Patrick A et al. (1998) East Coast Worm Meeting "Progress with the aph's"

Goutte C, Patrick A, Haduong J

[East Coast Worm Meeting, 1998]

We have been pursuing the enigmatic function of two genes, aph-1 and aph-2, which appear to be involved in embryonic cell-cell interactions that are mediated by glp-1. These two genes were isolated in the Priess lab based on mutant phenotypes that look indistinguishable from the embryonic mutant phenotypes caused by glp-1 mutations. The aph-2 gene encodes a novel protein whose only distinguishing feature is a signal sequence at the amino terminus. Based on immunolocalization, the APH-2 protein is present in embryos and localizes to the peripheral cell membranes of all blastomeres in the early embryo. Using APH-2 antibodies and western analysis we are able to detect the APH-2 protein in crude embryonic extracts and in solubilized gravid worms. We have used this assay to address the nature of the membrane-localization that is observed for APH-2. Our cell fractionation data suggest that APH-2 behaves as an integral membrane protein. We have followed APH-2 protein expression and size during development, and have found that APH-2 can be detected not only in embryos, but also in larvae and sterile adults, suggesting that APH-2 is present, and possibly active, during post-embryonic development. Efforts by our lab and the Priess lab are still underway to clone the aph-1 gene; its identity remains unknown. Two approaches have provided us with a hint as to what the specific role of aph-1 might be: immunostaining of aph-1 embryos and western analysis of aph-1 mutant worms both demonstrate that levels of APH-2 protein are severely reduced in aph-1 mutants. Thus, one simple interpretation would be that the aph-1 mutant phenotype results from a loss or instability of APH-2 protein. In order to identify what gene products might interact with the aph-1 or aph-2 products, we have used a weak aph-1 mutation to screen for extragenic suppressors. We have identified six mutations that rescue the embryonic lethality caused by this aph-1 mutation. Analysis of these suppressors is currently underway.

Patrick Narbonne et al. (2008) European Worm Meeting "Hypodermal LKB1/AMPK signalling is critical to dauer longevity"

Richard Roy, Patrick Narbonne

[European Worm Meeting, 2008]

The alteration of genes regulating dauer formation often has a dramatic. effect on adult lifespan. Dauer is a developmentally-arrested, non-feeding. stage that is long-lived and very resistant to harsh conditions. It is. therefore proposed that what confers adult lifespan extension in dauer-. constitutive mutants is the inappropriate expression of a plethora of dauer-. specific genes during adulthood. If the alteration of signalling downstream. of the same genes during dauer is responsible for the extreme longevity of. this stage, dauer-constitutive mutations should not extend dauer lifespan.. We therefore compared the lifespan of naturally-induced (crowding +. starvation) N2 dauers to that of daf-2 dauers and indeed found that reduced. insulin signalling does not extend dauer lifespan.. Mutations in the insulin pathway largely require aak-2 (an AMPK catalytic. subunit) activity to extend adult lifespan. Interestingly, aak-2 mutants. form morphologically normal dauer larvae, which however have a very short. lifespan. We observed similar effects in strd-1; par-4 mutants (AMPK. activators), as well as in aakb-1; aakb-2 mutants (AMPK regulatory. subunits), but not in aak-1 (the second AMPK catalytic subunit) animals. We. therefore believe that an aak-2-specific LKB1/AMPK cascade is necessary for. the extreme dauer longevity, likely through potentiating the effects of the. naturally reduced level of insulin signalling during this stage. To. localize the defect in LKB1/AMPK deficient dauer larvae, we attempted to. rescue the dauer lifespan of aak-2 mutants using tissue-specific promoters. that drive a wild type aak-2 cDNA. We tested intestinal (elt-2), neuronal. (unc-119), muscle (unc-54) and hypodermal (dpy-7) specific promoters to. restore aak-2 function in these tissues. Only hypodermal expression of the. aak-2 cDNA significantly rescued dauer lifespan. A major function of the. hypodermis is to act in energy storage and we found that aak-2 mutant. dauers rapidly deplete their stored lipids while these are maintained in. wild-type dauers. We are currently testing potential AMPK targets for their. ability to suppress energy storage defects and lethality of aak-2 mutant. dauers.

