Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex

Background: Alpha-adrenergic (α-AR) and cannabinoid CB (CB-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α-AR- and CB-R in vivo are unclear. This study investigated the roles of Gαi and Gαz proteins in mediating α-AR- and CB-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. Methods: Gαi or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T) MEK1, activated (p-S) MEK1/2, activated (p-T/Y) ERK1/2, p-S FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. Results: Increased (+ 88%) p-T MEK1 cortical density, with a concomitant reduction (–43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi or Gαz antisense ODNs. Contrastingly, WIN induced Gαi- and Gαz-independent upregulations of p-T MEK1 (+ 63%), p-S MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (− 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi or Gαz protein integrity. Conclusion: The results indicate that α-AR (UK), but not CB-R (WIN), agonists use Gαi and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.The study was supported by grants RTI2018-094414-A-I00 (AR-M), SAF2011-29918 (JAG-S), and RT 2018-093677-B-100 (PSB) from the Spanish Ministry of Science, Innovation and Universities (MCIU) and the European Regional Development Fund (ERDF). AR-M is a ‘Ramón y Cajal’ Researcher (grant RYC-2016-19282) supported by MCIU. JAG-S is a member of the Institut d’Estudis Catalans (Barcelona, Catalonia, Spain). None of the above funding agencies took part in the elaboration of the study, formulation of the hypotheses, or interpretation of the results