Learning can be either non-associative or associative, though the molecular mechanisms underlying both remain enigmatic. The nematode Caenorhabditis elegans can adapt to both the AWC sensed odorants benzaldehyde (Bnz) and isoamyl alcohol (IsoA) and reciprocally cross-adapt. Surprisingly, however, these four adaptation permutations actually represent two distinct forms of learning: non-associative habituation and associative learning by pairing with starvation. Conditioning to the single odorant IsoA leads to both associative and non-associative memory traces, which can be preferentially accessed by choice of a Bnz or IsoA retrieval stimulus, respectively. Furthermore, we show that the molecular mechanisms underlying each form of memory can be genetically double dissociated, with insulin signalling and egl-4 being required for associative learning and osm-9 and arr-1 being essential for IsoA olfactory habituation. This represents the first demonstration where the form of learning displayed after conditioning to a single stimulus is a function of the retrieval stimulus employed.

Learning can be either non-associative or associative, though the molecular mechanisms underlying both remain enigmatic. The nematode Caenorhabditis elegans can adapt to both the AWC sensed odorants benzaldehyde (Bnz) and isoamyl alcohol (IsoA) and reciprocally cross-adapt. Surprisingly, however, these four adaptation permutations actually represent two distinct forms of learning: non-associative habituation and associative learning by pairing with starvation. Conditioning to the single odorant IsoA leads to both associative and non-associative memory traces, which can be preferentially accessed by choice of a Bnz or IsoA retrieval stimulus, respectively. Furthermore, we show that the molecular mechanisms underlying each form of memory can be genetically double dissociated, with insulin signalling and egl-4 being required for associative learning and osm-9 and arr-1 being essential for IsoA olfactory habituation. This represents the first demonstration where the form of learning displayed after conditioning to a single stimulus is a function of the retrieval stimulus employed.

The over-expression of Down syndrome cell adhesion molecules (DSCAMs) is partially responsible for the mental retardation associated with Down syndrome. Previous work in our lab showed that a DSCAM homolog in C. elegans, Y32G9A.8, is expressed at all developmental stages and appears to be crucial for survival. In an effort to map the expression pattern, I used the Genome Sciences Centre’s primer design program (http://elegans.bcgsc.bc.ca/gfp_primers/) to design a GFP promoter fusion product that was used to monitor gene expression. The results indicate that Y32G9A.8 is expressed in the animal’s gut, suggesting that it may function in the worm’s innate immune response. I also designed a primer set to amplify the Y32G9A.8 transcript. RT-PCR of the entire Y32G9A.8 coding region resulted in a single product; there appears to be no alternative splicing. Although this gene shows homology to other N-CAMS, results indicate that this gene may function in the innate immune system of C. elegans.

All complex organisms possess a nervous system which they use to monitor environmental and internal stimuli. In higher vertebrates, the nervous system is comprised of billions of cells which form highly plastic neural networks from their synapses. These large neural circuits modulate complex behaviors. The nematode roundworm Caenorhabditis elegans uses a small but highly-interconnected nervous system to carry out complex behaviors. The nervous system of C. elegans is a tractable model to determine the effects of changes on a nervous system at the systemic, cellular, genetic, and molecular levels. The C. elegans male’s nervous system detects environmental conditions, mating cues, attractants, repellents, and the location and composition of possible food sources and integrates these inputs to compute the decision of whether or not to mate. Mating behavior in the C. elegans male is regulated at a number of steps by cholinergic signaling from various sensory and sensory-motor neurons, but a comprehensive model of how cholinergic signaling controls this circuit has not yet been elucidated. Previous studies have thoroughly dissected the cellular structure, neural connectivity, and signaling pathways of the male’s peripheral circuits located in the genital regions of the animal’s tail. However, no studies have been conducted to determine what role the cephalic cholinergic neurons have in regulating mating behavior. I hypothesized that cephalic cholinergic neurons exert regulatory control over the male-specific mating circuit. I inserted the transmembrane light-activated ion pore Channelrhodopsin-2 fused to YFP and expressed from the Punc-17small promoter into these neurons and selectively stimulated them using high-intensity blue light. Stimulation induced mating-like behaviors in the male tail consistent with behaviors seen during copulation with a hermaphrodite. Using behavioral assays, I demonstrated that these behaviors were male-specific and only occurred after direct stimulation in the absence of a hermaphrodite. Incidence of mating-like behaviors increased significantly as the worm aged, and the mating circuit retained a memory of the stimulus, indicated by the latency between stimulation and onset of mating-like behaviors. Brief food deprivation, which normally downregulates excitability of the mating circuit via UNC-103 ERG-like K+ channels, caused an unexpected increase in the number of blue light-stimulated behaviors displayed. Pharmacological assays using acetylcholine (ACh) agonists showed that stimulation of the cephalic cholinergic neurons increased propensity for spicule protraction in the presence of an ACh agonist, and partially restored the decline in spicule protraction associated with temporary food deprivation. I sought to identify the cephalic cholinergic neuron or neurons responsible for regulating mating-like behavior in the tail circuits. I looked for a reduction in mating-like behaviors after stimulation after removal of a cephalic cholinergic neuron pair via laser micro-ablation. Two cholinergic and chemosensory neuron pairs in the inner labial sensilla (IL2L/R and IL2VL/R) appear to generate and/or relay the signal that induces mating-like behaviors in the tail. I hypothesize that these neurons sense environmental cues before the male contacts a mate, and modulate lasting motivational changes within the male mating circuit.