Gonzales, Patrick K. (2013) International Worm Meeting "Transcriptome analysis of ALS-associated Mutants fust-1 and tdp-1."

Gonzales, Patrick K.

[International Worm Meeting, 2013]

ALS is an adult-onset neurodegenerative disorder characterized by premature degeneration of upper and lower motor neurons eventually leading to fatal paralysis. Approximately 10% of disease instances are familial, with several dominant mutations identified. TDP43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma), are two RNA binding proteins whose mutations are present in a subset of familial ALS cases. Interestingly, the two proteins share structural and functional similarities. Both contain RNA recognition motifs, have glycine rich regions, and both are associated with multiple steps of RNA processing including transcription, RNA splicing, RNA transport and translation. ALS-associated mutations in TDP-43 and FUS suggest defects in RNA processing could result in ALS. In order to understand which RNA processing defects are central to the disease process, it is important to determine which RNA transcripts depend on both proteins for correct abundance and processing. To answer this question we analyzed the transcriptome of strains deleted for the C. elegans homologs of TDP-43 and Fus, tdp-1 and fust-1. Deletion alleles, tdp-1(ok803) and fust-1(tm4439), are viable and display only mild phenotypes. Previous high throughput sequencing analysis from our lab showed that tdp-1(ok803) preferentially binds transcripts with potential dsRNA structure and limits this structure, particularly within introns. In this study we asked if fust-1 mutants have a similar expression profile as the tdp-1 mutant. We used high throughput sequencing of N2, tdp-1(ok803), and fust-1(tm4439) to characterize altered transcripts in both mutants from total RNA and from RNA captured using a dsRNA specific antibody (J2). The results of this comparison are reported.

Patrick Laurent et al. (2009) European Worm Neurobiology Meeting "The complex regulations of neuropeptide secretion in neurons."

Queelim Chng, Mario De Bono, Patrick Laurent

[European Worm Neurobiology Meeting, 2009]

Acting as hormones, neurotransmitter and/or modulator, neuropeptides maintain physiological homeostasis and influence behavioural patterns. When overexpressed, the neuropeptide FLP-18 causes a strong uncoordinated behaviour in C. elegans. With the aim to find new genes affecting peptidergic secretion, we screened for mutations suppressing or increasing oex-flp-18 phenotype. Many mutants suppress oex-flp-18 phenotype including the processing enzymes egl-3 and egl-21. Other mutants increase oex-flp-18 phenotype including nep-1, a degradating enzyme for neuropeptides. Neuropeptides, as well as bioamines, are packed into Dense Core Vesicles (DCV). This organelle biogenesis, trafficking and exocytosis in neurons is still poorly understood. A secondary assay, using a GFP-tagged neuropeptide expressed in the oxygen-sensing neurons, was designed to analyse DCV distribution and neuropeptide release in neurons for the active and inactive states. This assay disqualified several mutants from the primary screen. The distribution of DCV within neurons revealed mutants affecting cell body content, axonal content or synaptic localisation. Coelomocyte accumulation is another tool to quantify the releasing activity. We can cluster the phenotypical characteristics of several mutants together. One cluster contains genes reducing neuronal activity, like tax-4 mutant. Another cluster contains unc-64, unc-31, ric-4 and unc-18 known to affect the .docking. step (when DCV contact the plasma membrane before the fusion step). Other phenotypic clusters are proposed to contain genes affecting DCV biogenesis, trafficking, synaptic trapping, docking and fusion. One cluster showing less GFP-tagged neuropeptide in the cell body, the axon, and the coelomocytes is proposed to affect DCV biogenesis. It contains rab-2, ric-19 and some others. RIC-19 is an effecter of the small GTPase RAB-2. It is an atypical BAR-domain protein; a family of domain able to sense/bend the membranes curvature. The cell body content for IDA-1:GFP, a DCV membrane marker, is increased in rab-2 and ric-19 mutants. This observation and the colocalisation studies support a role for these genes posterior to the Trans-Golgi .sorting. step, but before export to axons, probably during a maturation step of the DCV. Interestingly, neuronal activity was observed to modify DCV exocytosis but also DCV localisation suggesting DCV trafficking to be affected by neuronal activity, another cluster of genes is proposed to affect this step. The fundamental role of neuropeptides and bioamines in physiology suggested a fine regulation of DCV secretion. Several of our results underlines new and complex regulations of DCV cell biology in neurons that only partially overlap with what is known for DCV in endocrine cells or for synaptic vesicles in neurons.

