonic GRN regulatory events., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1 , the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

Membrane remodeling by inflammatory mediators influences the function of sensory ion channels. The capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channel contributes to neurogenic inflammation and pain hypersensitivity, in part because of its potentiation downstream of phospholipase C-coupled receptors that regulate phosphoinositide lipid content. Here, we determined the effect of phosphoinositide lipids on TRPV1 function by combining genetic dissection, diet supplementation, and behavioral, biochemical, and functional analyses in Caenorhabditis elegans As capsaicin elicits heat and pain sensations in mammals, transgenic TRPV1 worms exhibit an aversive response to capsaicin. TRPV1 worms with low levels of phosphoinositide lipids display an enhanced response to capsaicin, whereas phosphoinositide lipid supplementation reduces TRPV1-mediated responses. A worm carrying a TRPV1 construct lacking the distal C-terminal domain features an enhanced response to capsaicin, independent of the phosphoinositide lipid content. Our results demonstrate that TRPV1 activity is enhanced when the phosphoinositide lipid content is reduced, and the C-terminal domain is key to determining agonist response in vivo ., Competing Interests: The authors declare no competing financial interests., (Copyright © 2021 Caires et al.)

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPR mt ) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPR mt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPR mt activation.

Nanoplastic exposure could cause toxicity to Caenorhabditis elegans at various aspects. Nevertheless, the effects of chronic exposure to nanoplastics remain largely unclear in nematodes. In this study, we employed C. elegans as an animal model to determine the effects of nanopolystyrene (30 nm) exposure from adult day-1 for 8-day. After the exposure, only 1000 μg/L nanopolystyrene reduced the lifespan. In contrast, nanopolystyrene ≥1 μg/L decreased locomotion behavior and activated oxidative stress. Meanwhile, in 10 μg/L nanopolystyrene exposed nematodes, both expression of SOD-3, a Mn-SOD, and autophagy induction as indicated by LGG-1:GFP expression were significantly increased. RNAi knockdown of daf-2 encoding an insulin receptor enhanced the autophagy induction, and RNAi knockdown of daf-16 encoding a FOXO transcriptional factor in insulin signaling pathway suppressed the autophagy induction in 10 μg/L nanopolystyrene exposed nematodes. Moreover, DAF-16 acted upstream of LGG-1, an ortholog of Atg8/LC3, to regulate the toxicity of nanopolystyrene toxicity in inducing ROS production and in decreasing locomotion behavior at adult day-9. Our data implied the potential toxicity of chronic exposure to nanoplastics at predicted environmental concentrations on organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

During development, cell fate decisions are often highly stochastic, but with the frequency of the different possible fates tightly controlled. To understand how signaling networks control the cell fate frequency of such random decisions, we studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. Using time-lapse microscopy to measure the single-cell dynamics of two key inhibitors of cell fusion, the Hox gene LIN-39 and Wnt signaling through the β-catenin BAR-1, we uncovered significant variability in the dynamics of LIN-39 and BAR-1 levels. Most strikingly, we observed that BAR-1 accumulated in a single, 1-4 ​h pulse at the time of the P3.p cell fate decision, with strong variability both in pulse slope and time of pulse onset. We found that the time of BAR-1 pulse onset was delayed relative to the time of cell fusion in mutants with low cell fusion frequency, linking BAR-1 pulse timing to cell fate outcome. Overall, a model emerged where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced. Our results highlight that timing of cell signaling dynamics, rather than its average level or amplitude, could play an instructive role in determining cell fate., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Nickel (Ni) is a ubiquitous metal in the environment with increasing industrial application. While environmental and occupational exposure to Ni compounds has been known to result in toxicities to several organs, including the liver, kidney, lungs, skin and gonads, neurotoxic effects have not been extensively investigated. In this present study, we investigated specific neuronal susceptibility in a C. elegans model of acute Ni neurotoxicity. Wild-type worms and worms expressing green fluorescent protein (GFP) in either cholinergic, dopaminergic or GABAergic neurons were treated with NiCl 2 for 1 h at the first larval (L1) stage. The median lethal dose (LD 50 ) was calculated to be 5.88 mM in this paradigm. Morphology studies of GFP-expressing worms showed significantly increasing degeneration of cholinergic, dopaminergic and GABAergic neurons with increasing Ni concentration. Significant functional changes in locomotion and basal slowing response assays reflected that cholinergic and dopaminergic neuronal function, respectively, were impaired due to Ni treatment. Interestingly, a small but significant number of worms exhibited shrinker phenotype upon Ni exposure but no loopy head foraging behaviour was observed suggesting that function of D-type GABAergic neurons of C elegans may be specifically attenuated while the RME subset of GABAergic neurons are not. GFP expression due to induction of glutathione S-transferase 4 (gst-4), a target of Nrf2 homolog skn-1, was increased in a P gst-4 ::GFP worm highlighting Ni-induced oxidative stress. RT-qPCR verified upregulation of this expression of gst-4 immediately after exposure. These data suggest that oxidative stress is associated with neuronal damage and altered behaviour due to developmental Ni exposure.

