An efficient behavioral screening platform classifies natural products and other chemical cues according to their chemosensory valence in C. elegans.

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction, and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multi-well plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals, but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

Combinatorial glucose, nicotinic acid and N-acetylcysteine therapy has synergistic effect in preclinical C. elegans and zebrafish models of mitochondrial complex I disease.

Mitochondrial respiratory chain disorders are empirically managed with variable antioxidant, cofactor and vitamin 'cocktails'. However, clinical trial validated and approved compounds, or doses, do not exist for any single or combinatorial mitochondrial disease therapy. Here, we sought to pre-clinically evaluate whether rationally designed mitochondrial medicine combinatorial regimens might synergistically improve survival, health and physiology in translational animal models of respiratory chain complex I disease. Having previously demonstrated that gas-1(fc21) complex I subunit ndufs2-/-C. elegans have short lifespan that can be significantly rescued with 17 different metabolic modifiers, signaling modifiers or antioxidants, here we evaluated 11 random combinations of these three treatment classes on gas-1(fc21) lifespan. Synergistic rescue occurred only with glucose, nicotinic acid and N-acetylcysteine (Glu + NA + NAC), yielding improved mitochondrial membrane potential that reflects integrated respiratory chain function, without exacerbating oxidative stress, and while reducing mitochondrial stress (UPRmt) and improving intermediary metabolic disruptions at the levels of the transcriptome, steady-state metabolites and intermediary metabolic flux. Equimolar Glu + NA + NAC dosing in a zebrafish vertebrate model of rotenone-based complex I inhibition synergistically rescued larval activity, brain death, lactate, ATP and glutathione levels. Overall, these data provide objective preclinical evidence in two evolutionary-divergent animal models of mitochondrial complex I disease to demonstrate that combinatorial Glu + NA + NAC therapy significantly improved animal resiliency, even in the face of stressors that cause severe metabolic deficiency, thereby preventing acute neurologic and biochemical decompensation. Clinical trials are warranted to evaluate the efficacy of this lead combinatorial therapy regimen to improve resiliency and health outcomes in human subjects with mitochondrial disease.

Juniperonic Acid Biosynthesis is Essential in Caenorhabditis Elegans Lacking Δ6 Desaturase (fat-3) and Generates New ω-3 Endocannabinoids.

In eukaryotes, the C20:4 polyunsaturated fatty acid arachidonic acid (AA) plays important roles as a phospholipid component, signaling molecule and precursor of the endocannabinoid-prostanoid axis. Accordingly, the absence of AA causes detrimental effects. Here, compensatory mechanisms involved in AA deficiency in Caenorhabditis elegans were investigated. We show that the ω-3 C20:4 polyunsaturated fatty acid juniperonic acid (JuA) is generated in the C. elegansfat-3(wa22) mutant, which lacks Δ6 desaturase activity and cannot generate AA and ω-3 AA. JuA partially rescued the loss of function of AA in growth and development. Additionally, we observed that supplementation of AA and ω-3 AA modulates lifespan of fat-3(wa22) mutants. We described a feasible biosynthetic pathway that leads to the generation of JuA from α-linoleic acid (ALA) via elongases ELO-1/2 and Δ5 desaturase which is rate-limiting. Employing liquid chromatography mass spectrometry (LC-MS/MS), we identified endocannabinoid-like ethanolamine and glycerol derivatives of JuA and ω-3 AA. Like classical endocannabinoids, these lipids exhibited binding interactions with NPR-32, a G protein coupled receptor (GPCR) shown to act as endocannabinoid receptor in C. elegans. Our study suggests that the eicosatetraenoic acids AA, ω-3 AA and JuA share similar biological functions. This biosynthetic plasticity of eicosatetraenoic acids observed in C. elegans uncovers a possible biological role of JuA and associated ω-3 endocannabinoids in Δ6 desaturase deficiencies, highlighting the importance of ALA.

Pre-clinical evaluation of cysteamine bitartrate as a therapeutic agent for mitochondrial respiratory chain disease.

