Numerous studies have shown that PUFA incorporation into the

Numerous studies have shown that ω-3 PUFA incorporation into the lipid raft can affect the distribution and function of protein in the lipid raft by altering raft composition [25], [26], [27]. For example, DHA treatment excluded phospholipase D (PLD) from lipid raft toward non-lipid raft, resulting in activation of PLD [28]. Additionally, DHA treatment on human breast cancer Sodium Picosulfate decreased epidermal growth factor receptor (EGFR) localization in lipid raft, accompanied by activation of EGFR [29]. Therefore, we hypothesized that chronic DHA treatment may exert its antidepressant effect by altering translocation of Gsα from lipid raft, accompanied by increased activity of adenylate cyclase that eventually triggers cell signaling pathways for neurogenesis. In the present study, we first showed that chronic treatment of C6 glioma cells with DHA, which mimics to antidepressant treatment, displaced Gsα from lipid raft and increased cAMP accumulation, suggesting that DHA treatment improves Gsα–adenylate cyclase–cAMP signaling transduction out of lipid raft. Furthermore, this study indicated that the translocation of Gsα from lipid raft induced by DHA was independent of disruption of lipid raft, whereas it increased the lipid raft level in a dose-dependent manner.
Materials and methods
Results
Discussion DHA is involved in mediating normal neuronal signaling and function. The deficiency of ω-3 PUFAs has been shown an association with an increased prevalence of depressive disorders [31], [32]. Despite the importance of ω-3 PUFAs in alleviating symptoms of depression, the molecular mechanism underlying the antidepressant effect of ω-3 PUFAs is not completely understood. Several previous studies showed that Gsα was one of the targets of antidepressant action, and chronic treatment with antidepressants increased the coupling between Gsα and adenylate cyclase [22], [33]. Later studies further confirmed that antidepressant treatment translocated Gsα from lipid raft into a non-lipid raft membrane fraction [23], [24], resulting in Gsα being more available for coupling with adenylate cyclase [24]. Therefore, it is suggested that the increased coupling between Gsα and adenylate cyclase may be the common mechanism for treatment of chronic antidepressant. In the present study, we showed that the chronic treatment of C6 cells with DHA decreased the content of Gsα in lipid raft, which is similar to the effect of desipramine on Gsα localization (Fig. 1A). We also observed that the total content of Gsα was not changed by chronic DHA treatment (Fig. 1B). To further support the effect of DHA on the localization of Gsα in lipid raft, confocal image analysis was used, showing that both DHA and desipramine decreased the colocalization of Gsα with lipid raft (Fig. 2). Lipid raft contains numerous signaling molecules such as receptors, G proteins, effector enzymes, and other membrane-associated proteins, thus it acts as a signaling platform [15], [34]. One study found that numerous antidepressants were concentrated in lipid raft after chronic treatments, suggesting that lipid raft may be the action target of antidepressant drugs [35]. It is noteworthy that ω-3 PUFAs can alter the structure and composition of lipid raft to displace protein from lipid raft fractions and affect subsequent downstream cell signaling [36], [37]. Therefore, the results of this study provide further evidence in support of lipid rafts being an important target for DHA to exert antidepressant effect. To confirm whether DHA treatment could facilitate activity of adenylate cyclase, intracellular cAMP levels in C6 cells were tested. The result showed that DHA treatment increased the intracellular cAMP level in a dose-dependent manner (Fig. 3A). Consistent with our findings, the deprivation of ω-3 PUFAs in rats has been previously shown a decrease in cAMP levels [38]. Furthermore, we observed that there was no change in the total membrane expression of ACVI (Fig. 3B), suggesting that enhanced activation of adenylate cyclase was not due to its increased expression. It has been previously demonstrated that Gsα signaling was inhibited in lipid raft, whereas augmented coupling between Gsα and adenylate cyclase occurs in non-raft fractions [18]. Additionally, immnoprecipitation experiments showed that the number of Gsα–adenylate cyclase complexes was increased by antidepressant treatment, which is responsible for the increased activity of adenylate cyclase [39]. In agreement with these evidences, our data supported the hypothesis that DHA treatment may facilitate the activation of adenylate cyclase by translocating Gsα from lipid raft to non-lipid raft fraction.