Cellular responsiveness to environment, including changes in extracellular matrix (ECM), is critical for normal processes such as development and wound healing, but can go awry, as in oncogenesis and fibrosis. One type of molecular pathway contributing to this responsiveness is the BMP signaling pathway. Owing to their broad and potent functions, BMPs and their pathways are regulated at multiple levels. In Caenorhabditis elegans , the BMP ligand DBL-1 is a regulator of body size. We previously showed that DBL-1/BMP signaling determines body size through transcriptional regulation of cuticle collagen genes. We now identify feedback regulation of DBL-1/BMP through analysis of four DBL-1-regulated collagen genes. Inactivation of any of these genes reduces DBL-1/BMP signaling, measured by a pathway activity reporter. Furthermore, depletion of these collagens reduces GFP::DBL-1 fluorescence and acts unexpectedly at the level of dbl-1 transcription. We conclude that cuticle, a specialized ECM, impinges on DBL-1/BMP expression and signaling. Interestingly, the feedback regulation of DBL-1/BMP signaling by collagens is likely to be contact independent due to physical separation of the cuticle from DBL-1-expressing cells in the ventral nerve cord. Our results provide an entry point into a novel regulatory mechanism for BMP signaling, with broader implications for mechanical regulation of gene expression.

Stochastic mechanisms diversify cell fate in organisms ranging from bacteria to humans [1-4]. In the anchor cell/ventral uterine precursor cell (AC/VU) fate decision during C. elegans gonadogenesis, two "α cells," each with equal potential to be an AC or a VU, interact via LIN-12/Notch and its ligand LAG-2/DSL [5, 6]. This LIN-12/Notch-mediated interaction engages feedback mechanisms that amplify a stochastic initial difference between the two α cells, ensuring that the cell with higher lin-12 activity becomes the VU while the other becomes the AC [7-9]. The initial difference between the α cells was originally envisaged as a random imbalance from "noise" in lin-12 expression/activity [6]. However, subsequent evidence that the relative birth order of the α cells biases their fates suggested other factors may be operating [7]. Here, we investigate the nature of the initial difference using high-throughput lineage analysis [10]; GFP-tagged endogenous LIN-12, LAG-2, and HLH-2, a conserved transcription factor that orchestrates AC/VU development [7, 11]; and tissue-specific hlh-2 null alleles. We identify two stochastic elements: relative birth order, which largely originates at the beginning of the somatic gonad lineage three generations earlier, and onset of HLH-2 expression, such that the α cell whose parent expressed HLH-2 first is biased toward the VU fate. We find that these elements are interrelated, because initiation of HLH-2 expression is linked to the birth of the parent cell. Finally, we provide a potential deterministic mechanism for the HLH-2 expression bias by showing that hlh-2 is required for LIN-12 expression in the α cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its β subunit (N-βNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-βNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The signal transmission in the nervous system operates through a sensitive balance between excitatory (E) inputs and inhibitory (I) responses. Imbalances in this system contribute to the development of pathologies such as seizures. In Caenorhabditis elegans, the locomotor circuit operates via the coordinated activity of cholinergic excitatory (E) and GABAergic inhibitory (I) transmission. Changes in E/I inputs can cause uncontrolled electrical discharges, mimicking the physiology of seizures. Molecules derived from 1,3,4-oxadiazole have been found to exhibit diverse biological activities, including anticonvulsant effect. In this work, we study the activity of the compound 2-[(4-methoxyphenylselenyl)methylthio]-5-phenyl-1,3,4-oxadiazole (MPMT-OX) in the GABAergic and cholinergic systems. We demonstrate that MPMT-OX reduced the locomotor activity of C. elegans with a normal balance between the E/I systems and increased the resistance to paralysis in worms exposed to pentylenetetrazol and aldicarb. MPMT-OX increased seizure resistance and assisted in the recovery of locomotor activity in worms with deletions in the genes unc-46, which regulates the transport of GABA into vesicles, and unc-49, which encodes the GABA A receptor. C. elegans with deletions in the unc-25 and unc-47 genes did not respond to treatment. Therefore, we suggest that the compound MPMT-OX upregulates GABAergic signaling in a manner dependent on the unc-25 gene, which is responsible for GABA synthesis, and unc-47, which encodes the vesicular GABA transporter., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Innovations in metazoan development arise from evolutionary modification of gene regulatory networks (GRNs). We report widespread cryptic variation in the requirement for two key regulatory inputs, SKN-1/Nrf2 and MOM-2/Wnt, into the C. elegans endoderm GRN. While some natural isolates show a nearly absolute requirement for these two regulators, in others, most embryos differentiate endoderm in their absence. GWAS and analysis of recombinant inbred lines reveal multiple genetic regions underlying this broad phenotypic variation. We observe a reciprocal trend, in which genomic variants, or knockdown of endoderm regulatory genes, that result in a high SKN-1 requirement often show low MOM-2/Wnt requirement and vice-versa, suggesting that cryptic variation in the endoderm GRN may be tuned by opposing requirements for these two key regulatory inputs. These findings reveal that while the downstream components in the endoderm GRN are common across metazoan phylogeny, initiating regulatory inputs are remarkably plastic even within a single species., Competing Interests: YT, CE, KC, EM, CW, CA, MA, TT, PJ, RS, JR No competing interests declared, (© 2019, Torres Cleuren et al.)

