Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB₁ (CB₁XS) or CB₂ (CB₂XS) 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, α/β-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB₁ or CB₂ 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 α/β hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB₁ receptor expression increased lengths of short (<30 μm) and long (>30 μm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gβγ inhibitor gallein, and β-arrestin inhibitor barbadin did not significantly alter long projection length in CB₁XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB₁ receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB₁ receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB₂ receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB₁ receptor signaling through Gi/o, Gβγ, and β-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.