Perillaldehyde alleviates polyQ-induced neurodegeneration through the induction of autophagy and mitochondrial UPR in Caenorhabditis elegans.

Huntington's disease (HD) is a fatal neurodegenerative disease associated with autophagy disorder and mitochondrial dysfunction. Here, we identified therapeutic potential of perillaldehyde (PAE), a monoterpene compound obtained from Perilla frutescens (L.) Britt., in the Caenorhabditis elegans (C. elegans) model of HD, which included lifespan extension, healthspan improvement, decrease in polyglutamine (polyQ) aggregation, and preservation of mitochondrial network. Further analyses indicated that PAE was able to induce autophagy and mitochondrial unfolded protein reaction (UPRmt) activation and positively regulated expression of associated genes. In lgg-1 RNAi C. elegans or C. elegans with UPRmt-related genes knockdown, the effects of PAE treatment on polyQ aggregation or rescue polyQ-induced toxicity were attenuated, suggesting that its neuroprotective activity depended on autophagy and UPRmt. Moreover, we found that pharmacological and genetic activation of UPRmt generally protected C. elegans from polyQ-induced cytotoxicity. Finally, PAE promoted serotonin synthesis by upregulating expression of TPH-1, and serotonin synthesis and neurosecretion were required for PAE-mediated UPRmt activation and its neuroprotective activity. In conclusion, PAE is a potential therapy for polyQ-related diseases including HD, which is dependent on autophagy and cell-non-autonomous UPRmt activation.

BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases.

Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3'→5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'→5' DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.

ACS-20/FATP4 mediates the anti-ageing effect of dietary restriction in C. elegans.

Dietary restriction is an effective anti-ageing intervention across species. However, the molecular mechanisms from the metabolic aspects of view are still underexplored. Here we show ACS-20 as a key mediator of dietary restriction on healthy ageing from a genetic screen of the C. elegans acyl-CoA synthetase family. ACS-20 functions in the epidermis during development to regulate dietary restriction-induced longevity. Functional transcriptomics studies reveal that elevated expression of PTR-8/Patched is responsible for the proteostasis and lifespan defects of acs-20. Furthermore, the conserved NHR-23 nuclear receptor serves as a transcriptional repressor of ptr-8 and a key regulator of dietary restriction-induced longevity. Mechanistically, a specific region in the ptr-8 promoter plays a key role in mediating the transcription regulation and lifespan extension under dietary restriction. Altogether, these findings identify a highly conserved lipid metabolism enzyme as a key mediator of dietary restriction-induced lifespan and healthspan extension and reveal the downstream transcriptional regulation mechanisms.

Pregnane X receptor agonist nomilin extends lifespan and healthspan in preclinical models through detoxification functions.

Citrus fruit has long been considered a healthy food, but its role and detailed mechanism in lifespan extension are not clear. Here, by using the nematode C. elegans, we identified that nomilin, a bitter-taste limoloid that is enriched in citrus, significantly extended the animals' lifespan, healthspan, and toxin resistance. Further analyses indicate that this ageing inhibiting activity depended on the insulin-like pathway DAF-2/DAF-16 and nuclear hormone receptors NHR-8/DAF-12. Moreover, the human pregnane X receptor (hPXR) was identified as the mammalian counterpart of NHR-8/DAF-12 and X-ray crystallography showed that nomilin directly binds with hPXR. The hPXR mutations that prevented nomilin binding blocked the activity of nomilin both in mammalian cells and in C. elegans. Finally, dietary nomilin supplementation improved healthspan and lifespan in D-galactose- and doxorubicin-induced senescent mice as well as in male senescence accelerated mice prone 8 (SAMP8) mice, and induced a longevity gene signature similar to that of most longevity interventions in the liver of bile-duct-ligation male mice. Taken together, we identified that nomilin may extend lifespan and healthspan in animals via the activation of PXR mediated detoxification functions.