O'Hern, Patrick J. et al. (2013) International Worm Meeting "A role for miRNA machinery at the neuromuscular junction?"

Hart, Anne, O'Hern, Patrick J.

[International Worm Meeting, 2013]

Synaptic signaling is strikingly similar across diverse species and studies of this process in C. elegans have broad significance. Ion channels, receptors, signaling molecules, and neurotransmitters coordinate rapid, high fidelity, trans-synaptic communication. Classically, Messenger RNAs (mRNAs) encoding synaptic proteins are likely regulated by microRNAs (miRNAs). Classically, miRNAs are a central part of the RNA-induced silencing complex (RISC) and bind to the complementary sequence of target mRNAs. Proteins in the miRNA pathway are also well conserved across species. Although individual miRNAs have been identified that regulate the NMJ, it is unknown whether perturbation of miRNA machinery proteins influences NMJ function. To study the function of miRNA pathway proteins at the neuromuscular junction, we used the drug aldicarb. Aldicarb inhibits synaptic acetycholinesterase and exposure leads to paralysis. The RISC components AIN-1, AIN-2, ALG-1, ALG-2, and CGH-1 as well as DCR-1, a well-characterized miRNA processing protein were examined. Loss-of-function of AIN-1;AIN-2 results in aldicarb hypersensitivity (Hic), whereas AIN-1, RDE-1, or CGH-1 loss-of-function resulted in aldicarb resistance (Ric). Surprisingly, single mutant strains for alg-1, alg-2, ain-2, or dcr-1 were indistinguishable from wild-type animals. While these results seem contradictory, they suggest broad and diverse roles for miRNA machinery at the NMJ. In the future, we plan to identify specific miRNAs to help explain these opposing phenotypes at the NMJ.

Patrick Dessi et al. (2003) International Worm Meeting "Expression pattern of the Exd/Pbx homologue ceh-20 in C.elegans"

Patrick Dessi, Thomas Burglin, Hiroshi KAGOSHIMA

[International Worm Meeting, 2003]

Homeodomain transcription factors called homeotic selector (HOX) proteins control the patterning process in all animal embryos. HOX proteins tend to bind target DNA with little specificity. Additional specificity is conferred by co-factor homeodomain proteins. Members of the TALE class of atypical homeodomain proteins act as HOX co-factors. The TALE members are subdivided into two families, PBC and MEIS, on the basis of their N terminal conserved domains. C.elegans contains the vertebrate pbx1 and Drosophila Extradenticle (Exd) orthologue, ceh-20, as well as a second PBC class homeobox gene, ceh-40. Previously we had examined the expression pattern using antibodies that showed ventral nerve cord expression. In addition, we saw weak expression in hypodermal and muscle nuclei. To confirm the expression and to look at its evolution dynamically during development, we have constructed ceh-20::gfp reporter constructs. We found widespread gfp expression in the embryo that progressively restricted to specific cells seen with the antibody. Expression in hypodermis was however clearer now with the GFP construct. We are currently performing 2-channel 4D microscopy to analyse the expression pattern during embryogenesis.