Di(2-ethylhexyl)phthalate (DEHP) is an ubiquitous and emerging contaminant that is widely present in food, agricultural crop, and the environment, posing a potential risk to human health. This study utilized the nematode Caenorhabditis elegans to decipher the toxic effects of early life exposure to DEHP on aging and its underlying mechanisms. The results showed that exposure to DEHP at 0.1 and 1.5 mg/L inhibited locomotive behaviors. In addition, DEHP exposure significantly shortened the mean lifespan of the worms and further adversely affected pharyngeal pumping rate and defecation cycle in aged worms. Moreover, DEHP exposure also further enhanced accumulation of age-related biomarkers including lipofuscin, lipid peroxidation, and intracellular reactive oxygen species in aged worms. In addition, exposure to DEHP significantly suppressed gene expression of hsp-16.1, hsp-16.49, and hsp-70 in aged worms. Further evidences showed that mutation of genes involved in insulin/IGF-1-like signaling (IIS) pathway (daf-2, age-1, pdk-1, akt-1, akt-2, and daf-16) restored lipid peroxidation accumulation upon DEHP exposure in aged worms, whereas skn-1 mutation resulted in enhanced lipid peroxidation accumulation. Therefore, IIS and SKN-1 may serve as an important molecular basis for DEHP-induced age-related declines in C. elegans. Since IIS and SKN-1 are highly conserved among species, the age-related declines caused by DEHP exposure may not be exclusive in C. elegans, leading to adverse human health consequences due to widespread and persistent DEHP contamination in the environment., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Animal responses to thermal stimuli involve intricate contributions of genetics, neurobiology and physiology, with temperature variation providing a pervasive environmental factor for natural selection. Thermal behavior thus exemplifies a dynamic trait that requires non-trivial phenotypic summaries to appropriately capture the trait in response to a changing environment. To characterize the deterministic and plastic components of thermal responses, we developed a novel micro-droplet assay of nematode behavior that permits information-dense summaries of dynamic behavioral phenotypes as reaction norms in response to increasing temperature (thermal tolerance curves, TTC)., Results: We found that C. elegans TTCs shift predictably with rearing conditions and developmental stage, with significant differences between distinct wildtype genetic backgrounds. Moreover, after screening TTCs for 58 C. elegans genetic mutant strains, we determined that genes affecting thermosensation, including cmk-1 and tax-4, potentially play important roles in the behavioral control of locomotion at high temperature, implicating neural decision-making in TTC shape rather than just generalized physiological limits. However, expression of the transient receptor potential ion channel TRPA-1 in the nervous system is not sufficient to rescue rearing-dependent plasticity in TTCs conferred by normal expression of this gene, indicating instead a role for intestinal signaling involving TRPA-1 in the adaptive plasticity of thermal performance., Conclusions: These results implicate nervous system and non-nervous system contributions to behavior, in addition to basic cellular physiology, as key mediators of evolutionary responses to selection from temperature variation in nature.

The present study was performed to evaluate the neurobehavioural deficit induced by nonylphenol (NP), a well-known xenobiotic chemical. The neurotoxic mechanism from oxidative stress and serotonin-related progress was also investigated. Caenorhabditis elegans was exposed at different levels of NP ranging from 0 to 200 μg L -1 for 10 days. The results revealed that from a relatively low concentration (i.e., 10 μg L -1 ), significant effects including decreased head thrashes, body bends and forging behaviour could be observed, along with impaired learning and memory behaviour plasticity. The level of reactive oxygen species (ROS) in head was significantly elevated with the increase of NP concentrations from 10 to 200 μg L -1 . Through antioxidant experiment, the oxidative damage caused by NP restored to some extent. At a NP concentration of 200 μg L -1 , the significant increased expression of stress-related genes, including sod-1, sod-3, ctl-2, ctl-3 and cyp-35A2 gene, was observed from integrated gene expression profiles. In addition, in comparison with wild-type N2 worms, the ROS accumulation was increased significantly with the mutation of sod-3. Tryptophan hydroxylase (TPH) in ADF and NSM neurons sharply decreased at the concentrations of 10-200 μg L -1 . The transcription of TPH synthesis-related genes and serotonin-related genes were both suppressed, including tph-1, cat-1, cat-4, ser-1, and mod-5. Overall, these results indicated that NP could induce neurotoxicity on Caenorhabditis elegans through excessive induction of ROS and disturbance synthesis of serotonin. The conducted research opened up new avenues for more effective exploration of neurotoxicity caused by NP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Humans are exposed simultaneously to a variety of neurotoxic agents, including manganese (Mn) and methylmercury (MeHg). Therefore, the study of combined exposures to toxicants is timely. This work aimed to study changes in cholinergic system focusing on acetylcholinesterase (ace-2), monoaminergic system focusing on vesicular monoamine transporter (VMAT, cat-1) expression, to address changes in antioxidant enzymatic systems, namely, the expression of superoxide dismutase (sod-3 and sod-4) and catalase (ctl-3), as well as worm reproduction and locomotion. C. elegans in the L1 larval stage were exposed to Mn, MeHg or both. All analyses were done 24 h after the end of exposure, except for behavior and reproduction tests that were assessed in L4 larval stage worms. The values obtained for lethal dose 50% (LD 50 ) were 17.78 mM for Mn and 30.63 μM for MeHg. It was observed that body bends, pharyngeal pumping and brood size decreased in worms exposed to metals when undergoing combined exposures. Relative mRNA content of ace-2, cat-1, sod-3, sod-4 and ctl-3 was increased at the highest concentration of the interaction (50 mM Mn + 50 μM MeHg). Cholinergic degeneration was observed in all groups co-exposed to both metals. Notably, combined exposure to metals was more toxic to the worms than when exposed to a single metal., (Copyright © 2018 Elsevier B.V. All rights reserved.)

