Endosomal CB1 stimulates neuronal differentiation of Neuro‐2A cells

Abstract

In recent years many states have legalized marijuana use for medical and recreational purposes, which emphasizes the need to better understand the actions of this chemical on the human cells. The main psycho-active component of marijuana, Δ9-tetrahydrocannabinol (THC), exerts its biological effects through two receptors named cannabinoid type-1 receptor (CB1R) and cannabinoid type-2 receptor (CB2R). Both receptors belong to one of the largest families of proteins from human genome, named G protein-coupled receptors (GPCR), which has over 700 members and represents the target for about 30% of the therapeutic agents available today. CB1R is the main receptor in the central nervous system and this receptor stimulation is responsible for the behavioral responses observed after marijuana use. In contrast to the majority of GPCRs which are localized exclusively at the plasma membrane (cell surface), CB1 is present not only at the plasma membrane but also in the endosomes. The subcellular localization of this receptor is under the control of Rab5 GTP-ase, which stimulates its traffic from the plasma membrane to the endosomes and Rab4 GTP-ase, which has an opposite effect. Noteworthy, cannabinoid-like molecules as THC, due to their high lipophilicity may activate both plasma membrane and endosomal CB1R. CB1R stimulation has been shown to stimulate neuronal differentiation of different neuroblastoma cell lines, but the pool of receptors involved in this effect remains unknown. To answer this question, in the present work we manipulate Rab5 GTP-ase cellular levels by transfection in Neuro-2A cells. We found that in control Neuro-2A cells, CP55,940 (100 nM, a CB1R agonist) increased the number of differentiated cells to 259.18 ± 17.62 % compared to control . Rab5 GTP-ase transfection alone enhanced the number of differentiated cells to 171.42 ± 10.28 %, and these effects were further augmented by treatment with CP55,940 to 455.10 ± 27.82 % (p<0.05 from three independent transfections. Our preliminary data that CB1 receptors with endosomal localization are more important in stimulation of neuronal differentiation.