<p>Cadmium is a human carcinogen and a persistent environmental pollutant of increasing concern. Yet, the exact molecular targets of cadmium toxicity and the molecular mechanisms by which cadmium influences gene expression have not been fully elucidated. Therefore, the characterization of cadmium-inducible genes will provide a better understanding of the underlying mechanism involved in sensing cadmium-stress and the subsequent signaling pathways important for cellular defense against cadmium toxicity. To this end, we characterized two cadmium-responsive genes of no known biological function from the nematode Caenorhabditis elegans (C. elegans), numr-1 and numr-2.</p><p>Expression analysis of numr-1 and numr-2 revealed the same temporal and spatial expression patterns of both genes in the absence and presence of metal treatment. In the absence of metal, constitutive expression of numr-1/-2 was developmentally regulated. When adult animals were exposed to metal, numr-1/-2 expression dramatically increased. We show that worms overexpressing numr-1/-2 were more resistant to metal stress and longer lived than control animals; whereas reducing numr-1/-2 activity resulted in increased sensitivity to metal exposure. Furthermore, in the absence of metal, the two numr-1 mutant alleles, tm2775 and ok2239, exhibited decreased muscular functions. The molecular characterization of numr-1 and numr-2 also revealed that the expression of these two genes, at least in part, was regulated by changes in intracellular calcium concentrations ([Ca2+]i). This finding lead us to reevaluate the role of calcium mobilization in cadmium-induced transcription. </p><p>While several studies have indicated that exposure to cadmium resulted in increased [Ca2+]i, the mechanism by which cadmium can effect [Ca2+]i and concurrent effects on gene expression remain poorly understood. Therefore, we investigated the effects of low-level cadmium exposure, sufficient to induce transcription of cadmium-responsive genes, on the regulation of [Ca2+]i. In these studies, we utilized the protein-based calcium sensor YC 3.60 stably expressed in a HEK293 cell line. YC 3.60 is insensitive to cadmium ions, and thus is useful to monitor changes in [Ca2+]i following cadmium treatment. Exposing HEK293 cells to 1-30 µM cadmium was sufficient to induce transcription of cadmium-responsive genes such as metallothionein. Cadmium exposure from 1-10 µM had no effect on cell viability, [Ca2+]i mobilization, or increased transcriptional activity of calcium-responsive genes. In contrast, exposure to 30 µM cadmium significantly decreased cell viability, reduced intracellular calcium stores, and significantly altered the transcriptional activity of calcium-responsive genes. Taken together, these data indicate that low-level cadmium exposures (1-10 µM) can induce transcription of cadmium-responsive genes such as metallothionein independent of [Ca2+]i mobilization. </p><p>To gain further insight into the mechanistic relationship between cadmium and calcium we investigated the effects of cadmium exposure on the defecation cycle of C. elegans. Defecation is a highly rhythmic behavior that is regulated by calcium oscillations. We found that low-level cadmium exposures, sufficient to induce expression of cadmium-responsive genes such as numr-1/-2, significantly shortened the defecation cycle but did not alter the rhythm of the cycle or the magnitude of the intestinal calcium oscillations. Modulation of lipid metabolism in C. elegans results in a similar shortened defecation cycle, whereas modulation of [Ca2+]i results in lengthened and arrhythmic defection cycles, suggesting that the mechanism by which cadmium alters defecation is independent of [Ca2+]i mobilization.</p><p>In summary, the data in this work demonstrates that low-level cadmium exposure induces expression of cadmium-responsive genes independent of calcium mobilization. Thus, modulation of intracellular calcium is unlikely the primary mechanism by which cadmium regulates transcription at low-levels of exposure.</p> / Dissertation