Patrick Narbonne et al. (2007) International Worm Meeting "The LKB1/AMPK cascade functions in the hypodermis to regulate dauer lifespan."

Patrick Narbonne, Richard Roy

[International Worm Meeting, 2007]

The genetic basis underlying C. elegans adult lifespan regulation is emerging from studies carried out in several laboratories. It appears that, among others, genes regulating dauer formation such as those involved in insulin signaling, have a dramatic effect on adult lifespan. Dauer is a developmentally-arrested, non-feeding stage that is long-lived and very resistant to harsh conditions. It is proposed that what confers lifespan extension in insulin signaling pathway mutants results from the inappropriate expression of a plethora of dauer-specific genes that are regulated by DAF-16, the downstream transcription factor of the insulin signaling cascade, during adulthood. Although largely accepted, the theory that daf-16 indeed mediates the extreme longevity of the dauer larva remains difficult to test because daf-16 mutants are dauer defective. In addition to daf-16 activity, mutations in the insulin pathway largely require AMPK activity to extend adult lifespan. Interestingly, aak-2 (an AMPK catalytic subunit) mutants form morphologically normal dauer larvae, which however have a very short lifespan. While aak-1 (the second AMPK catalytic subunit) null mutants do not show such a phenotype, we find that double mutant combinations between par-4/LKB1 (an AMPK activator) and strd-1 (an LKB1 co-factor), or between both of the AMPK beta regulatory subunit isoforms (aakb-1 and aakb-2) also severely reduce dauer lifespan. We therefore believe that an aak-2 specific LKB1/AMPK cascade is necessary for the extreme dauer longevity, likely through potentiating the effects of the naturally reduced level of insulin signaling during this stage. To localize the defect in LKB1/AMPK deficient dauer larvae, we have attempted to rescue the dauer lifespan of aak-2 mutants using tissue-specific promoters that drive a wild type aak-2 cDNA. We used the sur-5 promoter as a positive control for ubiquitous somatic aak-2 cDNA expression, which largely rescued dauer lifespan. We used intestinal (elt-2), neuronal (unc-119), muscle (unc-54) and hypodermal (dpy-7) specific promoters to restore aak-2 function in these tissues. Only hypodermal expression of the aak-2 cDNA significantly rescued dauer lifespan, albeit only partially. Since the co-injection of these four constructs does not enhance the rescue above that observed with the injection of the hypodermal construct alone, we believe that aak-2 must be required in additional tissues/cells. While we are confident that the somatic gonad or germline are not necessary for dauer lifespan since their ablation does not phenocopy aak-2 mutants, we are currently testing alternative tissues/cells, such as the excretory cell and coelomocytes for potential complementary rescue.

Phillips, Patrick et al. (2015) International Worm Meeting "Caenorhabditis Intervention Testing Program: Gateway to the Fountain of Youth."

Guo, Max, Lithgow, Gordon, Driscoll, Monica, Phillips, Patrick, Harke, Jailynn

[International Worm Meeting, 2015]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institution effort, sponsored by the National Institute of Aging, designed to screen bioactive compounds for their ability to extend lifespan and enhance health using nematodes as a model system for the potential effects of background genetic variation. The Intervention Testing Program (ITP), began assaying pharmacological interventions for lifespan extension in mice in the early 2000s [1]. However, with a median lifespan over two years, this project has decreased capacity to test multiple compounds in a robust manner [2]. The CITP capitalizes on the short lifespan of hermaphroditic C. elegans, C. briggsae and C. tropicalis. Selected natural isolates represent a biologically and geographically diverse population, yielding a total of 22 test strains. A compound effective across a diverse worm panel increases the likelihood of potency across other species, including heterogeneous human populations.Reproducibility marks a key goal of the CITP; the collaborative group generated detailed standardized protocols to minimize among and within lab variability. A data center facilitates near-instantaneous and global access to all CITP data. Compound candidates are selected based on the scientific community's recommendations and advice from the project's steering committee.A subset of strains has already undergone initial testing of four compound interventions: (R) alpha-lipoic acid, Aspirin, Quercetin, and NP1. Resveratrol, propyl gallate, and Thioflavine T are to be tested shortly. In addition to manual lifespan experiments, each lab built over 20 automated lifespan machines which use flatbed scanner technology [3], allowing for high throughput and precise longevity measurements. Mid- and late-age health assays are also underway.Initial results indicate significant differences in among-isolate variation in longevity within species (e.g., C. elegans low variation, C. briggsae high variation), as well as strain- and species-specific differences in longevity responses to compound treatments.1. Warner et al (2000) Mech Ageing Dev 115:199-2072. Miller et al (2007) Aging Cell 6:565-5753. Stroustrup et al (2013) Nature Methods, 10:665-670.

