Adenylosuccinate lyase deficiency affects neurobehavior via perturbations to tyramine signaling in Caenorhabditis elegans.

Adenylosuccinate lyase deficiency is an ultrarare congenital metabolic disorder associated with muscle weakness and neurobehavioral dysfunction. Adenylosuccinate lyase is required for de novo purine biosynthesis, acting twice in the pathway at non-sequential steps. Genetic models can contribute to our understanding of the etiology of disease phenotypes and pave the way for development of therapeutic treatments. Here, we establish the first model to specifically study neurobehavioral aspects of adenylosuccinate lyase deficiency. We show that reduction of adsl-1 function in C. elegans is associated with a novel learning phenotype in a gustatory plasticity assay. The animals maintain capacity for gustatory plasticity, evidenced by a change in their behavior in response to cue pairing. However, their behavioral output is distinct from that of control animals. We link substrate accumulation that occurs upon adsl-1 deficiency to an unexpected perturbation in tyrosine metabolism and show that a lack of tyramine mediates the behavioral changes through action on the metabotropic TYRA-2 tyramine receptor. Our studies reveal a potential for wider metabolic perturbations, beyond biosynthesis of purines, to impact behavior under conditions of adenylosuccinate lyase deficiency.

A Caenorhabditis elegans model of adenylosuccinate lyase deficiency reveals neuromuscular and reproductive phenotypes of distinct etiology.

Inborn errors of purine metabolism are rare syndromes with an array of complex phenotypes in humans. One such disorder, adenylosuccinate lyase deficiency (ASLD), is caused by a decrease in the activity of the bi-functional purine biosynthetic enzyme adenylosuccinate lyase (ADSL). Mutations in human ADSL cause epilepsy, muscle ataxia, and autistic-like symptoms. Although the genetic basis of ASLD is known, the molecular mechanisms driving phenotypic outcome are not. Here, we characterize neuromuscular and reproductive phenotypes associated with a deficiency of adsl-1 in Caenorhabditis elegans. We demonstrate that adsl-1 function contributes to regulation of spontaneous locomotion, that adsl-1 functions acutely for proper mobility, and that aspects of adsl-1-related dysfunction are reversible. Using pharmacological supplementation, we correlate phenotypes with distinct metabolic perturbations. The neuromuscular defect correlates with accumulation of a purine biosynthetic intermediate whereas reproductive deficiencies can be ameliorated by purine supplementation, indicating differing molecular mechanisms behind the phenotypes. Because purine metabolism is highly conserved in metazoans, we suggest that similar separable metabolic perturbations result in the varied symptoms in the human disorder and that a dual-approach therapeutic strategy may be beneficial.

Correction: The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

[This corrects the article DOI: 10.1371/journal.ppat.1009350.].

Noncanonical necrosis in 2 different cell types in a Caenorhabditis elegans NAD+ salvage pathway mutant.

Necrosis was once described as a chaotic unregulated response to cellular insult. We now know that necrosis is controlled by multiple pathways in response to many different cellular conditions. In our pnc-1 NAD+ salvage deficient Caenorhabditis elegans model excess nicotinamide induces excitotoxic death in uterine-vulval uv1 cells and OLQ mechanosensory neurons. We sought to characterize necrosis in our pnc-1 model in the context of well-characterized necrosis, apoptosis, and autophagy pathways in C. elegans. We confirmed that calpain and aspartic proteases were required for uv1 necrosis, but changes in intracellular calcium levels and autophagy were not, suggesting that uv1 necrosis occurs by a pathway that diverges from mec-4d-induced touch cell necrosis downstream of effector aspartic proteases. OLQ necrosis does not require changes in intracellular calcium, the function of calpain or aspartic proteases, or autophagy. Instead, OLQ survival requires the function of calreticulin and calnexin, pro-apoptotic ced-4 (Apaf1), and genes involved in both autophagy and axon guidance. In addition, the partially OLQ-dependent gentle nose touch response decreased significantly in pnc-1 animals on poor quality food, further suggesting that uv1 and OLQ necrosis differ downstream of their common trigger. Together these results show that, although phenotypically very similar, uv1, OLQ, and touch cell necrosis are very different at the molecular level.

Overactive EGF signaling suppresses a C. elegans pnc-1 egg-laying phenotype independent of known signaling mediators.