A sphingolipid-mTORC1 nutrient-sensing pathway regulates animal development by an intestinal peroxisome relocation-based gut-brain crosstalk.

The mTOR-dependent nutrient-sensing and response machinery is the central hub for animals to regulate their cellular and developmental programs. However, equivalently pivotal nutrient and metabolite signals upstream of mTOR and developmental-regulatory signals downstream of mTOR are not clear, especially at the organism level. We previously showed glucosylceramide (GlcCer) acts as a critical nutrient and metabolite signal for overall amino acid levels to promote development by activating the intestinal mTORC1 signaling pathway. Here, through a large-scale genetic screen, we find that the intestinal peroxisome is critical for antagonizing the GlcCer-mTORC1-mediated nutrient signal. Mechanistically, GlcCer deficiency, inactive mTORC1, or prolonged starvation relocates intestinal peroxisomes closer to the apical region in a kinesin- and microtubule-dependent manner. Those apical accumulated peroxisomes further release peroxisomal-ß-oxidation-derived glycolipid hormones that target chemosensory neurons and downstream nuclear hormone receptor DAF-12 to arrest the animal development. Our data illustrate a sophisticated gut-brain axis that predominantly orchestrates nutrient-sensing-dependent development in animals.

Tangeretin promotes lifespan associated with insulin/insulin-like growth factor-1 signaling pathway and heat resistance in Caenorhabditis elegans.

Tangeretin is a polymethoxylated flavonoid naturally occurred in citrus fruits with many pharmacological activities, such as anti-inflammatory, antiproliferative, and neuroprotective properties. A previous study reported that tangeretin-enriched orange extract could prolong the lifespan in Caenorhabditis elegans. However, the antiaging effect of tangeretin remains uncertain. In this study, we used the model organism C. elegans to conduct a lifespan test, observed the aging-related functional changes of nematodes, the fluorescence changes of stress-related proteins (DAF-16 and HSP-16.2) and its response to stress assay, and monitored the effect of tangeretin on the mRNA expression levels. The results showed that tangeretin supplementation (30 and 100 µM) extended the mean lifespan, slowed aging-related functional declines, and increased the resistance against heat-shock stress. Furthermore, tangeretin upregulated the mRNA expression of daf-16, hsp-16.2, and hsp-16.49, promoted the nuclear localization of DAF-16, and enhanced the fluorescence intensity of HSP-16.2, while it had no effect on the lifespan of daf-2, age-1, and daf-16 mutants. The current findings suggest that tangeretin can significantly extend the lifespan and enhance heat stress tolerance in an insulin/insulin-like growth factor signaling dependent manner.

Monomethyl branched-chain fatty acid mediates amino acid sensing upstream of mTORC1.

Animals have developed various nutrient-sensing mechanisms for survival under fluctuating environmental conditions. Although extensive cell-culture-based analyses have identified diverse mediators of amino acid sensing upstream of mTOR, studies using animal models to examine intestine-initiated amino acid sensing mechanisms under specific physiological conditions are lacking. Here, we developed a Caenorhabditis elegans model to examine the impact of amino acid deficiency on development. We discovered a leucine-derived monomethyl branched-chain fatty acid and its downstream metabolite, glycosphingolipid, which critically mediates the overall amino acid sensing by intestinal and neuronal mTORC1, which in turn regulates postembryonic development at least partly by controlling protein translation and ribosomal biogenesis. Additional data suggest that a similar mechanism may operate in mammals. This study uncovers an amino-acid-sensing mechanism mediated by a lipid biosynthesis pathway.

Saturated very long chain fatty acid configures glycosphingolipid for lysosome homeostasis in long-lived C. elegans.

