Cannabidiol Mediates Glycogen Synthase Kinase-3β Signalling In Cerebellar Granule Neurons

Carolyn Tanner, Leyu Shi, Cecilia Hillard. Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin, 53226, USA

Glycogen synthase kinase 3β (GSK-3β) is a constitutively active kinase, expressed throughout the brain. It is a component of a number of signaling pathways including those that determine cell fate in embryonic development and modulate the regulation of cell survival and death (Jope and Johnson, 2004). Over recent years, data have accumulated linking dysregulated GSK-3β signalling with bipolar disorder (BPD) (Jope and Roh, 2006). One reported mechanism by which lithium, a current treatment for BPD, works is via inhibition of GSK-3β activation (Klein and Melton, 1996). Thus, hyperactivation of GSK-3β could contribute to the etiology of the disorder. Epidemiological studies have demonstrated a significant co-morbidity between cannabis use and BPD (Regier et al, 1990). Cannabidiol (CBD) is present in high amounts in some strains of cannabis sativa, however, unlike Δ9-tetrahydrocannabinol (Δ9-THC), CBD is non-psychoactive and does not display significant binding to either of the classified cannabinoid receptors, CB1 or CB2 (Pertwee et al, 2010).

The aim of this study was to investigate the effects of CBD on GSK-3β activity in cerebellar granule neurons (CGN) isolated from neonatal Sprague-Dawley rats (described in Nogueron et al, 2001), utilizing a kinase activity assay and western blotting techniques. As the activation state of GSK-3β is regulated by phosphorylation at serine 9, the ratio of phosphorylated GSK-3β (pGSK-3β) to total GSK-3β was measured using western blotting.

CGN exposed to CBD for 6 hours exhibited a concentration-related decrease in pGSK-3β/GSK-3β; significant effects were observed at 1 µM, 3 µM and 10 µM CBD (P<0.01, one-way ANOVA with Dunnett’s test). The effect of 1 µM CBD was time dependent, with significant effects at incubation times of 40 min (P<0.05) and longer (P<0.01, one-way ANOVA with Dunnett’s test). CBD-mediated changes in GSK-3β phosphorylation were accompanied by an increase in GSK-3β enzymatic activity (P<0.01, one-way ANOVA with Dunnett’s test), via a decrease in serine 9 phosphorylation. Several kinases can phosphorylate GSK-3β at serine 9, including PKA. We tested the hypothesis that CBD can reduce pGSK-3β/GSK-3β via inhibition of PKA activity. Supporting this, incubation of CGN with 1 µM CBD for 2 hours resulted in complete loss of the phosphorylation at serine 133 of cAMP response element binding (CREB) protein (P<0.001, unpaired t-test). The CBD effect was pertussis toxin sensitive (P<0.01, two-way ANOVA with Bonferroni’s test) and CBD inhibited forskolin-induced increases in pGSK-3β/GSK-3β.

The data demonstrate that in CGN, CBD had potent inhibitory effects on a GPCR / Gi/o / PKA / GSK-3β pathway that resulted in the enhancement of GSK-3β activity through inhibition of PKA-dependent phosphorylation. The target at which CBD exerts these effects is currently unknown. These findings suggest that the exposure of the brain to CBD could have important implications during critical developmental periods and could contribute to the mechanism by which cannabis use predisposes individuals with other risk factors to develop BPD.

Jope and Johnson, (2004). Trends Biochem Sci 29, 95-102

Jope and Roh, (2006). Curr Drug Targets 7, 1421-1434

Klein and Melton, (1996). Proc. Natl. Acad. Sci. 93, 8455-8459.

Nogueron et al, (2001). J Neurochem. 79, 371-381

Pertwee et al, (2010). Curr Med Chem. 17, 1360-1381

Prickaerts et al, (2006). J. Neurosci. 26(35), 9022-9029

Regier et al, (1990). Jama. 264(19), 2511-2518

Supported by NIH grant DA026996