Lyons_2020_Mol.Cell.Neurosci__103566

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB(1) (CB(1)XS) or CB(2) (CB(2)XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, alpha/beta-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB(1) or CB(2) expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and alpha/beta hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB(1) receptor expression increased lengths of short (<30 microns) and long (>30 microns) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gbetagamma inhibitor gallein, and beta-arrestin inhibitor barbadin did not significantly alter long projection length in CB(1)XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB(1) receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB(1) receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB(2) receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB(1) receptor signaling through Gi/o, Gbetagamma, and beta-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.