The contents of numerous membrane lipids change upon ageing. However, it is unknown whether and how any of these changes are causally linked to lifespan regulation. Acyl chains contribute to the functional specificity of membrane lipids. In this study, working with C. elegans, we identified an acyl chain-specific sphingolipid, C22 glucosylceramide, as a longevity metabolite. Germline deficiency, a conserved lifespan-extending paradigm, induces somatic expression of the fatty acid elongase ELO-3, and behenic acid (22:0) generated by ELO-3 is incorporated into glucosylceramide for lifespan regulation. Mechanistically, C22 glucosylceramide is required for the membrane localization of clathrin, a protein that regulates membrane budding. The reduction in C22 glucosylceramide impairs the clathrin-dependent autophagic lysosome reformation, which subsequently leads to TOR activation and longevity suppression. These findings reveal a mechanistic link between membrane lipids and ageing and suggest a model of lifespan regulation by fatty acid-mediated membrane configuration.

A Potent Anti-SpuE Antibody Allosterically Inhibits Type III Secretion System and Attenuates Virulence of Pseudomonas Aeruginosa.

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria.

A Tet/Q Hybrid System for Robust and Versatile Control of Transgene Expression in C. elegans.

Binary gene regulatory tools such as the Tetracycline (Tet)-controlled transcription system have revolutionized genetic research in multiple organisms, but their applications to the worm remain very limited. Here we report that the canonical Tet system is largely inactive in the worm but can be adapted for the worm by introducing multiple modifications, a crucial one being the use of the transcription activation domain from the fungal Q binary system. The resultant Tet/Q hybrid system proves more robust and flexible than either of its precursors, enabling elaborate modes of transgene manipulation previously hard to achieve in the worm, including inducible intersectional regulation and, in combination with the Q system, independent control of distinct transgenes within the same cells. Furthermore, we demonstrated, as an example of its applications, that the hybrid system can tightly and efficiently control Cre expression. This study establishes Tet/Q as a premier binary system for worm genetic research.

Current advances in the functional studies of fatty acids and fatty acid-derived lipids in C. elegans.

Fatty acids and fatty acid-derived lipids (FAs/FADLs) play essential roles in many living organisms, including contributions to membrane structure and signaling transduction. Aberrant metabolism of FAs/FADLs often causes diseases and health problems. However, the detailed mechanistic studies of specific FAs/FADLs in vivo are limited. C. elegans has been an effective model system for FA/ FADL studies due to its powerful genetics and conserved lipid biosynthetic pathways. The recently developed high-throughput analytic tools also enable sophisticated profiling of lipids molecules in C. elegans, which is critical for understanding their specific functions. Here we review a subset of current advances in FA/FADL functional studies in C. elegans.

Intestinal apical polarity mediates regulation of TORC1 by glucosylceramide in C. elegans.

TORC1 (target of rapamycin complex 1) plays a central role in regulating growth, development, and behavior in response to nutrient cues. We previously showed that leucine-derived monomethyl branched-chain fatty acids (mmBCFAs) and derived glucosylceramide promote intestinal TORC1 activity for post-embryonic development and foraging behavior in Caenorhabditis elegans. Here we show that clathrin/adaptor protein 1 (AP-1)-dependent intestinal apical membrane polarity and polarity-dependent localization of the vacuolar-type H(+)-ATPase (V-ATPase) mediate the impact of the lipid pathway on intestinal TORC1 activation. Moreover, NPRL-3 represses mmBCFA-dependent intestinal TORC1 activity at least partly by regulating apical membrane polarity. Our results provide new insights into TORC1 regulation by lipids and membrane polarity in a specific tissue.

A Lipid-TORC1 Pathway Promotes Neuronal Development and Foraging Behavior under Both Fed and Fasted Conditions in C. elegans.