Cysteamine bitartrate is a US Food and Drug Administration-approved therapy for nephropathic cystinosis also postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionarily distinct species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in Caenorhabditis elegans gas-1(fc21) RC complex I (NDUFS2-/-) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 µm concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) Danio rerio vertebrate zebrafish disease models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways was greater in RC disease human cells than in C. elegans, with similar rescue in both models of downregulated ribosomal and proteasomal pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.

High-throughput BioSorter quantification of relative mitochondrial content and membrane potential in living Caenorhabditis elegans.

Mitochondrial respiratory chain disease is caused by a wide range of individually rare genetic disorders that impair cellular energy metabolism. While fluorescence microscopy analysis of nematodes fed MitoTracker Green (MTG) and tetramethylrhodamine ethyl ester (TMRE) can reliably quantify relative mitochondrial density and membrane potential, respectively, in C. elegans models of mitochondrial dysfunction, it is a tedious process with limitations in the number and age of animals that can be studied. A novel, large particle, flow cytometry-based method reported here accelerates and automates the relative quantitation of mitochondrial physiology in nematode populations. Relative fluorescence profiles of nematode populations co-labeled with MTG and TMRE were obtained and analyzed by BioSorter (Union Biometrica). Variables tested included genetic mutation (wild-type N2 Bristol versus nuclear-encoded respiratory chain complex I mutant gas-1(fc21) worms), animal age (day 1 versus day 4 adults), classical respiratory chain inhibitor and uncoupler effects (oligomycin, FCCP), and pharmacologic therapy duration (24h versus 96h treatments with glucose or nicotinic acid). A custom MATLAB script, which can be run on any computer with MATLAB runtime, was written to automatically quantify and analyze results in large animal populations. BioSorter analysis independently validated relative MTG and TMRE changes that we had previously performed by fluorescence microscopy in a variety of experimental conditions, with notably greater animal population sizes and substantially reduced experimental time. Older, fragile animal populations that are difficult to study by microscopy approaches were readily amenable to analysis with the BioSorter method. Overall, this high-throughput method enables efficient relative quantitation of in vivo mitochondrial physiology over time in a living animal in response to gene mutations and candidate therapies, which can be used to accelerate the translation of basic research into optimization of clinical therapies for mitochondrial disease.

Aberrant autolysosomal regulation is linked to the induction of embryonic senescence: differential roles of Beclin 1 and p53 in vertebrate Spns1 deficiency.

Spinster (Spin) in Drosophila or Spinster homolog 1 (Spns1) in vertebrates is a putative lysosomal H+-carbohydrate transporter, which functions at a late stage of autophagy. The Spin/Spns1 defect induces aberrant autolysosome formation that leads to embryonic senescence and accelerated aging symptoms, but little is known about the mechanisms leading to the pathogenesis in vivo. Beclin 1 and p53 are two pivotal tumor suppressors that are critically involved in the autophagic process and its regulation. Using zebrafish as a genetic model, we show that Beclin 1 suppression ameliorates Spns1 loss-mediated senescence as well as autophagic impairment, whereas unexpectedly p53 deficit exacerbates both of these characteristics. We demonstrate that 'basal p53' activity plays a certain protective role(s) against the Spns1 defect-induced senescence via suppressing autophagy, lysosomal biogenesis, and subsequent autolysosomal formation and maturation, and that p53 loss can counteract the effect of Beclin 1 suppression to rescue the Spns1 defect. By contrast, in response to DNA damage, 'activated p53' showed an apparent enhancement of the Spns1-deficient phenotype, by inducing both autophagy and apoptosis. Moreover, we found that a chemical and genetic blockage of lysosomal acidification and biogenesis mediated by the vacuolar-type H+-ATPase, as well as of subsequent autophagosome-lysosome fusion, prevents the appearance of the hallmarks caused by the Spns1 deficiency, irrespective of the basal p53 state. Thus, these results provide evidence that Spns1 operates during autophagy and senescence differentially with Beclin 1 and p53.

Supplement timing of cranberry extract plays a key role in promoting Caenorhabditis elegans healthspan.