When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 ( Drosophila tsunagi and mago nashi ) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage., (Copyright © 2018 Gauvin et al.)

In this study, the possible molecular mechanisms of zearalenone (ZEA)-induced reproductive and developmental toxic effects in Caenorhabditis elegans (C. elegans) were investigated. Differential gene expression profiles were identified, and 171, 245, and 3149 genes were down- or up-regulated (>2.0 fold) in 10, 20, and 40 μg/ml ZEA treated groups, respectively, as compared to untreated controls. Pathway specific mapping showed that the major differentially expressed genes were collagen synthetic pathways regulating genes, col-121 and dpy-17. Real-time PCR reconfirmation of key genes, related to cuticle collagen synthetic pathway, found dramatic changes in the expression of the genes dpy-31, sqt-3, col-121, and dpy-17 following exposure to ZEA (40 μg/ml), which indicated the significance of these genes in ZEA-induced toxicity. Cuticle collagen plays many key roles in the development and reproduction of C. elegans. The hypersensitive responses in transgenic and mutant worms also confirmed the roles of these genes in lethality and reproductive response to ZEA exposure, which indicates that ZEA blocked the normal collagen processing and cuticle formation. Taken together, our results demonstrate that disruption of the collagen biosynthetic pathway might be a key mechanism in ZEA-induced reproductive and developmental toxic effects in C. elegans., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The sorting nexin (SNX) family proteins play an essential role in vesicular transport, cell signaling, and membrane remodeling. The SNX members SNX1/2 and SNX5/6 form dimers, and mediate endosome-to-trans Golgi network (TGN) transport through coordinating cargo selection and membrane remodeling. It is well-known how a SNX-BAR protein forms a homodimer; however, it is less clear how a heterodimer is formed. Here a detailed expression and purification protocol of the SNX1/SNX6 complex, from both worm and human, is described. Keys to the successful protein production include co-expression of both genes, and inclusion of glycerol in the protein buffer. Solution studies suggest that SNX1 and SNX6 form a 1:1 heterodimer. The production of a large amount, high quality of the SNX1/SNX6 complex provides a basis for future biochemical and structural studies of the complex, and in vitro reconstitution of SNX1/SNX6-mediated transport., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in the gene encoding Tau (MAPT-microtubule-associated protein tau) cause a group of neurodegenerative diseases called tauopathies. A recently identified Tau variant, p.A152T, has been reported as a risk factor for frontotemporal dementia-related disorders and Alzheimer disease. However, the mechanism for the pathologies still remain poorly understood. Transgenic Caenorhabditis elegans expressing mutant 2N4R-Tau A152T (Tau AT ) panneuronally show locomotor defects, neurodegeneration and accelerated aging. Here we report that, in Tau AT animals, the glutamatergic nervous system is at a high risk of progressive neuronal loss. We present genetic data that this loss occurs predominantly through necrosis. The neuronal loss is caused by several determinants, such as altered adenylyl cyclase (type AC9) pathway, prevalence of excitotoxicity-like conditions, aging-related factors and finally dyshomeostasis of intracellular calcium (Ca 2+ ). The study provides novel insights into the mechanisms involved in selective loss of glutamatergic neurons in a Tau AT tauopathy model which could point to new therapeutic targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mutations in the microtubule-associated protein tau (MAPT) gene have been linked to a heterogeneous group of progressive neurodegenerative disorders commonly called tauopathies. From patients with frontotemporal lobar degeneration with distinct atypical clinical phenotypes, we recently identified two new mutations on the same codon, in position 363 of the MAPT gene, which resulted in the production of Val-to-Ala (tau V363A ) or Val-to-Ile (tau V363I ) mutated tau. These substitutions specifically affected microtubule polymerization and propensity of tau to aggregate in vitro suggesting that single amino acid modification may dictate the fate of the neuropathology. To clarify whether tau V363A and tau V363I affect protein misfolding differently in vivo driving certain phenotypes, we generated new transgenic C. elegans strains. Human 2N4R tau carrying the mutation was expressed in all the neurons of worms. The behavioral defects, misfolding and proteotoxicity caused by the tau V363A and tau V363I mutated proteins were compared to that induced by the expression of wild-type tau (tau wt ). Pan-neuronal expression of human 2N4R tau WT in worms resulted in a neuromuscular defect with characteristics of a neurodegenerative phenotype. This defect was worsened by the expression of mutated proteins which drive distinct neuronal dysfunctions and synaptic impairments involving, in transgenic worms expressing tau V363A (V363A) also a pharyngeal defect never linked before to other mutations. The two mutations differently affected the tau phosphorylation and misfolding propensities: tau V363I was highly phosphorylated on epitopes corresponding to Thr231 and Ser202/Thr205, and accumulated as insoluble tau assemblies whereas tau V363A showed a greater propensity to form soluble oligomeric assemblies. These findings uphold the role of a single amino acid substitution in specifically affecting the ability of tau to form soluble and insoluble assemblies, opening up new perspectives in the pathogenic mechanism underlying tauopathies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Environmental temperature acclimation is essential to animal survival, yet thermoregulation mechanisms remain poorly understood. We demonstrate cold tolerance in Caenorhabditis elegans as regulated by paired ADL chemosensory neurons via Ca 2+ -dependent endoribonuclease (EndoU) ENDU-2. Loss of ENDU-2 function results in life span, brood size, and synaptic remodeling abnormalities in addition to enhanced cold tolerance. Enzymatic ENDU-2 defects localized in the ADL and certain muscle cells led to increased cold tolerance in endu-2 mutants. Ca 2+ imaging revealed ADL neurons were responsive to temperature stimuli through transient receptor potential (TRP) channels, concluding that ADL function requires ENDU-2 action in both cell-autonomous and cell-nonautonomous mechanisms. ENDU-2 is involved in caspase expression, which is central to cold tolerance and synaptic remodeling in dorsal nerve cord. We therefore conclude that ENDU-2 regulates cell type-dependent, cell-autonomous, and cell-nonautonomous cold tolerance., Competing Interests: The authors declare no conflict of interest.

Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans , we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ. SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans ; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology., (Copyright © 2018 Sharma, Li et al.)

In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern in which the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation.

Acetaminophen (APAP) overdose-induced hepatotoxicity is the most commonly cause of drug-induced liver failure characterized by oxidative stress, mitochondrial dysfunction, and cell damage. Therapeutic efficacy of omega-3 polyunsaturated fatty acids (n-3 PUFA) in several models of liver disease is well documented. However, the impacts of n-3 PUFA on APAP hepatotoxicity are not adequately addressed. In this study, the fat-1 transgenic mice that synthesize endogenous n-3 PUFA and wild type (WT) littermates were injected intraperitoneally with APAP at the dose of 400 mg/kg to induce liver injury, and euthanized at 0 h, 2 h, 4 h and 6 h post APAP injection for sampling. APAP overdose caused severe liver injury in WT mice as indicated by serum parameters, histopathological changes and hepatocyte apoptosis, which were remarkably ameliorated in fat-1 mice. These protective effects of n-3 PUFA were associated with regulation of the prolonged JNK activation via inhibition of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase kinase 4 (MKK4) pathway. Additionally, the augment of endogenous n-3 PUFA reduced nuclear factor kappa B (NF-κB) - mediated inflammation response induced by APAP treatment in the liver. These findings indicate that n-3 PUFA has potent protective effects against APAP-induced acute liver injury, suggesting that n-3 dietary supplement with n-3 PUFA may be a potential therapeutic strategy for the treatment of hepatotoxicity induced by APAP overdose., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Manganese (Mn) is an essential trace element for physiological functions since it acts as an enzymatic co-factor. Nevertheless, overexposure to Mn has been associated with a pathologic condition called manganism. Furthermore, Mn has been reported to affect lipid metabolism by mechanisms which have yet to be established. Herein, we used the nematode Caenorhabditis elegans to examine Mn's effects on the dopaminergic (DAergic) system and determine which transcription factors that regulate with lipid metabolism are affected by it. Worms were exposed to Mn for four hours in the presence of bacteria and in a liquid medium (85 mM NaCl). Mn increased fat storage as evidenced both by Oil Red O accumulation and triglyceride levels. In addition, metabolic activity was reduced as a reflection of decreased oxygen consumption caused by Mn. Mn also affected feeding behavior as evidenced by decreased pharyngeal pumping rate. DAergic neurons viability were not altered by Mn, however the dopamine levels were significantly reduced following Mn exposure. Furthermore, the expression of sbp-1 transcription factor and let-363 protein kinase responsible for lipid accumulation control was increased and decreased, respectively, by Mn. Altogether, our data suggest that Mn increases the fat storage in C. elegans, secondary to DAergic system alterations, under the control of SBP-1 and LET-363 proteins., (Copyright © 2018. Published by Elsevier B.V.)

Monoamines and neuropeptides often modulate the same behavior, but monoaminergic-peptidergic crosstalk remains poorly understood. In Caenorhabditis elegans, the adrenergic-like ligands, tyramine (TA) and octopamine (OA) require distinct subsets of neuropeptides in the two ASI sensory neurons to inhibit nociception. TA selectively increases the release of ASI neuropeptides encoded by nlp-14 or nlp-18 from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively, and OA selectively increases the release of ASI neuropeptides encoded by nlp-9 asymmetrically, from only the synaptic/perisynaptic region of the right ASI axon. The predicted amino acid preprosequences of genes encoding either TA- or OA-dependent neuropeptides differed markedly. However, these distinct preprosequences were not sufficient to confer monoamine-specificity and additional N-terminal peptide-encoding sequence was required. Collectively, our results demonstrate that TA and OA specifically and differentially modulate the release of distinct subsets of neuropeptides from different subcellular sites within the ASIs, highlighting the complexity of monoaminergic/peptidergic modulation, even in animals with a relatively simple nervous system.

