iPLA2 activity in isolated energized RBM
Brain tissue exhibits high specific oxygen uptake, has low antioxidative defense capacity, is rich in PUFA (Cabezas et al. 2012), and expresses a high level of iPLA2 (Yang et al. 1999; Balboa et al. 2002). These features prompted us to consider brain mitochondria as a suitable object for examining the physiological functions of iPLA2. In addition, previously we studied primary astrocytes from a mouse disease model of INAD, which is caused by a spontaneous VIA iPLA2 mutation. There we could show that the VIA iPLA2 plays a central role for the proper Ca2+ homeostasis (Strokin et al. 2012). These results confirm that iPLA2 isoforms are a promising pharmacological target for treatment of neurodegenerative diseases. This view seems to be substantiated by the hypothesis, assuming that iPLA2 contributes in situ to mild uncoupling of mitochondria, a process which attenuates Δψm-dependent RET-associated O2•− generation. This study addresses the role of the enzymatic activity of iPLA2 in the control of ROS generation by brain mitochondria. Oxidative stress, the bad side of the cellular ROS generation, has been discussed in numerous reports as the cause or enhancing factor for the pathological development of neurodegenerative diseases.
Here, we demonstrate firstly that iPLA2 is active in intact, energized native brain mitochondria (Fig. 1). In contrast it has been reported that intact mitochondria have little or no activity of iPLA2 (Rauckhorst et al. 2014). Nevertheless, iPLA2 exhibits activity without exposure to oxidants, in contrast to what has been claimed before (Jezek et al. 2010; Jaburek et al. 2013), and without collapse of Δψm (Gadd et al. 2006).
To explain the discrepancy between data reporting that iPLA2 is active in energized mitochondria (Kinsey et al. 2007b) and the claim that activation of iPLA2 requires mitochondrial deenergization, it is helpful to consider the methods used for assessing iPLA2 activity. The method used by the Schnellmann group (Kinsey et al. 2007b) measured iPLA2 activity in mitochondria using a radioactively labeled synthetic phospholipid substrate. However, the Pfeiffer group measured the accumulated FFA derived from hydrolysis of endogenous mitochondrial phospholipids (Gadd et al. 2006; Rauckhorst et al. 2014). Since in intact, energized mitochondria liberated FFAs are used largely for membrane remodeling, an enrichment of mitochondria with FFAs can be seen only after the interruption of the ATP supply. In conclusion, iPLA2 activity seems to be indeed present in isolated native brain mitochondria.
BEL has detrimental influence on energy-dependent functions of mitochondria indicated by decrease in CRC and content of reduced glutathione in mitochondria
We tested BEL as an inhibitor of iPLA2 (Ackermann et al. 1994), to suppress the liberation of FFA from membrane phospholipids by iPLA2 activity. If we assume that resting brain mitochondria are slightly uncoupled and that this uncoupling is because of the iPLA2 activity, it can be expected that the iPLA2 inhibitor BEL increases the RET-driven O2•− generation. Indeed, we found that BEL-treated brain mitochondria oxidizing the RET-supporting succinate show higher H2O2 release (Fig. 4). However, this slightly higher H2O2 release cannot be explained by an improved coupling, which would result from treatment of brain mitochondria with BEL. In contrast, the Δψm of the IMM is reduced compared with that in untreated brain mitochondria. Depolarization is shown by the lower uptake of safranine O as well as, indirectly, by the increased resting respiration (Fig. 5). Interestingly, BEL-induced mitochondrial depolarization was also reported for mitochondria in situ in PC12 cells (Ma et al. 2011). BEL, a lipophilic and bulky chemical compound, is likely to disturb the membrane structure after enrichment in the IMM, and consequently, membrane properties, which could lower Δψm by inducing membrane leakage. Finally, deenergizing effects of BEL on the mitochondria are indicated by the CRC decline (Fig. 6).
In addition, BEL-treated brain mitochondria enhanced ROS generation (Fig. 4). We propose that a lowered mitochondrial antioxidative defense contributes to this ROS increase. This idea is based on the observation that the BEL treatment of brain mitochondria results in partial depletion of glutathione in the mitochondrial matrix (Fig. 7), which is in the range 6–10 mM (Giulivi and Cadenas 1998). Glutathione depletion might be related to reactive elements in the BEL molecule structure. The reactive carbon–bromine bond reacts with a cysteine residue (651Cys) in the proximity of serine (Jenkins et al. 2013). The reactive lactone ring structure covalently binds serine-465Ser in the catalytic center of iPLA2, thereby inactivating the enzyme. This recently reported reactivity of BEL encourages us to speculate that BEL depletes mitochondria from glutathione by forming adducts with glutathione.