Consumption of nutraceuticals is a major and potent dietary intervention for delaying aging. As the timing of administration is critical for the efficacy of bioactive compounds in medicine, the effectiveness of nutraceuticals may also be dramatically affected by the timing of supplementation. Cranberry exact (CBE), rich in polyphenols, is consumed as a nutraceutical, and possesses anti-aging properties. Here, we examined the influence of timing on the beneficial effects of CBE supplementation in C. elegans. The prolongevity effect of CBE in different aged worms, young adults, middle-age adults, and aged adults, was determined. Early-start intervention with CBE prolonged the remaining lifespan of worms of different ages more robustly than late-start intervention. The effectiveness of CBE on stress responses and physiological behaviors in different aged worms was also investigated. The early-start intervention prominently promoted motility and resistance to heat shocks and V. cholera infection, especially in aged worms. Together, these findings suggest that the timing of CBE supplementation critically influences its beneficial effects on C. elegans lifespan and healthspan. It is of interest to further investigate whether the similar results would occur in humans.

Influence of planktonic and sessile Listeria monocytogenes on Caenorhabditis elegans.

Listeria monocytogenes is the etiologic agent of listeriosis, a food-borne disease affecting humans and a variety of animals. In order to combat this pathogen, it is crucial to have an understanding of its natural interplay with the environment. For this reason, the free soil nematode Caenorhabditis elegans was focused upon because of its shared natural habitat with Listeria and its potential as a model organism for Listeria pathogenesis. Previous studies have generated some contradictory results on Listeria's ability to kill C. elegans, making additional interaction studies such as this more attractive. In our study, we carried out a series of killing assays in a systematic manner using different Listeria strains under different growth conditions. In addition to studying the effects of planktonic cells, we examined the interaction between C. elegans and sessile listerial cells. Our findings suggest that, rather than causing infection and death, L. monocytogenes may extend the life span of C. elegans. This indicates that Listeria is not pathogenic to C. elegans. We also found that C. elegans can feed and ingest sessile cells, as well as carry the pathogen in its gut, implying that C. elegans could be a vehicle for L. monocytogenes spread in the environment.

The longevity effect of cranberry extract in Caenorhabditis elegans is modulated by daf-16 and osr-1.

Nutraceuticals are known to have numerous health and disease preventing properties. Recent studies suggest that extracts containing cranberry may have anti-aging benefits. However, little is known about whether and how cranberry by itself promotes longevity and healthspan in any organism. Here we examined the effect of a cranberry only extract on lifespan and healthspan in Caenorhabditis elegans. Supplementation of the diet with cranberry extract (CBE) increased the lifespan in C. elegans in a concentration-dependent manner. Cranberry also increased tolerance of C. elegans to heat shock, but not to oxidative stress or ultraviolet irradiation. In addition, we tested the effect of cranberry on brood size and motility and found that cranberry did not influence these behaviors. Our mechanistic studies indicated that lifespan extension induced by CBE requires the insulin/IGF signaling pathway and DAF-16. We also found that cranberry promotes longevity through osmotic stress resistant-1 (OSR-1) and one of its downstream effectors, UNC-43, but not through SEK-1, a component of the p38 MAP kinase pathway. However, SIR-2.1 and JNK signaling pathways are not required for cranberry to promote longevity. Our findings suggest that cranberry supplementation confers increased longevity and stress resistance in C. elegans through pathways modulated by daf-16 and osr-1. This study reveals the anti-aging property of widely consumed cranberry and elucidates the underpinning mechanisms.

Nutraceutical interventions for promoting healthy aging in invertebrate models.

Aging is a complex and inevitable biological process that is associated with numerous chronically debilitating health effects. Development of effective interventions for promoting healthy aging is an active but challenging area of research. Mechanistic studies in various model organisms, noticeably two invertebrates, Caenorhabditis elegans and Drosophila melanogaster, have identified many genes and pathways as well as dietary interventions that modulate lifespan and healthspan. These studies have shed light on some of the mechanisms involved in aging processes and provide valuable guidance for developing efficacious aging interventions. Nutraceuticals made from various plants contain a significant amount of phytochemicals with diverse biological activities. Phytochemicals can modulate many signaling pathways that exert numerous health benefits, such as reducing cancer incidence and inflammation, and promoting healthy aging. In this paper, we outline the current progress in aging intervention studies using nutraceuticals from an evolutionary perspective in invertebrate models.