Patrick McGrath (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Analysis of the neural circuitry required for the omega turn"

Patrick McGrath

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

C. elegans displays many complicated types of behavior, such as various taxis behaviors, social behavior, and learning behavior. These behaviors manifest themselves through the modulation of the C. elegans movement, either through modulation of speed, reversal frequency, or omega turn frequency. Here, we present preliminary analysis of the omega turn. The omega turn is characterized by a large change in direction of the worm and is coupled to long reversals. An omega turn can be induced through multiple stimuli, and we analyze the properties and neurons required for the omega turn for each of the different stimuli.

Patrick Narbonne et al. (2005) International Worm Meeting "Regulation of germline proliferation and lifespan during dauer development"

Patrick Narbonne, Richard Roy

[International Worm Meeting, 2005]

During adverse environmental conditions, C. elegans larvae can enter a diapause-like stage called "dauer", and three parallel signalling pathways are implicated in this decision (insulin-like, TGF-, or cGMP). The dauer larva is specialized for long-term survival, presumably as a result of profound metabolic changes, as well as the establishment and maintenance of cell cycle quiescence throughout the animal, including the cells of the germline. Surprisingly, the Notch ligand (LAG-2) is strongly expressed in the distal tip cells throughout the dauer stage, suggesting that the germ cells presumably receive signals to proliferate, yet they nevertheless arrest without entering the default meiotic program. We reasoned therefore that dauer-inducing signals repress mitosis downstream of the Notch ligand/receptor interaction, while the latter may continue to block entry into meiosis.From a genetic screen designed to identify these potential regulatory genes, we have characterized one mutant (aak-2) in which the dauer larvae show pronounced germline hyperplasia when induced by either of the three signalling pathways mentioned above. In glp-1 (Notch receptor) mutants, germ cells enter meiosis prematurely, but arrest in pachytene during dauer. However, in glp-1; aak-2 dauer larvae, germ cells enter meiosis and complete spermatogenesis during dauer development, indicating that aak-2 not only suppresses germ cell mitosis, but also blocks progression through meiosis during dauer development. From our genetic analysis, we found that daf-18/PTEN and par-4/LKB1, the loss of which causes cancer in humans (Cowden and Peutz-Jeghers syndromes, respectively), are also required to suppress germline proliferation during dauer formation triggered by either reduced insulin-like or TGF- signalling.A striking, independent phenotype of aak-2 dauer larvae is that they are short-lived. aak-2 encodes a C. elegans homolog of the mammalian AMP-activated kinase alpha-2 catalytic subunit, a metabolic "master switch", suggesting that the longevity of the dauer larva may require a substantial metabolic change that is largely triggered by aak-2. LKB1, is a major activator of AMPK and reduced par-4 activity partially phenocopies and enhances aak-mediated defects, suggesting that both proteins may cooperate downstream of daf-18 in insulin-like and TGF- signalling-dependent germline regulation, providing genetic evidence that defective AMPK activation may participate in tumorigenesis in patients affected with Cowden or Peutz-Jeghers syndromes.