Nicotinamide recycling is critical to the development and function of Caenorhabditis elegans. Excess nicotinamide in a pnc-1 nicotinamidase mutant causes the necrosis of uv1 and OLQ cells and a highly penetrant egg laying defect. An EGF receptor (let-23) gain-of-function mutation suppresses the Egl phenotype in pnc-1 animals. However, gain-of-function mutations in either of the known downstream mediators, let-60/ Ras or itr-1, are not sufficient. Phosphatidylcholine synthesis is neither required nor sufficient, in contrast to its role in the let-23gf rescue of uv1 necrosis. The mechanism behind the let-23gf suppression of the pnc-1 Egl phenotype is unknown.

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans.

Intestinal epithelial cells are subject to attack by a diverse array of microbes, including intracellular as well as extracellular pathogens. While defense in epithelial cells can be triggered by pattern recognition receptor-mediated detection of microbe-associated molecular patterns, there is much to be learned about how they sense infection via perturbations of host physiology, which often occur during infection. A recently described host defense response in the nematode C. elegans called the Intracellular Pathogen Response (IPR) can be triggered by infection with diverse natural intracellular pathogens, as well as by perturbations to protein homeostasis. From a forward genetic screen, we identified the C. elegans ortholog of purine nucleoside phosphorylase pnp-1 as a negative regulator of IPR gene expression, as well as a negative regulator of genes induced by extracellular pathogens. Accordingly, pnp-1 mutants have resistance to both intracellular and extracellular pathogens. Metabolomics analysis indicates that C. elegans pnp-1 likely has enzymatic activity similar to its human ortholog, serving to convert purine nucleosides into free bases. Classic genetic studies have shown how mutations in human purine nucleoside phosphorylase cause immunodeficiency due to T-cell dysfunction. Here we show that C. elegans pnp-1 acts in intestinal epithelial cells to regulate defense. Altogether, these results indicate that perturbations in purine metabolism are likely monitored as a cue to promote defense against epithelial infection in the nematode C. elegans.

Animal Model Contributions to Congenital Metabolic Disease.

Genetic model systems allow researchers to probe and decipher aspects of human disease, and animal models of disease are frequently specifically engineered and have been identified serendipitously as well. Animal models are useful for probing the etiology and pathophysiology of disease and are critical for effective discovery and development of novel therapeutics for rare diseases. Here we review the impact of animal model organism research in three examples of congenital metabolic disorders to highlight distinct advantages of model system research. First, we discuss phenylketonuria research where a wide variety of research fields and models came together to make impressive progress and where a nearly ideal mouse model has been central to therapeutic advancements. Second, we review advancements in Lesch-Nyhan syndrome research to illustrate the role of models that do not perfectly recapitulate human disease as well as the need for multiple models of the same disease to fully investigate human disease aspects. Finally, we highlight research on the GM2 gangliosidoses Tay-Sachs and Sandhoff disease to illustrate the important role of both engineered traditional laboratory animal models and serendipitously identified atypical models in congenital metabolic disorder research. We close with perspectives for the future for animal model research in congenital metabolic disorders.

Nicotinamide Inhibits Aphid Fecundity and Impacts Survival.

Nicotinamide (NAM) alters behavior in C. elegans and Drosophila, serving as an agonist of TRPV channels affecting sensory neurons and mimicking the mode of action of insecticides used to control phloem-feeding insects. The impact of NAM on green peach aphid (Myzus persicae) behaviors was assessed in artificial diet assays and foliar applications to Arabidopsis plants. Aphids feeding on artificial diets supplemented with NAM impaired stylet movement causing feeding interruptions and ultimately starvation and death. Aphid feeding behaviors were negatively impacted on NAM sprayed plants at concentrations as low as 2.5 mM leading to increased mortality. In choice assays with NAM sprayed leaves aphids showed clear preference for untreated control leaves. NAM is an intermediate in the NAD salvage pathway that should accumulate in nicotinamidase (nic) mutants. LC-MS analysis showed NAM accumulates 60-fold in nic-1-1 Arabidopsis mutants as compared with Col-0. Aphid reproductive potential was significantly decreased on nic-1-1 mutant plants, resulting in a smaller colony size and arrested population development. The results support the hypothesis that dietary NAM causes behavioral changes in aphids, including altered feeding, reduced reproduction, and increased mortality. NAM is thought to bind to TRPV channels causing overstimulation of sensory neurons in the aphid feeding apparatus.