<p>Basement membranes are dense, sheet-like forms of extracellular matrix that</p><p>surround the epithelial tissues of metazoan organisms. While these structures are</p><p>critical for epithelial support and tissue organization, basement membranes also pose</p><p>formidable barriers to most cells. However, certain specialized cells are able to breach</p><p>these barriers and move between tissues. Acquisition of cell invasive behavior by some</p><p>tumor cells is thought be an important step in cancer progression. Due to the clear basic</p><p>and clinical importance of understanding the mechanisms underlying cell invasion</p><p>through basement membranes, cell invasive behaviors has been an area of intense study.</p><p>In this work I examine a developmentally regulated model of cell invasive behavior in</p><p>the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels</p><p>basement membrane to connect two epithelial tissues, the uterus and vulva. Using this</p><p>model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.</p> / Dissertation

The nervous system coordinates a sequence of muscle movements to give rise to animal behaviors. In complex invertebrates or lab-studied vertebrates, due to the large number of cells in their nervous systems and the complexities of their behaviors, it is difficult to address how circuits process information to direct each motor output of the behavior. In this dissertation, I used the Caenorhabditis elegans male copulation behavior as a model to address how a compact circuit coordinates different behavioral programs. Insertion of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial mating. However, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the C. elegans male cloaca is positioned over the hermaphrodite’s vulva as he attempts to insert his copulatory spicules repetitively. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, I show that contraction of the male oblique muscles is required to sustain genital contact between the sexes. These muscles are innervated by the postcloacal sensilla (p.c.s.) sensory/motor neurons, which secret ACh to activate the levamisole-sensitive AChR and the ACR-16-containing ionotropic AChR on the oblique muscles. For spicules to rhythmically thrust during genital contact, activity of the oblique muscles and the gubernacular muscles is transmitted to the spicule protractor muscles instantaneously via gap junctions between these muscles and causes shallow protractor contractions. The rhythmic protractor contractions eventually switch to sustained contraction, as the SPC sensory-motor neurons integrate information of spicule position at the vulva with inputs from the hook and cloacal sensilla. The ERG-like K+ channel, UNC-103, which decreases the spicule circuit excitability, is likely to set a threshold requirement for integration of these inputs, so that sustained spicule muscle contraction is not stimulated by fewer inputs. In addition, I demonstrate that a cholinergic signaling pathway mediated by a muscarinic acetylcholine receptor, GAR-3, is used to enhance the ionotropic AChRs-mediated fast synaptic transmission in the copulation circuit. GAR-3 is expressed in multiple cells of the copulation circuit, but mainly in the cholinergic p.c.s. neurons and SPC neurons. Activation of GAR-3 is coupled to Gαq to trigger downstream signal transduction events that modulate neurotransmitter release from these neurons. Males with a loss-of-function allele of the gar-3 gene are defective in inserting their spicules into the hermaphrodite’s vulva efficiently. Since the p.c.s. neurons regulate the male’s contact with the hermaphrodite’s vulva, and the SPC neurons are required for spicule insertion during mating, GAR-3 probably facilitates male mating behavior via enhancing synaptic transmission from these neurons to their postsynaptic partners.

