A Dissertation Entitled Uncovering Cannabinoid Signaling in C. Elegans
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A Dissertation Entitled Uncovering Cannabinoid Signaling in C. elegans: A New Platform to Study the Effects of Medicinal Cannabis By Mitchell Duane Oakes Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology ________________________________________ Dr. Richard Komuniecki, Committee Chair _______________________________________ Dr. Bruce Bamber, Committee Member ________________________________________ Dr. Patricia Komuniecki, Committee Member ________________________________________ Dr. Robert Steven, Committee Member ________________________________________ Dr. Ajith Karunarathne, Committee Member ________________________________________ Dr. Jianyang Du, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2018 Copyright 2018, Mitchell Duane Oakes This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Uncovering Cannabinoid Signaling in C. elegans: A New Platform to Study the Effects of Medical Cannabis By Mitchell Duane Oakes Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo August 2018 Cannabis or marijuana, a popular recreational drug, alters sensory perception and exerts a range of medicinal benefits. The present study demonstrates that C. elegans exposed to cannabinoids (CBs) exhibit a “dazed and confused” phenotype, with delayed responses to aversive stimuli, decreased feeding rates, slowed locomotion and increased unproductive turning. These CB-dependent responses are mediated by distinct signaling pathways. For example, C. elegans synthesizes the endogenous CBs, 2- arachidonoylglycerol (2-AG) and anandamide (AEA), and contains a canonical CB signaling system, involving the human CB1 orthologue, NPR-19. The CB-dependent inhibition of feeding and nociception, as measured by pharyngeal pumping and the initiation of aversive responses to the noxious repellant, 1-octanol, respectively, are absent in npr-19 null animals and can be rescued by the expression of human CB1 driven by the npr-19 promoter. Similarly, inhibiting the breakdown of 2-AG or AEA mimics their addition and also inhibits feeding and nociception. Importantly, CBs directly activate NPR-19 after heterologous expression in Xenopus laevis oocytes with nM iii affinities similar to those human CB1, confirming NPR-19 as the canonical cannabinoid receptor in C. elegans. The CB-dependent inhibition of nociceptive responses also requires a D1-like dopamine receptor, DOP-1. Importantly, the CB-dependent inhibition of nociceptive responses is still present in cat-2 null animals that lack a tyrosine hydroxylase, a key enzyme required for dopamine (DA) biosynthesis, suggesting that CBs activate DOP-1 directly. At higher CB concentrations, the inhibition of nociceptive responses also requires the α2-adrenergic-like octopamine receptor, OCTR-1, and the 5-HT1-like serotonin receptor, SER-4, suggesting a complex interaction between octopaminergic and serotonergic signaling. In fact, CBs activate OCTR-1 directly after heterologous expression in Xenopus laevis oocytes, but does not activate SER-4, suggesting that requirement for SER-4 to mediate CB-dependent inhibition of nociception is indirect. In contrast, CBs also signal through NPR-19-independent pathways to inhibit locomotion and increase turning behavior, including reversals and omega turns. These NPR-19-independent signaling pathways require multiple TRP channels and the release of both serotonin (5-HT) and DA from subsets of monoaminergic neurons. For example, CB-dependent locomotory inhibition is dramatically reduced in tph-1 and cat-2 null animals that encode key enzymes required for 5-HT and DA biosynthesis, respectively, and in ser-4 and dop-4 null animals that encode a 5-HT1-like 5-HT receptor and a D1-like DA receptor, respectively. CB-stimulated 5-HT release activates SER-4 in the two key AIB interneurons, leading to “locomotory confusion” and transient paralysis. CB- dependent locomotory inhibition is also absent in cat-4 null animals that encode an orthologue of the human GTP cyclohydrolase 1 and contains reduced levels of both 5-HT iv and DA. CB-dependent locomotory inhibition is rescued in cat-4 null animals by incubation in either 5-HT or DA, suggesting that the overstimulation of either 5-HT or DA signaling compensates for the absence of the other. Importantly, CBs also inhibit locomotion in remodeled, chimeric mutant animals designed to detect acute release of either 5-HT or DA. For example, the Gαo-coupled 5-HT (SER-4) and DA (DOP-3) receptors were expressed in the cholinergic motor neurons of 5-HT receptor quintuple null animals (ser-5;ser-4;mod-1;ser-7 ser-1; 5-HT quints) and DA receptor quadruple null animals (dop-2; dop-4 dop-1 dop-3; DA quads) that lack most, if not all, 5-HT and DA receptors, respectively. Theoretically, any CB-dependent monoamine release in these animals will activate its cognate receptor on the motorneurons, inhibit acetylcholine release onto the muscle and inhibit locomotion. As predicted, neither 5-HT nor DA have any effect on locomotory rate in the 5-HT quint and DA quad mutant, respectively, but rapidly inhibit locomotion following the selective expression of the appropriate inhibitory Gαo-coupled monoamine receptor in the cholinergic motorneurons. CBs also increase reversal and the frequency of omega turn in an NPR-19-independent manner through a pathway requiring 5-HT, but not DA signaling. Using a reverse genetics approach, our lab found that CB-dependent locomotory inhibition is absent in ocr-1, ocr-4, osm-9 and trp-4 null animals that lack functional TRP channel subunits. ocr-1, ocr-4, osm-9 encode TRPV-1 like channel subunits expressed in a number of sensory neurons and trp-4 the pore-forming subunit of a mechanosensitive TRPN (nompC) channel expressed in the dopaminergic neurons. Similarly, 2-aminoethoxydiphenol (2-APB), a non-selective TRP channel blocker also inhibits CB-dependent locomotory inhibition. More importantly, 2-APB also inhibits CB- v dependent locomotory inhibition in the monoamine receptor mutants expressing Go- coupled monoamine receptors in the motorneurons in a system designed to measure acute monoamine release, suggesting that CB-dependent TRP channel activation is required for monoamine release and locomotory inhibition. Interestingly, the temporal pattern of 2- AG inhibition differs significantly in the 5-HT/TRPV-1 and DA/TRP4 -deficient signaling mutants. For example, in mutants with disrupted TRPV-1 or serotonergic signaling, 2-AG-dependent inhibition was initially delayed, but eventually these mutant animals began to slow (25% at 30 min). In contrast, in mutants with disrupted DA or TRP-4 signaling, 2-AG never inhibited locomotion and, in fact, 2-AG rapidly (<30 sec) and significantly (>25%) stimulated locomotion in these mutants, suggesting the TRP-4 might be modulating DA release. This study highlights the advantages of studying cannabinoid signaling in a genetically-tractable, whole-animal model and might also explain the proposed anthelmintic properties of Cannabis, given that the CB-dependent locomotory inhibition mimics the “locomotory confusion” phenotype previously proposed as a potential anthelmintic target by our lab. These studies provide insights into the potential effects of Cannabis on monoaminergic signaling and suggest a role for CBs in activating the dopaminergic-mediated reward system and anxiety/depression in humans. vi I would like to dedicate the work in this study to my mentor Dr. Richard Komuniecki. None of the work in this project would be possible if he had not allowed me to independently explore a topic I was interested in and turn it into a very successful project. I do not think many, if any other PIs would trust a new graduate student to stop working on a project that had been on going in the lab for more than 15 years to start a new project on a controversial subject that was not even thought to exist in our model organism. Most importantly, I cannot thank Dr. Komuniecki enough for teaching me how to think critically, analyze carefully and to never stop asking questions. To me, learning to think critically is unequivocally the most important skill I have gained in my Ph. D and will undoubtedly help me become a successful researcher and veterinarian in the future. v Acknowledgements A special thank you to my advisor Dr. Richard Komuniecki for transforming me from a naïve and unfocused undergraduate into the passionate, thinking scientist I am today. I cannot thank Dr. Komuniecki enough for the constant mentoring over the past 5 years, inside the lab and out. I am extremely grateful for his patience toward my countless idiosyncrasies and allowing me to explore my many strange hobbies including culturing tardigrades, growing plants and keeping a lot of fish in the lab. A heartfelt thank you to my lab mate and now lifelong friend Dr. Wen Jing Law, who contributed greatly to the studies in this project. I am extremely grateful to him for performing the locomotory studies used in this study, teaching me everything I know about the art of keeping fish and most importantly, for being my ‘red pill’. I will be forever grateful to you for opening my eyes to the world and pulling me from the matrix. I would also like to thank my former lab mate