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

NAD+ biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD+ biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD+ biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD+ biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD+ biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD+ from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD+ levels and partially reversed NAD+-dependent phenotypes caused by mutation of pnc-1, which encodes a nicotinamidase required for NAD+ salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD+ homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD+ biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD+ biosynthesis creates novel possibilities for manipulating NAD+ biosynthetic pathways, which is key for the future of therapeutics.

Zinc deficiency reduces fertility in C. elegans hermaphrodites and disrupts oogenesis and meiotic progression.

Zinc is necessary for successful gametogenesis in mammals; however the role of zinc in the gonad function of non-mammalian species has not been investigated. The genetic tractability, short generation time, and hermaphroditic reproduction of the nematode C. elegans offer distinct advantages for the study of impaired gametogenesis as a result of zinc deficiency. However the phenotypic reproductive effects arising from zinc restriction have not been established in this model. We therefore examined the effect of zinc deficiency on C. elegans reproduction by exposing worms to the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethane-1,2-diamine (TPEN). Treatment began at the early larval stage and continued until reproductive senescence. TPEN treatment reduced the total number of progeny produced by C. elegans hermaphrodites compared with control subjects, with the largest difference in output observed 48h after larval stage 4. At this time-point, zinc deficient worms displayed fewer embryos in the uterus and disorganized oocyte development when observed under DIC microscopy. DAPI staining revealed impaired oogenesis and chromosome dynamics with an expanded region of pachytene stage oocytes extending into the proximal arm of the gonad. This phenotype was not seen in control or zinc-rescue subjects. This study demonstrates that reproduction in C. elegans is sensitive to environmental perturbations in zinc, indicating that this is a good model for future studies in zinc-mediated subfertility. Aberrant oocyte development and disruption of the pachytene-diplotene transition indicate that oogenesis in particular is affected by zinc deficiency in this model.

Nicotinamide is an endogenous agonist for a C. elegans TRPV OSM-9 and OCR-4 channel.

TRPV ion channels are directly activated by sensory stimuli and participate in thermo-, mechano- and chemo-sensation. They are also hypothesized to respond to endogenous agonists that would modulate sensory responses. Here, we show that the nicotinamide (NAM) form of vitamin B3 is an agonist of a Caenorhabditis elegans TRPV channel. Using heterologous expression in Xenopus oocytes, we demonstrate that NAM is a soluble agonist for a channel consisting of the well-studied OSM-9 TRPV subunit and relatively uncharacterized OCR-4 TRPV subunit as well as the orthologous Drosophila Nan-Iav TRPV channel, and we examine stoichiometry of subunit assembly. Finally, we show that behaviours mediated by these C. elegans and Drosophila channels are responsive to NAM, suggesting conservation of activity of this soluble endogenous metabolite on TRPV activity. Our results in combination with the role of NAM in NAD+ metabolism suggest an intriguing link between metabolic regulation and TRPV channel activity.

Epidermal Growth Factor Receptor Cell Survival Signaling Requires Phosphatidylcholine Biosynthesis.

Identification of pro-cell survival signaling pathways has implications for cancer, cardiovascular, and neurodegenerative disease. We show that the Caenorhabditis elegans epidermal growth factor receptor LET-23 (LET-23 EGFR) has a prosurvival function in counteracting excitotoxicity, and we identify novel molecular players required for this prosurvival signaling. uv1 sensory cells in the C. elegans uterus undergo excitotoxic death in response to activation of the OSM-9/OCR-4 TRPV channel by the endogenous agonist nicotinamide. Activation of LET-23 EGFR can effectively prevent this excitotoxic death. We investigate the roles of signaling pathways known to act downstream of LET-23 EGFR in C. elegans and find that the LET-60 Ras/MAPK pathway, but not the IP3 receptor pathway, is required for efficient LET-23 EGFR activity in its prosurvival function. However, activation of LET-60 Ras/MAPK pathway does not appear to be sufficient to fully mimic LET-23 EGFR activity. We screen for genes that are required for EGFR prosurvival function and uncover a role for phosphatidylcholine biosynthetic enzymes in EGFR prosurvival function. Finally, we show that exogenous application of phosphatidylcholine is sufficient to prevent some deaths in this excitotoxicity model. Our work implicates regulation of lipid synthesis downstream of EGFR in cell survival and death decisions.