Modulation of iPLA2 is no suitable cellular tool to diminish oxidative stress
Generally, modulation of the mitochondrial O2•− generation by iPLA2-derived FFA should depend on the nature of the oxidized substrate. With FADH2-donating substrates, such as succinate, RET is important for energized mitochondria (Chance and Hollunger 1961) and the RET-associated O2•− generation is attenuated by uncoupling (Loschen et al. 1971; Korshunov et al. 1997). We used low micromolar DHA concentrations to mimic increased levels of iPLA2-liberated FFA. The release of DHA, but also of other PUFAs, is controlled by the activity of the VIB iPLA2 isoform in neuronal cells [e.g., (Strokin et al. 2007)]. DHA stimulates uncoupled respiration, as shown in Fig. 2. However, when RBM were treated with the ANT inhibitor CAT, the stimulation of respiration by DHA was significantly lower. This indicates a major contribution of the ANT in mediating FFA-dependent uncoupling. Previous work (Schönfeld 1990) shows that FFA-dependent uncoupling cannot completely be attributed to ANT. Residual uncoupling by DHA in CAT-treated mitochondria has been connected with several proteins of the inner membrane, like glutamate-aspartate carriers, phosphate carriers, and UCPs (Samartsev et al. 1997; Jezek et al. 1998; Zackova et al. 2000).
Nevertheless, we conclude that mainly the ANT mediates DHA-linked attenuation of the O2•− generation, because it becomes restored by the ANT inhibitor CAT (Fig. 3a and b). Binding of the CAT to ANT suppresses the ability of the ANT to enhance the protonophoric activity of FFA. In case of CAT application, Δψm increased and, consequently the Δψm-driven RET-based O2•− generation gets higher.
However, GTP or GDP, which inhibit UCPs (Wojtczak et al. 2011; Cardoso et al. 2013; Jaburek et al. 2013), did not influence the DHA-linked attenuation of the RET-driven O2•− generation (Fig. 3a and b). This demonstrates that the DHA-linked uncoupling is mediated mostly by the ANT and, importantly, in brain mitochondria a participation of UCPs-assisted fatty acid cycling is unlikely. Nevertheless, some reports describe that UCP2, which is expressed in the brain might be responsible for diverse pathologic changes in neurodegenerative diseases (Sullivan et al. 2003; Deierborg et al. 2008). Moreover, it has been proposed that UCP2 has a neuroprotective role in cerebral stroke (Mattiasson et al. 2003; Mehta and Li 2009). However, a recent study strongly indicates that such action of UCP2 cannot be attributed to UCP2-mediated mild uncoupling (Shabalina et al. 2014).
In addition, it is important to remember that FADH2-donating substrates (fatty acids) are poorly used as brain fuel, as discussed recently in a topical review (Schönfeld and Reiser 2013). Furthermore, it is assumed that only RET is sensitive to Δψm (Murphy 2009; Brand 2010) and a Δψm-sensitive RET-linked ROS generation is exclusively observed during mitochondrial succinate oxidation (Shabalina et al. 2014).
The strict dependence of the energy metabolism of brain tissue on glucose degradation makes it very likely that FFA modulate the O2•− generation by brain mitochondria, which oxidize NADH-donating substrates. Thus, it is important to understand, to which extent iPLA2-liberated FFA can affect FET-dependent ROS generation. We found a two-fold increase of H2O2 release, when DHA (10 nmol/mg of protein) was added to RBM oxidizing glutamate plus malate (Fig. 3c). This increase is most likely because of deregulated FET, thereby supporting the one-electron transfer to molecular O2, to form O2•−.
Two recent studies, where mitochondria isolated from heart, lung and spleen tissue were exposed to oxidants (H2O2 or tert-butylhydroperoxide), to activate the iPLA2, proposed an iPLA2-associated stimulation of mitochondrial respiration (Jezek et al. 2010; Jaburek et al. 2013). These mitochondria responded to the addition of oxidants with rapidly increased respiration, which was not found with mitochondria pre-treated with BEL. However, it should be stressed that this reported increase in resting respiration was minimal, only about 10%.
In conclusion, we demonstrate that exogenous DHA causes ANT-dependent mild uncoupling of brain mitochondria. This mild uncoupling partly abolishes RET-dependent ROS release from mitochondria. However, we find no evidence for an involvement of UCPs in the DHA-induced uncoupling. Moreover, even assuming that high succinate concentrations would be available for oxidation by mitochondria in neural cells, the decreased Δψm connected with the ATP turnover in neural cells is sufficient to abolish the succinate-driven RET-related ROS generation (Votyakova and Reynolds 2001).
Moreover, we propose that iPLA2 does not generate high FFA levels in energized mitochondria. Energized mitochondria supply ATP. ATP prevents FFA accumulation in mitochondria owing to promoting their reesterification. Thus, we rule out that mitochondrial iPLA2 has a role in the modulation of ROS production in brain mitochondria.
Nevertheless, iPLA2 has been shown to attenuate the detrimental consequences of oxidative stress, the peroxidation of lipids (Kinsey et al. 2007a, 2008). We attribute this beneficial activity to the ability of iPLA2 to hydrolyze oxidatively damaged membrane phospholipids, a precondition for the membrane repair.
Moreover, we here demonstrate for the first time that pharmacological concentrations of the iPLA2 inhibitor BEL exert detrimental influences on energy-dependent functions of mitochondria. Such detrimental activities of BEL should be taken into consideration for interpreting the effects of BEL on mitochondrial depolarization or apoptosis triggered by mitochondrial permeability transition. Finally, the present findings describing the activity of iPLA2 in maintenance of mitochondrial functions have to be connected to the role of iPLA2 in neurodegenerative diseases, such as INAD.