Understanding how metabolites contribute to anaplerosis, antioxidant effects, and hormetic pathways during aging is fundamental to creating supplements and dietary habits that may decrease age-associated disease and decline, thus improving the quality of life in old age. In order to uncover metabolic pathways that delay aging, the effects of large sets of metabolites associated with mitochondrial function on lifespan were investigated. Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in C. elegans. Addition of fumarate and succinate also extended lifespan and all three metabolites activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductaseF48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Each of the twenty amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine were found to extend lifespan at least to a small extent at one or more of the 3 concentrations tested with serine, histidine, and proline showing the largest effects. In most cases, amino acid supplementation did not extend lifespan in eat-2 worms, a model of dietary restriction or in daf-16, sir-2.1, rsks-1 (S6 kinase), or aak-2 (AMPK) longevity pathway mutants or in worms fed RNAi to skn-1, the C. elegans Nrf2 homolog. Serine and tryptophan addition further protected worms from Alzheimer’s amyloid-beta toxicity. Tryptophan and its catabolites nicotinic acid, picolinic acid, and NAD further induced a broad heat shock response. These results indicate that dietary amino acid imbalance and amino acid catabolism affect organismal longevity. The ketone body beta-hydroxybutyrate (βHB) is a histone deacetylase (HDAC) inhibitor and has been shown to be protective in many disease models, but its effects on aging are not well studied. Therefore we determined the effect of βHB supplementation on the lifespan of C. elegans. βHB supplementation extended mean lifespan by approximately 20%. RNAi knockdown of HDACs hda-2 or hda-3 also increased lifespan and further prevented βHB-mediated lifespan extension. βHB-mediated lifespan extension required the DAF-16/FOXO and SKN-1/Nrf longevity pathways, the sirtuin SIR-2.1, and the AMP kinase subunit AAK-2. βHB did not extend lifespan in a genetic model of dietary restriction indicating that βHB is likely functioning through a similar mechanism. βHB addition also upregulated ΒHB dehydrogenase activity and increased oxygen consumption in the worms. RNAi knockdown of F55E10.6, a short chain dehydrogenase and SKN-1 target gene, prevented the increased lifespan and βHB dehydrogenase activity induced by βHB addition, suggesting that F55E10.6 functions as an inducible βHB dehydrogenase. Furthermore, βHB supplementation delayed Alzheimer's amyloid-beta toxicity and decreased Parkinson's alpha-synuclein aggregation. The results indicate that D-βHB extends lifespan through inhibiting HDACs and through the activation of conserved stress response pathways. Aging is a progressive disease caused by the time dependent decline of an organism and is the primary risk factor for many human ailments, including heart disease, cancer, and Alzheimer’s disease. Uncovering metabolic pathways and metabolites that delay the onset of age-related decline was the primary drive of this investigation.

Alzheimer disease is characterized by the initial degeneration of a subset of cholinergic neurons. This pattern of degeneration can be triggered by overexpression of the amyloid precursor protein (APP) gene in humans. Interestingly, APP is widely expressed; it is therefore unclear why only certain cholinergic neurons are vulnerable to degeneration. We show that widespread expression of the human APP gene in the nematode Caenorhabditis elegans also induces age-dependent apoptotic degeneration of select cholinergic neurons. Identical results were obtained by overexpressing the orthologous worm gene apl-1. The pattern of neurodegeneration matched the cell-autonomous accumulation of APP protein in vulnerable neurons and could be activated cell-non-autonomously by distinct portions of APP. Vulnerability to APP accumulation and degeneration depended inversely on the level of ASK1/p38MAPK innate-immune signaling in cholinergic neurons. Lastly, we identify a compound P7C3 that blocks entrance to apoptosis caused by APP or immunodeficiency. Our results suggest that immunosenescence sculpts the cellular pattern of neurodegeneration by APP. / text

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of aquatic protozoa. It exhibits a distinct dimorphic lifecycle that alternates between vegetative replicative form (RF) and infectious cyst-like form (CLF). Inadvertent inhalation of aerosolized CLFs by immunocompromised individuals leads to an infection in alveolar macrophages causing Legionnaires' disease. To further study a Legionella infection the use of the multicellular organism Caenorhabditis elegans was done. Differential Interference Contrast (DIC) microscopy of live L. pneumophila infected nematodes shows Legionella-containing vacuoles (LCVs) with motile forms. Transmission Electron Microscopy (TEM) defined the ultrastructure of L. pneumophila forms found in the primary infection site of the intestinal lumen and the secondary infection site in the gonadal tissues. These findings suggest the possible intracellular replication cycle of Legionella occurring in the gonadal tissues of the nematode. Providing insight and a plausible evolutionary origin of the ability of L. pneumophila to manipulate the macrophage innate immune system. / October 2014