Astrocytes support neuronal functions by releasing various gliotransmitters and energy substrates, maintaining homeostasis within the CNS [1,2] and regulating neuronal plasticity [3]. They are responsible for about 20% of total oxygen consumption in the brain [4] and carry out many ATP-dependent processes, including the removal of glutamate and potassium ions from the extracellular fluid, the metabolism of glutamate to glutamine followed by its export across the cell membrane [5], as well the synthesis and breakdown of glycogen [6,7]. ATP is also one of the most important gliotransmitters released by Ca2+-dependent exocytosis [8,9]. Moreover, ATP appears to be a predominant extracellular signalling molecule, since the activation of metabotropic purinergic P2Y receptors in glial cells results in IP3-mediated Ca2+ release [10] and the propagation of Ca2+ transients, as demonstrated in cultured astrocytes [11] and in the animal brain in vivo [12]. Astrocytes rely heavily on the glycolytic pathway for energy production [13,14], however, this fact does not diminish the role of the mitochondria. It has been demonstrated that mitochondrial dysfunction may shift astrocytes toward their pro-inflammatory phenotype [15], which in turn may enhance neurodegeneration [16,17].

Mitochondria are dynamic multifunctional organelles, that are closely compartmented by lipid double bilayer membranes. The inner mitochondrial membrane (IMM) possesses various embedded respiratory enzyme complexes named the electron transport chain (ETC), as well as an O2-dependent ATP production system [18]. The ETC creates a proton motive force composed of a pH gradient (chemical gradient) and an electric potential across the IMM, used by F1F0-ATP synthase to produce ATP from ADP [19]. The IMM has a unique phospholipid composition containing 15–20% cardiolipin, apart from common phospholipids [20]. Cardiolipin is a four-acyl chain diphosphatidylglycerol that promotes the formation of high IMM curvature [21]. This enables respiratory enzyme complex clustering [18], which augments the efficiency of electron channeling for optimal OXPHOS [22,23]. In addition to oxidative metabolism, mitochondria play an important role in intracellular Ca2+ homeostasis and signalling. They shape Ca2+ fluctuations [24], and modulate calcium-dependent processes such as gliotransmission [3,24]. Furthermore, the mitochondria remain the site of the ultimate phase of cellular catabolism and regulate the intracellular redox state. Under unfavorable conditions for the cell, such as ischemia, toxicity, oxidative or endoplasmic reticulum stress, the mitochondrial membrane potential (MMP) collapses and cytochrome c is released from the mitochondria, which activates the apoptotic machinery [25].

The long-chain polyunsaturated n-3 fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5), commonly named omega-3 PUFAs, can be provided through a diet [26] or are synthetized from the essential linolenic acid (C18:3 n-3) in the hepatocytes through sequences of a hydrocarbon chain elongation and desaturation by elongases and desaturases [27,28]. DHA is the most abundant n-3 PUFA in neuronal membranes and it has considerable impact on neuronal survival and differentiation [29,30]. Unlike neurons, astrocytes do not contain a high level of DHA in their membranes, but they express FADS2 [31] and have the potential capacity to synthesize DHA. However, the ability of astrocytes to elongate a 20-carbon chain to 24-carbon PUFAs and then one β-oxidation cycle to produce 22-carbon PUFA is questionable [32]. Introducing n-3 PUFAs into membrane phospholipids has many effects on molecular cell metabolism and signalling. It has been demonstrated that DHA readily replaces C18:2 in mitochondrial cardiolipin that results in changing the rate of mitochondrial respiration and activation of mitochondria-dependent apoptosis [33]. EPA, the second n-3 PUFA produced by sea algae and some bacteria, is absent in the neuronal and astrocyte membranes under physiological conditions. However, it is introduced into membrane phospholipids after cell culture in medium enriched with n-3 PUFA [34], or into the brain after supplementation of animal diet with fish oil [35].

Since astrocytes do not contain a significant amount of n-3 PUFAs their role in astrocyte function has not been extensively studied. Begum et al. demonstrated that DHA delayed a depletion of Ca2+ from ER and reduced the ER stress markers levels in astrocytes [36]. Recently, Yu et al. reported that DHA and EPA enhanced GFAP expression and growth factor production in astrocytes differentiated from neuronal stem cells generated from a patient with major depressive disorder, and their effect may mimic the effects of antidepressants [37]. Our earlier studies indicate that in astrocytes, both n-3 PUFAs have a powerful antioxidative effect via Nrf2 up-regulation [34], the inhibition of immunoproteasome assembly [38], and the anti-inflammatory effect of DHA, in part through inhibition of NF-κB activation [39]. To fully understand the effect of n-3 PUFAs on astrocyte functionality, the aim of the present study was to determine the effect of DHA and EPA on MMP, ATP production and apoptosis activation. Our findings indicate that EPA has a stronger effect than DHA in elevating the MMP and reducing apoptosis, also after uncoupling of the ETC by CCCP. These effects were not accompanied by a significant change in the intracellular ATP levels. Interestingly, in EPA-incubated astrocytes the cardiolipin level was increased in a dose-dependent manner.