Exchange proteins directly activated by cAMP (Epac) proteins are implicated in a wide range of cellular functions including oxidative stress and cell survival. Mitochondrial-dependent oxidative stress has been associated with progressive neuronal death underlying the pathology of many neurodegenerative diseases. The role of Epac modulation in neuronal cells in relation to cell survival and death, as well as its potential effect on mitochondrial function, is not well established. In immortalized hippocampal (HT-22) neuronal cells, we examined mitochondria function in the presence of various Epac pharmacological modulators in response to oxidative stress due to ferroptosis. Our study revealed that selective pharmacological modulation of Epac1 or Epac2 isoforms, exerted differential effects in erastin-induced ferroptosis conditions in HT-22 cells. Epac1 inhibition prevented cell death and loss of mitochondrial integrity induced by ferroptosis, while Epac2 inhibition had limited effects. Our data suggest Epac1 as a plausible therapeutic target for preventing ferroptosis cell death associated with neurodegenerative diseases.

It has been shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF) - a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, it was also shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study therefore aimed at investigating whether metformin protects against DF-induced toxicity via cAMP-EPACs. Primary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression was determined by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging. Metformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction which was demonstrated by decreased oxygen consumption rate, an increased ROS production and a reduced MnSOD level, were all reversed by metformin in an EPAC-dependent manner. Metformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2.