Acoustofluidic Rotational Manipulation of Cells and Organisms Using Oscillating Solid Structures.

A polydimethylsiloxane microchannel featuring sidewall sharp-edge structures and bare channels, and a piezoelement transducer is attached to a thin glass slide. When an external acoustic field is applied to the microchannel, the oscillation of the sharp-edge structures and the thin glass slide generate acoustic streaming flows which in turn rotate single cells and C. elegans in-plane and out-of-plane.

Rotational manipulation of single cells and organisms using acoustic waves.

The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans.

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD(+) consumers to resynthesize NAD(+), resulting in a reduction in global NAD(+) bioavailability. We manipulate NAD(+) levels to demonstrate that a minor deficit in NAD(+) availability is incompatible with a normal pace of gonad development. The NAD(+) deficit compromises NAD(+) consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD(+) biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD(+) salvage biosynthesis for the purposes of inhibiting tumor growth.

An NAD(+) biosynthetic pathway enzyme functions cell non-autonomously in C. elegans development.

BACKGROUND: Disruption of cellular metabolite levels can adversely impact development. Specifically, loss-of-function of the C. elegans NAD(+) salvage biosynthesis gene PNC-1 results in an array of developmental phenotypes. Intriguingly, PNC-1 and its functional equivalent in vertebrates are secreted, but the contributions of the extracellular enzymes are poorly understood. We sought to study the tissue-specific requirements for PNC-1 expression and to examine the role of the secreted isoform. RESULTS: A thorough analysis of PNC-1 expression did not detect expression in tissues that require PNC-1 function. Limited expression of both the secreted and intracellular PNC-1 isoforms provided function at a distance from the tissues with phenotypes. We also find that the secreted isoform contributes to in vivo PNC-1 activity. Furthermore, uv1 cell survival has the most stringent requirements in terms of PNC-1 expression pattern or level. CONCLUSIONS: Using careful promoter analysis and a restricted expression approach, we have shown that both the secreted and the intracellular PNC-1 isoforms function cell non-autonomously, and that the PNC-1a isoform is functionally relevant in vivo. Our work suggests a model where PNC-1 function is provided cell non-autonomously by a mix of intra and extracellular activity, most likely requiring NAD(+) salvage metabolite transport between tissues.

Necrosis in C. elegans.

To use Caenorhabditis elegans to study the mechanisms for initiation and execution of necrosis, the experimentalist should be familiar with the established models of necrosis in C. elegans and the genetic and molecular tools available. We present a summary of two contrasting models for studying necrosis in C. elegans and outline the methods for scoring necrosis in each. These methods are useful for the study of necrosis under other conditions in C. elegans and for comparative studies both between established and new necrosis models. We also present a list of the genetic and drug tools available for perturbing pathways known to be important for initiation or execution of necrosis and describe their use in C. elegans. Specifically, we outline methods to inhibit autophagy, to perturb calcium homeostasis, and to disrupt lysosomal function in the C. elegans system.

A postmitotic function and distinct localization mechanism for centralspindlin at a stable intercellular bridge.

Centralspindlin, a complex composed of the subunits ZEN-4 and CYK-4, recruits and regulates proteins that modulate the actin cytoskeleton to promote cleavage furrow formation and progression during cytokinesis. The ZEN-4 subunit is a kinesin that is proposed to function primarily by bundling microtubules and promoting transport of the complex to the midzone. ZEN-4 and CYK-4 are mutually dependent for localization to the midzone during cytokinesis. Once at the midzone, the CYK-4 subunit functions to recruit actin regulators and the scaffold anillin as well as to regulate RhoA and Rac via its intrinsic GAP domain, ultimately promoting actomyosin contractile ring assembly. We have revealed a distinct mechanism for centralspindlin localization and function at a stable, postmitotic intercellular bridge in the Caenorhabditis elegans gonad. Loss of zen-4 or cyk-4 function disrupts germ cell progression postmitotically. In contrast to the localization and recruitment relationships during mitosis, centralspindlin is maintained at the intercellular bridge by anillin, and CYK-4 is localized independently of ZEN-4 but not vice versa. We present evidence that centralspindlin function at the rachis bridge involves ZEN-4 action on the microtubules as opposed to the regulation of the actin cytoskeleton mediated by CYK-4 during cytokinesis.

Morphogenesis of the vulva and the vulval-uterine connection.

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus.