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Cannabinoid Receptor Signaling in Human SH-SY5Y Neuroblastoma Cell Viability, Proliferation Rate, and Extension Length

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abstract
We created clonal cell lines that stably overexpress either CB1 (CB1XS) or CB2 receptor (CB2XS) in human neuroblastoma SH-SY5Y as a model to investigate the role of cannabinoid signaling in neurite extension, proliferation, apoptosis, and cell viability. The mRNA of the enzymatic components of the endogenous cannabinoid system diacylglycerol lipase, monoacylglycerol lipase, alpha/beta-hydrolase domain containing 6, alpha/beta-hydrolase domain containing 12, N-acyl phosphatidylethanolamine phospholipase D, and fatty acid amide hydrolase are present in SH-SY5Y cells. Activity of these enzymes produced endocannabinoid ligands, resulting in a steady state concentration of 2-arachidonoylglycerol of 135 pmol per gram of protein and anandamide of 0.82 pmol per gram protein. Application of tetrahydrolipstatin, an inhibitor of diacylglycerol lipase activity, decreased 2-arachidonoylglycerol concentration to 15 pmol per gram of protein while anandamide remained at 0.79 pmol per gram of protein. Oxotremorine M, an agonist of Gq coupled muscarinic acetylcholine receptors, mobilized calcium from intracellular stores to stimulate diacylglycerol lipase, increasing 2-arachidonoylglycerol concentration to 564 pmol per gram of protein. A 2-arachidonoylglycerol hydrolysis enzyme inhibitor cocktail increased 2-arachidonoylglycerol to 2,140 pmol per gram protein. Transfection resulted in a stable increase of CB1 receptor mRNA by 11 fold increase in abundance in the CB1XS cell line and of CB2 receptor mRNA by 14 fold increase in abundance in the CB2XS cell line. Cannabinoid receptor overexpression did not alter the mRNA abundance of the enzyme components of the endogenous cannabinoid system in SH-SY5Y cells. Parental SH-SY5Y cells had an average neurite length per nuclei of 0.7 μm per nuclei. Stable increased expression of CB2 receptor in three clonal cell lines did not consistently change extension length, and resulted in a maximal neurite length per nuclei increase to 1.69 μm. Stable increased expression of CB1 receptor increased the abundance of one soma long neuritic extensions from 0.7 to 6.0 micrometers per nuclei per field. This CB1 stimulated increase in neurite length was not constitutive receptor activity as it was halved by tetrahydrolipstatin inhibition of diacylglycerol lipase enzymatic production of the endogenous cannabinoid ligand 2-arachidonoylglycerol. Neurite extension length was halved with a p of 0.07 by gallein inhibition of G protein beta gamma dependent signaling. Stable increased expression of CB1 receptor increased the mRNA abundance of growth cone associated protein 43 and ST8 Alpha-N-Acetyl-Neuraminide Alpha-2,8-Sialyltransferase 2 mRNA and decreased integrin alpha 1 mRNA. Stable increased expression of CB2 receptor increased neural cell adhesion molecule and synaptotagmin 1 mRNA. We identified the endogenous cannabinoid enzyme monoacylglycerol lipase as a novel target of retinoic acid mRNA transcription in the SH-SY5Y neuroblastoma model of neuron differentiation and neurite extension. Overexpression of cannabinoid receptors did not alter the viability, apoptosis rate, or proliferation rate of cells relative to parental or empty vector SH-SY5Y.
subject
apoptosis
endocannabinoid
neurite
neuroblastoma
proliferation
synaptogenesis
contributor
Lyons, Erica Lynn (author)
Howlett, Allyn C (committee chair)
Parks, John (committee member)
Chen, Rong (committee member)
Thomas, Brian (committee member)
date
2019-05-24T08:35:51Z (accessioned)
2019 (issued)
degree
Molecular Medicine and Translational Science (discipline)
2019-11-23 (liftdate)
embargo
2019-11-23 (terms)
identifier
http://hdl.handle.net/10339/93988 (uri)
language
en (iso)
publisher
Wake Forest University
title
Cannabinoid Receptor Signaling in Human SH-SY5Y Neuroblastoma Cell Viability, Proliferation Rate, and Extension Length
type
Dissertation

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