Amygdalar mTORC2-PKC Epsilon Interactions in Alcohol Use Disorder: A Rodent Model

Abstract

Protein Kinase C epsilon (PKCɛ) modulates alcohol use disorder-related behavioural changes through interactions with various receptors in the brain, especially in the amygdala. Prior to activation, PKCɛ is phosphorylated at its active loop by phosphoinositide dependent kinase 1 (PDK1), and at last at Serine729 (Ser729) either through autophosphorylation or by mammalian target of rapamycin complex 2 (mTORC2) as reported by recent in vitro studies using cardiomyocytes. The mTORC2 activities are escalated in the dorsal medial striatum following binge ethanol intake. Therefore, this study was undertaken to investigate the changes in expressions of mTORC2 components such as mTOR and SAPK-interacting protein 1 (Sin1), and co expressions of mTOR with Sin1, PKCɛ and phosphorylated PKCɛ (Ser729) during various stages of ethanol exposure. Male Wistar rats (n=30) were allocated into control, acute ethanol (EtOH), chronic ethanol, ethanol withdrawal (EW), and EW + EtOH. The control and acute ethanol groups were given Modified Liquid Diet (MLD) without ethanol for 27 days. On day 28, the control rats were given normal saline, whereas the rats assigned to acute ethanol group were given ethanol (2.5 g/kg, 20% v/v) via intraperitoneal route. For chronic group, MLD without ethanol was given for initial 7 days and followed by gradual introduction of ethanol at 2.4% (3 days), 4.8% (3 days) and 7.2% (14 days). The rats assigned to EW and EW + EtOH groups, were fed with MLD as described for chronic group. However, on day 28, ethanol was withdrawn and replaced with isocaloric MLD. At the 6th hour from the last ethanol intake, EW and EW + EtOH groups were administered with normal saline and acute ethanol challenge 2.5 g/kg, respectively. All the animals were euthanised one hour following the last intraperitoneal administration of drug. The rats’ sera collected for serum ethanol analysis, and amygdala for gene and protein expression analysis. The chronic ethanol group recorded the highest serum ethanol level, followed by EW + EtOH, EW, and acute ethanol groups. The qPCR analysis revealed mRNA levels of mTOR, Sin1, and PKCɛ to be upregulated in the amygdala of EW and EW + EtOH rats. On contrary, chronic ethanol intake significantly reduced the gene expression of Sin1 with minimal changes to mTOR and PKCɛ levels. Western Blot analysis recorded a significant increase in the protein expression of mTOR, Sin1, PKCɛ, and phosphorylated PKCɛ (Ser729) in the chronic ethanol, EW, and EW + EtOH groups. Protein expression analysis using co-Immunoprecipitation (co-IP) with mTOR and target proteins showed a significant upregulation in the mTOR/PKCɛ and mTOR/phospho-PKCɛ (Ser729) expression in the acute ethanol group. Whereas, significant downregulation in co-IP expression of mTOR/Sin1, mTOR/PKCɛ, and mTOR/phosphor-PKCɛ (Ser729) in the chronic ethanol, EW, and EW + EtOH rats. Our results indicate that significant increase in phosphorylation of PKCɛ noticed during the late stages of alcohol use disorder (AUD) is not directly mediated by mTORC2, but due to increased reserve pool of PKCɛ available to be phosphorylated. Whereas, during acute ethanol exposure, mTORC2 may interact more directly with PKCɛ, however the interaction has little effects on phosphorylation of PKCɛ at Ser729.