The neuroprotective effect of lithium in cannabinoid dependence is mediated through modulation of cyclic AMP, ERK1/2 and GSK-3β phosphorylation in cerebellar granular neurons of rat

Document Type: Research article


1 Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Pharmacology and Applied Medicine Department of Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran


Lithium (Li), a glycogen synthase kinase-3β (GSK-3β) inhibitor, has used to attenuate the
cannabinoid-induced dependence/withdrawal signs, but molecular mechanisms related to this are
unclear. Recent studies indicate the involvement of upstream extracellular signal kinase1/2 (ERK1/2)
and downstream GSK-3β pathways in the development of cannabinoid-induced dependence. This
is mediated through cannabinoid receptor 1 (CB1) enriched in cerebellar granular neurons (CGNs).
Accordingly, the present study aimed to investigate the mechanism of modulatory/neuroprotective
effects of Li on a cannabinoid agonist (WIN 55,212-2 (WIN))-induced dependence, through
quantitative analysis of some involved proteins such as ERK1/2, GSK-3β and related signaling
pathways including their phosphorylated forms; and cAMP level as the other molecular mechanisms
leading to dependence, in CGNs model. The CGNs were prepared from 7-day-old Wistar rat pup in a
12-well plate, pretreated with Li (1mM) and an ERK1/2 inhibitor SL327 (SL, 10 μM). The WIN (1
μM) was added 30 minutes prior to treatment and AM251 (AM, 1 μM), as a cannabinoid antagonist
was co-treated with WIN. The cAMP level, as an indicator of cannabinoid-induced dependence, was
measured by ELISA following forskolin (FSK) stimulation. Western blot analyses determined the
phosphorylated forms of ERK1/2 (p-ERK1/2), GSK-3β (p-GSK-3β) as well as their total expressions
in various treatment times and doses in CGNs. WIN alone could down regulate the cAMP/p-ERK1/2
cascade compared to AM treatment. However, P-GSK-3β was up-regulated with Li and WIN or
with SL and Li pretreatment to AM-induced cellular response, which was the highest 60 minutes
after CGNs exposure. Results further suggested the potential role of Li pretreatment to diminish
the development of cannabinoid-induced dependence/neuronal injury through possible mechanisms
of modulating the cAMP/p-ERK1/2 cascade independent of p-GSK-3β signaling pathway in-vitro.


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