Self-avoidance allows sister dendrites from the same neuron to form non-redundant coverage of the sensory territory and is important for neural circuitry functions. Here, we report an unexpected, cell-autonomous role of the Wnt-secretory factor MIG-14/Wntless in mediating dendrite self-avoidance in the C. elegans multidendritic PVD neurons. Similar findings in Drosophila suggest that this novel function of Wntless is conserved. The mig-14 mutant shows defects in dendrite self-avoidance, and ectopic MIG-14 expression triggers dendrite repulsion. Functions of dendrite self-avoidance and Wnt secretion could be mapped to distinct MIG-14 domains, indicating that these two functions of MIG-14 are genetically separable, consistent with lack of self-avoidance defects in the Wnt mutants. We further demonstrate that MIG-14 engages Wiskott-Aldrich syndrome protein (WASP)-dependent actin assembly to regulate dendrite self-avoidance. Our work expands the repertoire of self-avoidance molecules and uncovers a previously unknown, Wnt-independent function of MIG-14/Wntless., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Focused ultrasound has been shown to stimulate excitable cells, but the biophysical mechanisms behind this phenomenon remain poorly understood. To provide additional insight, we devised a behavioral-genetic assay applied to the well-characterized nervous system of Caenorhabditis elegans nematodes. We found that pulsed ultrasound elicits robust reversal behavior in wild-type animals in a pressure-, duration-, and pulse protocol-dependent manner. Responses were preserved in mutants unable to sense thermal fluctuations and absent in mutants lacking neurons required for mechanosensation. Additionally, we found that the worm's response to ultrasound pulses rests on the expression of MEC-4, a DEG/ENaC/ASIC ion channel required for touch sensation. Consistent with prior studies of MEC-4-dependent currents in vivo , the worm's response was optimal for pulses repeated 300-1000 times per second. Based on these findings, we conclude that mechanical, rather than thermal, stimulation accounts for behavioral responses. Further, we propose that acoustic radiation force governs the response to ultrasound in a manner that depends on the touch receptor neurons and MEC-4-dependent ion channels. Our findings illuminate a complete pathway of ultrasound action, from the forces generated by propagating ultrasound to an activation of a specific ion channel. The findings further highlight the importance of optimizing ultrasound pulsing protocols when stimulating neurons via ion channels with mechanosensitive properties. SIGNIFICANCE STATEMENT How ultrasound influences neurons and other excitable cells has remained a mystery for decades. Although it is widely understood that ultrasound can heat tissues and induce mechanical strain, whether or not neuronal activation depends on heat, mechanical force, or both physical factors is not known. We harnessed Caenorhabditis elegans nematodes and their extraordinary sensitivity to thermal and mechanical stimuli to address this question. Whereas thermosensory mutants respond to ultrasound similar to wild-type animals, mechanosensory mutants were insensitive to ultrasound stimulation. Additionally, stimulus parameters that accentuate mechanical effects were more effective than those producing more heat. These findings highlight a mechanical nature of the effect of ultrasound on neurons and suggest specific ways to optimize stimulation protocols in specific tissues., (Copyright © 2018 the authors 0270-6474/18/383081-11$15.00/0.)

Ischemic stroke is a devastating neurological disorder and one of the leading causes of death and serious disability in adults. Adult neural stem cell (NSC) replacement therapy is a promising treatment for both structural and functional neurological recovery. However, for the treatment to work, adult NSCs must be protected against hypoxic-ischemic damage in the ischemic penumbra. In the present study, we aimed to investigate the neuroprotective effects of the mfat-1 transgene on cobalt chloride (CoCl 2 )-induced hypoxic-ischemic injury in cultured adult NSCs as well as its underlying mechanisms. The results show that in the CoCl 2 -induced hypoxic-ischemic injury model, the mfat-1 transgene enhanced the viability of adult NSCs and suppressed CoCl 2 -mediated apoptosis of adult NSCs. Additionally, the mfat-1 transgene promoted the proliferation of NSCs as shown by increased bromodeoxyuridine labeling of adult NSCs. This process was related to the reduction of reactive oxygen species. Quantitative real-time polymerase chain reaction and Western blot analysis revealed a much higher expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream genes (HO-1, NQO-1, GCLC). Taken together, our findings show that the mfat-1 transgene restored the CoCl 2 -inhibited viability and proliferation of NSCs by activating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signal pathway to inhibit oxidative stress injury. Further investigation of the function of the mfat-1 transgene in adult protective mechanisms may accelerate the development of adult NSC replacement therapy for ischemic stroke., (© 2017 Wiley Periodicals, Inc.)