Food deprivation suppresses animal growth and development but spares the systems essential for foraging. The mechanisms underlying this selective development, and potential roles of lipids in it, are unclear. When C. elegans hatch in a food-free environment, postembryonic growth and development stall, but sensory neuron differentiation and neuronal development required for food responses continue. Here, we show that monomethyl branched-chain fatty acids (mmBCFAs) and their derivative, d17iso-glucosylceramide, function in the intestine to promote foraging behavior and sensory neuron maturation through both TORC1-dependent and -independent mechanisms. We show that mmBCFAs impact the expression of a subset of genes, including ceh-36/Hox, which we show to play a key role in mediating the regulation of the neuronal functions by this lipid pathway. This study uncovers that a lipid pathway promotes neuronal functions involved in foraging under both fed and fasting conditions and adds critical insight into the physiological functions of TORC1.

Exploring developmental and physiological functions of fatty acid and lipid variants through worm and fly genetics.

Lipids are more than biomolecules for energy storage and membrane structure. With ample structural variation, lipids critically participate in nearly all aspects of cellular function. Lipid homeostasis and metabolism are closely related to major human diseases and health problems. However, lipid functional studies have been significantly underdeveloped, partly because of the difficulty in applying genetics and common molecular approaches to tackle the complexity associated with lipid biosynthesis, metabolism, and function. In the past decade, a number of laboratories began to analyze the roles of lipid metabolism in development and other physiological functions using animal models and combining genetics, genomics, and biochemical approaches. These pioneering efforts have not only provided valuable insights regarding lipid functions in vivo but have also established feasible methodology for future studies. Here, we review a subset of these studies using Caenorhabditis elegans and Drosophila melanogaster.

A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans.

Regulation of animal development in response to nutritional cues is an intensely studied problem related to disease and aging. While extensive studies indicated roles of the Target of Rapamycin (TOR) in sensing certain nutrients for controlling growth and metabolism, the roles of fatty acids and lipids in TOR-involved nutrient/food responses are obscure. Caenorhabditis elegans halts postembryonic growth and development shortly after hatching in response to monomethyl branched-chain fatty acid (mmBCFA) deficiency. Here, we report that an mmBCFA-derived sphingolipid, d17iso-glucosylceramide, is a critical metabolite in regulating growth and development. Further analysis indicated that this lipid function is mediated by TORC1 and antagonized by the NPRL-2/3 complex in the intestine. Strikingly, the essential lipid function is bypassed by activating TORC1 or inhibiting NPRL-2/3. Our findings uncover a novel lipid-TORC1 signaling pathway that coordinates nutrient and metabolic status with growth and development, advancing our understanding of the physiological roles of mmBCFAs, ceramides, and TOR. DOI:http://dx.doi.org/10.7554/eLife.00429.001.

Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

Cloning and characterization of a gene encoding glutathione-regulated potassium-efflux system protein KefKL from the endosymbiont Wolbachia.

The maternally inherited intracellular symbiont Wolbachia is well known for inducing a variety of reproductive and developmental abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, feminization of genetic males and male killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these abnormalities have not yet been determined. In our study, representational difference analysis (RDA) was used to analyze the genomic difference between different Wolbachia strains. A gene encoding glutathione-regulated potassium-efflux system protein KefKL from Wolbachia in Drosophila simulans Riverside (w Ri) was isolated. The homologous genes from Wolbachia in Drosophila melanogaster yw67c23 (wMel) and Wolbachia in Drosophila melanogaster CantonS (wMelCS) were also cloned and sequenced. Sequence analysis showed that these deduced amino acid sequences contained two important motifs: Na+/H+ antiportor and NAD binding domain, which shared conserved sequences among different strains. Considering the crucial function of KefKL for ionic homeostasis, this gene might play an important role in Wolbachia physiology. Further study indicated that there was no homologue detected from Wolbachia in Drosophila simulans DSW/Mau (wMa) and Wolbachia in Drosophila simulans Noumea (wNo). Whether Wolbachia contained KefKL (or the homologous gene) was consistent with the phylogenetic studies using wsp sequences, which showed that wMa and wNo were grouped into one branch, while w Ri, wMel and wMelCS were more closely related.