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[
West Coast Worm Meeting,
2004]
Parkinson's disease (PD) is a slowly progressive neurodegenerative disease characterized by the selective loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). Although the origin of pathogenesis of the DA neurons in idiopathic PD remains unclear, corollary evidence suggests both genetic and xenobiotic contributions. In some familial forms of PD, alleles of alpha-synuclein have been linked to the disease, and epidemiological studies involving pesticides (e.g., paraquot) and exposure to heavy metals (e.g., Fe +3 , Al +3 , Cu +2 ) show an increased risk for PD. Furthermore, accumulation of these heavy metals has been found in the SNpc in PD brains. In rodents and man, DA transporters (DATs) constitute the molecular gateway through which many exogenous neurotoxins (eg., 6-OHDA, methamphetamine, and MPP + ) enter these neurons and affect lesions reminiscent of the DA neuron pathology of PD. DATs can also physically interact with alpha-synuclein, and pharmacological blockade of DAT can protect DA neurons from endogenous and environmental toxins. We have previously shown that in the nematode C. elegans the DA neurons can be selectively damaged by exposure to 6-OHDA. We now show that chronic exposure to Fe +3 , Al +3 , or Cu +2 causes DA neuron specific degeneration. Furthermore, 6-OHDA toxicity is amplified by chronic exposure to these metals. We also show that heavy metals can amplify the neurotoxicty conferred by alpha-synuclein. Finally we show that the pesticide paraquot confers DA neuron degeneration. This system will allow us a facile test to examine the role that these metals and xenobiotics play in the degeneration of these neurons.
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[
International Worm Meeting,
2005]
Manganese (Mn2+) neurotoxicity and amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) resemble a number of aspects of the dopamine (DA) neuron degenerating disorder Parkinson's disease (PD). PD, ALS-PDC, and Mn2+ neurotoxicity are characterized by motor deficits and damage to the substantia nigra and other basal ganglia nuclei, and dopamine or its metabolites are believed to contribute to the disorder. The pre-synaptic protein ?-synuclein has also been proposed to contribute to the pathogenesis of each of the disorders, and occupational exposure to Mn2+ may predispose individuals to PD. Furthermore ALS-PDC, a chronic and always fatal disease that has a very high prevalence among the native people of Guam, Kii-peninsula of Japan, and Western New Guinea, is believed to have a strong environmental component that contributes to the progression of the illness, although to date, none has been convincingly identified. Despite intensive research within the past several decades, the molecular determinants involved in both these disorders have yet to be elucidated. We have previously developed a PD model using C. elegans to dissect and characterize the molecular components involved in DA neuron degeneration. We have shown that the nematode C. elegans DA neurons can be selectively damaged by exposure of whole animals to the parkinsonian-inducing neurotoxin 6-hydroxydopamine (6-OHDA) or expression of wild-type or mutant human -synuclein. We now show that the DA neurons are sensitive to brief exposures to Mn2+, and Mn2+ amplifies the 6-OHDA-induced DA neuron cell death. We have purified and identified compound(s) indigenous to a plant species in the territories described above that confer DA neuron cell death in C. elegans. Finally, we have identified several molecular transporters that play roles in the DA neuron degeneration. This system will allow us a facile test to examine the role that Mn2+ and xenobiotics play in the degeneration of DA neurons.
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[
European Worm Meeting,
2006]
Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.
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Ferkey, Denise, Bethke, Mary, Gottschalk, Alexander, L'Etoile, Noelle, Nagpal, Jatin, Schneider, Martin, Krzyzanowski, Michelle, Woldemariam, Sarah
[
International Worm Meeting,
2015]
cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.
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[
European Worm Meeting,
2006]
Martijn Dekkers1, Michelle Teng2, John McCafferty, Gert Jansen1. We use C. elegans to study the molecular and cellular mechanisms of salt perception, using behavioural assays and calcium imaging. We discriminate three distinct responses to NaCl: First, attraction to NaCl concentrations ranging from 0.1 to 200 mM. Second, avoidance of higher concentrations. Third, avoidance of an otherwise attractive NaCl concentration after prolonged exposure. We call this latter behaviour gustatory plasticity. Previous studies have shown that chemo attraction to NaCl is mediated primarily by ASE, and to a lesser extent by ASI, ADF and ASG, and avoidance of high concentrations of NaCl is mediated by ASH (Bargmann & Horvitz, 1991). In our lab we have identified 85 proteins and five pairs of gustatory neurons that mediate gustatory plasticity. Based on our results we propose a model in which prolonged exposure to 100 mM of NaCl, elicits a signal from the ASE neurons, leading to sensitisation of the avoidance signalling ASI, ADF, ADL and ASH neurons. This results in avoidance of low concentrations of NaCl.. In an effort to identify the roles of the individual cells in gustatory plasticity we expressed either a TRP channel or a G-Protein Coupled Receptor (GPCR) in the neurons that have been implicated in gustatory plasticity. This allows us to specifically activate those cells. The TRP channel that we use is the mammalian capsaicin receptor VR-1. Normally C. elegans does not respond to capsaicin. Previously it has been shown that expression of VR-1 in the ASH neurons results in avoidance of capsaicin (Tobin et al 2002). We have generated animals that express the VR-1 receptor in the ASE, ASI, ADL and ADF neurons. We are currently testing their responses to capsaicin and the effects of preexposure to NaCl on this response, using behavioural assays.. The GPCRs that we have chosen are the mouse SSTR-2 somatostatin receptor and the human CCR-5 chemokine receptor. We have expressed these receptors in the ASH cells, and tested the responses in a novel avoidance assay. We found that the transgenic animals display specific avoidance behaviour to the ligands of the receptors, indicating that these GPCRs are integrated into the endogenous C. elegans signalling machinery, which is remarkable, given the evolutionary distance between the species. We are now making constructs to express these GPCRs in the other cells to assess their role in gustatory plasticity.