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

Maintenance of protein homeostasis, or proteostasis, is crucial for organismal health. Disruption of proteostasis can lead to the accumulation of protein aggregates, which are associated with aging and many human diseases such as Alzheimer's disease [1-3]. Through analysis of the C. elegans host response to intracellular infection, we describe here a novel response pathway that enhances proteostasis capacity and appears to act in parallel to well-studied proteostasis pathways. These findings are based on analysis of the transcriptional response to infection by the intracellular pathogen Nematocida parisii [4]. The response to N. parisii is strikingly similar to the response to infection by the Orsay virus, another natural intracellular pathogen of C. elegans, and is distinct from responses to extracellular pathogen infection [4-6]. We have therefore named this common transcriptional response the intracellular pathogen response (IPR), and it includes upregulation of several predicted ubiquitin ligase complex components such as the cullin cul-6. Through a forward genetic screen we found pals-22, a gene of previously unknown function, to be a repressor of the cul-6/cullin gene and other IPR gene expression. Interestingly, pals-22 mutants have increased thermotolerance and reduced levels of stress-induced polyglutamine aggregates, likely due to upregulated IPR gene expression. We found the enhanced stress resistance of pals-22 mutants to be dependent on cul-6, suggesting that pals-22 mutants have increased activity of a CUL-6/cullin-containing ubiquitin ligase complex. pals-22 mutant phenotypes appear independent of the well-studied heat shock and insulin signaling pathways, indicating that the IPR is a distinct pathway that protects animals from proteotoxic stress., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Due to environmental insult or innate genetic deficiency, protein folding environments of the mitochondrial matrix are prone to dysregulation, prompting the activation of a specific organellar stress-response mechanism, the mitochondrial unfolded protein response (UPR MT ). In Caenorhabditis elegans , mitochondrial damage leads to nuclear translocation of the ATFS-1 transcription factor to activate the UPR MT After short-term acute stress has been mitigated, the UPR MT is eventually suppressed to restore homeostasis to C. elegans hermaphrodites. In contrast, and reflective of the more chronic nature of progressive neurodegenerative disorders such as Parkinson's disease (PD), here, we report the consequences of prolonged, cell-autonomous activation of the UPR MT in C. elegans dopaminergic neurons. We reveal that neuronal function and integrity decline rapidly with age, culminating in activity-dependent, non-apoptotic cell death. In a PD-like context wherein transgenic nematodes express the Lewy body constituent protein α-synuclein (αS), we not only find that this protein and its PD-associated disease variants have the capacity to induce the UPR MT , but also that coexpression of αS and ATFS-1-associated dysregulation of the UPR MT synergistically potentiate dopaminergic neurotoxicity. This genetic interaction is in parallel to mitophagic pathways dependent on the C. elegans PINK1 homolog, which is necessary for cellular resistance to chronic malfunction of the UPR MT Given the increasingly recognized role of mitochondrial quality control in neurodegenerative diseases, these studies illustrate, for the first time, an insidious aspect of mitochondrial signaling in which the UPR MT pathway, under disease-associated, context-specific dysregulation, exacerbates disruption of dopaminergic neurons in vivo , resulting in the neurodegeneration characteristic of PD. SIGNIFICANCE STATEMENT Disruptions or alterations in the activation of pathways that regulate mitochondrial quality control have been linked to neurodegenerative diseases due in part to the central role of mitochondria in metabolism, ROS regulation, and proteostasis. The extent to which these pathways, including the mitochondrial unfolded protein response (UPR MT ) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to compounding stressors. Furthermore, therapeutic strategies that aim to induce these pathways may benefit from increased study into cellular responses that arise from long-term or ectopic stimulation, especially in neuronal compartments. By demonstrating the detrimental consequences of prolonged cellular activation of the UPR MT , we provide evidence that this pathway is not a universally beneficial mechanism because dysregulation has neurotoxic consequences., (Copyright © 2017 the authors 0270-6474/17/3711085-16$15.00/0.)

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

Intermittent fasting (IF) is a dietary restriction regimen that extends the lifespans of Caenorhabditis elegans and mammals by inducing changes in gene expression. However, how IF induces these changes and promotes longevity remains unclear. One proposed mechanism involves gene regulation by microRNAs (miRNAs), small non-coding RNAs (∼22 nucleotides) that repress gene expression and whose expression can be altered by fasting. To test this proposition, we examined the role of the miRNA machinery in fasting-induced transcriptional changes and longevity in C. elegans We revealed that fasting up-regulated the expression of the miRNA-induced silencing complex (miRISC) components, including Argonaute and GW182, and the miRNA-processing enzyme DRSH-1 (the ortholog of the Drosophila Drosha enzyme). Our lifespan measurements demonstrated that IF-induced longevity was suppressed by knock-out or knockdown of miRISC components and was completely inhibited by drsh-1 ablation. Remarkably, drsh-1 ablation inhibited the fasting-induced changes in the expression of the target genes of DAF-16, the insulin/IGF-1 signaling effector in C. elegans Fasting-induced transcriptome alterations were substantially and modestly suppressed in the drsh-1 null mutant and the null mutant of ain-1 , a gene encoding GW182, respectively. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting. These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme DRSH-1, play an important role in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The germ cells of multicellular organisms protect their developmental potential through specialized mechanisms. A shared feature of germ cells from worms to humans is the presence of nonmembrane-bound, ribonucleoprotein organelles called germ granules. Depletion of germ granules in Caenorhabditis elegans ( i.e. , P granules) leads to sterility and, in some germlines, expression of the neuronal transgene unc-119 :: gfp and the muscle myosin MYO-3 Thus, P granules are hypothesized to maintain germ cell totipotency by preventing somatic development, although the mechanism by which P granules carry out this function is unknown. In this study, we performed transcriptome and single molecule RNA-FISH analyses of dissected P granule-depleted gonads at different developmental stages. Our results demonstrate that P granules are necessary for adult germ cells to downregulate spermatogenesis RNAs and to prevent the accumulation of numerous soma-specific RNAs. P granule-depleted gonads that express the unc-119 :: gfp transgene also express many other genes involved in neuronal development and concomitantly lose expression of germ cell fate markers. Finally, we show that removal of either of two critical P-granule components, PGL-1 or GLH-1, is sufficient to cause germ cells to express UNC-119::GFP and MYO-3 and to display RNA accumulation defects similar to those observed after depletion of P granules. Our data identify P granules as critical modulators of the germline transcriptome and guardians of germ cell fate., (Copyright © 2017 by the Genetics Society of America.)

