Effects And Signal Transduction Mechanism Of Cannabinoid Receptors On Neurite Outgrowth Of Differentiated Neural Stem Cells

We focused our study on the effects of cannabinoid receptor on neurite outgrowth of neurons coming from mouse neural stem cell in vitro and the possible signal transduction mechanism.We firstly cultured neural stem cells (NSCs) in suspension in serum-free medium containing basic fibroblast growth factor(bFGF) and epidermal growth factor (EGF). The NSCs are floated as multi-cell aggregates called neurospheres in vitro. The neurospheres which are nestin positive can be passaged and can differentiate to neurons and glia cells. In order to study the effects of cananbinoid receptors on neurite outgrowth, we induced the NSCs to differentiate mostly into neurons.The endogenous cannabinoid system has been found widely expressed in various tissues and organs of mammalian. To date, two major cannabinoid receptors have been described, CB1 receptors (CB1R) and CB2 receptors (CB2R). CB1R is present mainly in CNS and modulate kinds of neurophysiology functions. The CB2R is thought to be expressed only in peripheral system. However, it has been reported recently that CB2R is also localized in different cell populations in CNS. To do so, we investigated the presence of the CB1R and CB2R mRNAs in the neurons, astrocytes, NSCs and differentiated cells from NSCs by RT-PCR. We found that CB1R and CB2R mRNAs were expressed by all the kinds of these cells.Subsequently, We studied the effects of WIN55212-2(WIN), a mixed CB1R/CB2R agonist, on neurite outgrowth of differentiated cells from NSCs in vitro. Through the neurite outgrowth assay, WIN had been showed to improve neurite outgrowth in a concentration-dependent manner. To better characterize the involvement of each single CB receptor in WIN-enhanced neurite outgrowth, we co-incubated the differentiated cells with WIN and either the CB1R antagonist SR141716A or the CB2R antagonist AM630. As a result, both of SR141716A and AM630 were able to significantly decrease the average length of neurite treated with WIN. It suggusted that WIN enhanced the neurite outgrowth by activating both CB1R and CB2R.To delineate how WIN enhanced neurite outgrowth, we examined the role of different signal pathways in this effect. Western blot analysis data suggested that WIN treatment induced activation of both ERK and STAT3. Neurite outgrowth assay showed that PI3K inhibitor Wortmannin could completely inhibit WIN-induced neurite outgrowth, and PKC inhibitor Go6983 could partly inhibit the effects of WIN, while the PKA inhibitor H89, ERK1/2 inhibitor U0126 and JAK/STAT3 inhibitor AG490 couldn't block WIN-improved neurite outgrowth. Taken together, our results demonstrate that WIN enhanced neurite outgrowth in differentiated cells from neural stem cells mainly through PI3K and PKC signaling pathway.