The acquisition of a specific cellular identity is usually paralleled by a restriction of cellular plasticity. Whether and how these two processes are coordinated is poorly understood. Transcription factors called terminal selectors activate identity-specific effector genes during neuronal differentiation to define the structural and functional properties of a neuron. To study restriction of plasticity, we ectopically expressed C. elegans CHE-1, a terminal selector of ASE sensory neuron identity. In undifferentiated cells, ectopic expression of CHE-1 results in activation of ASE neuron type-specific effector genes. Once cells differentiate, their plasticity is restricted and ectopic expression of CHE-1 no longer results in activation of ASE effector genes. In striking contrast, removal of the respective terminal selectors of other sensory, inter-, or motor neuron types now enables ectopically expressed CHE-1 to activate its ASE-specific effector genes, indicating that terminal selectors not only activate effector gene batteries but also control the restriction of cellular plasticity. Terminal selectors mediate this restriction at least partially by organizing chromatin. The chromatin structure of a CHE-1 target locus is less compact in neurons that lack their resident terminal selector and genetic epistasis studies with H3K9 methyltransferases suggest that this chromatin modification acts downstream of a terminal selector to restrict plasticity. Taken together, terminal selectors activate identity-specific genes and make non-identity-defining genes less accessible, thereby serving as a checkpoint to coordinate identity specification with restriction of cellular plasticity.

Environmental conditions experienced during animal development are thought to have sustained impact on maturation and adult lifespan. Here we show that in the model organism C. elegans developmental rate and adult lifespan depend on larval population density, and that this effect is mediated by excreted small molecules. By using the time point of first egg laying as a marker for full maturity, we found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Population density-dependent acceleration of development (Pdda) was dramatically enhanced in fatty acid β-oxidation mutants that are defective in the biosynthesis of ascarosides, small-molecule signals that induce developmental diapause. In contrast, Pdda is abolished by synthetic ascarosides and steroidal ligands of the nuclear hormone receptor DAF-12. We show that neither ascarosides nor any known steroid hormones are required for Pdda and that another chemical signal mediates this phenotype, in part via the nuclear hormone receptor NHR-8. Our results demonstrate that C. elegans development is regulated by a push-pull mechanism, based on two antagonistic chemical signals: chemosensation of ascarosides slows down development, whereas population-density dependent accumulation of a different chemical signal accelerates development. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans , and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Scorpion venom could be a useful treatment for a variety of diseases, such as cancer, epilepsy and analgesia. BmKTX is a polypeptide extracts from scorpion venom (PESV), which have attracted much attention from researchers in recent years. mBmKTX is a mutant polypeptide according to the amino acid sequence of BmKTX. We expressed it with the vector pGEX-4T-1 in Escherichia coli, and Caenorhabditis elegans were used as the animal model and fed with the strains. In this study, the expression of pGEX-mBmKTX was analyzed by SDS-PAGE, and GST-mBmKTX purified from pGEX-mBmKTX as a glutathione S-transferase (GST)-tagged fusion protein is approximately 30kDa. The secondary structure prediction shows that mBmKTX is mainly composed of approximately 13% β-sheet and 86% loop. A food clearance assay and brood size assay indicated that the worms fed pGEX-mBmKTX ate more and had greater fecundity than those fed the empty vector. A lifespan analysis demonstrated that mBmKTX could significantly prolong the lifespan of C. elegans, with an increase of 22.5% compared with the control. Behavioral assays confirmed that mBmKTX had no influence on the locomotion of C. elegans. In addition, microarray analysis and quantitative real-time PCR demonstrated that there are 320 differentially expressed genes, 182 of which are related to reproduction, growth and lifespan. In conclusion, the data suggested that mBmKTX has potential utility for increasing fecundity and animal survival., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks., Competing Interests: The authors have declared that no competing interests exist.

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations., Competing Interests: The authors declare no competing of interests.

Safeguarding the proteome is central to the health of the cell. In multi-cellular organisms, the composition of the proteome, and by extension, protein-folding requirements, varies between cells. In agreement, chaperone network composition differs between tissues. Here, we ask how chaperone expression is regulated in a cell type-specific manner and whether cellular differentiation affects chaperone expression. Our bioinformatics analyses show that the myogenic transcription factor HLH-1 (MyoD) can bind to the promoters of chaperone genes expressed or required for the folding of muscle proteins. To test this experimentally, we employed HLH-1 myogenic potential to genetically modulate cellular differentiation of Caenorhabditis elegans embryonic cells by ectopically expressing HLH-1 in all cells of the embryo and monitoring chaperone expression. We found that HLH-1-dependent myogenic conversion specifically induced the expression of putative HLH-1-regulated chaperones in differentiating muscle cells. Moreover, disrupting the putative HLH-1-binding sites on ubiquitously expressed daf-21(Hsp90) and muscle-enriched hsp-12.2(sHsp) promoters abolished their myogenic-dependent expression. Disrupting HLH-1 function in muscle cells reduced the expression of putative HLH-1-regulated chaperones and compromised muscle proteostasis during and after embryogenesis. In turn, we found that modulating the expression of muscle chaperones disrupted the folding and assembly of muscle proteins and thus, myogenesis. Moreover, muscle-specific over-expression of the DNAJB6 homolog DNJ-24, a limb-girdle muscular dystrophy-associated chaperone, disrupted the muscle chaperone network and exposed synthetic motility defects. We propose that cellular differentiation could establish a proteostasis network dedicated to the folding and maintenance of the muscle proteome. Such cell-specific proteostasis networks can explain the selective vulnerability that many diseases of protein misfolding exhibit even when the misfolded protein is ubiquitously expressed., Competing Interests: The authors have declared that no competing interests exist.

Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like C.elegans protein DAF-28. DAF-28(R37C) is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype-deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C) animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations., Competing Interests: The authors have declared that no competing interests exist.