Although the molecular mechanisms of most neurodegenerative disorders remain elusive, neuronal apoptosis has been reported in Parkinson's disease (PD), Huntington's chorea and Alzheimer's disease (Cohen, 2000). 6-Hydroxydopamine (6-OHDA) is a selective catecholaminergic neurotoxin, and is widely used to study the death of catecholaminergic cells. 6-OHDA can be formed from dopamine by nonenzymatic hydroxylation in the presence of Fe²⁺ and H2O2 (Linert et al., 1996). Dopamine turnover is elevated in the brain during PD (Kopin, 1985). Enzymatic oxidation of dopamine by the peroxidase/H2O2 system also leads to the production of 6-OHDA in oxidized quinonoid form (Napolitano et al., 1995). The 6-OHDA and auto-oxidation of dopamine produce semiquinones and quinones that are capable of generating radicals (Graham 1978; Kumar et al. 1995). Dopamine and its oxidative products are likely to promote apoptosis through the oxidative damage of mitochondria by radical-induced lipid peroxidation (Berman and Hastings 1999; Choi et al. 1999; He et al. 2000; Tatton and Olanow 1999). An experiment in vivo showed that 6-OHDA increased malondialdehyde and conjugated dienes, whereas it decreased antioxidants in corpus striatum (Kumar et al., 1995). Thus, PD might develop by the selective degeneration of nigrostriatal neurons through apoptosis induced by the auto-oxidation of dopamine and its metabolites.#

Mitochondria can release apoptosis inducing factors by membrane permeability transition (MPT) (Cai et al. 1998; He and Lemasters 2002). The classic type of MPT (CMPT) is characterized by the following events: (1) the requirement of Ca²⁺ and biological energy, (2) mitochondrial membrane depolarization and swelling, (3) inhibition by cyclosporin A (CsA) and (4) regulation by Bcl-2 family proteins. In addition, nonclassic type MPT has also been reported, which is insensitive to CsA and Ca²⁺, and occurs without swelling (Sultan and Sokolove, 2001). Furthermore, recent studies have indicated that MPT is the consequence of thiol oxidation of the preexisting membrane proteins (Kowaltowski et al., 2001). Moreover, the oxidation of protein dithiols in adenine nucleotide transporter was required to open MPT that was sensitive to antioxidant (Sakurai et al., 2001). As for the role of mitochondria in 6-OHDA-induced apoptosis, it has been reported that CsA blocks 6-OHDA-induced Ca²⁺ efflux from mitochondria (Reichman et al., 1994), and that 6-OHDA induces the release of cytochrome c from the mitochondria in PC12 cells (Ha et al., 2003). Furthermore, 6-OHDA induced the mitochondrial swelling and depolarization of mitochondrial membrane potential (Kim et al. 2001; Lee et al. 2002). These findings suggested that mitochondrial MPT might be involved in the 6-OHDA-induced apoptosis of the cells.#

Elevated levels of intracellular cAMP have been reported to protect neuronal cells from apoptosis stimulated by various agents. Treatment with cell-permeable cAMP analog prevents nerve growth factor withdrawal-induced chromatin condensation of intact rat superior cervical ganglion neurons (Neame et al., 1998) and protects PC12 cells from proteasome inhibitor-induced apoptosis (Rideout et al., 2001). The mechanisms responsible for the protective action of cAMP against apoptosis include the synthesis of antiapoptotic proteins, the inactivation of proapoptotic proteins, and phosphatidylinositol 3-kinase-dependent Akt activation. Although it has been reported that a cell permeable cAMP analog also protects cells from 6-OHDA toxicity (Yamada et al., 1997), its mechanism is not clear.#

Serine/threonine kinase Akt serves as a multifunctional regulator of apoptotic cell death and cell growth. With respect to neuronal cell function, Akt has been shown to be required for the prevention of apoptosis and the promotion of cell survival through the phosphorylation of proapoptotic Bad (Datta et al., 1997) and procaspase-9 (Cardone et al., 1998). Recently, it has also been reported that p38 MAPK is induced in the 6-OHDA-induced apoptosis (Choi et al., 2004). To get a better insight into the molecular mechanism of neuronal cell apoptosis induced by dopamine metabolites, we investigated the mechanism of 6-OHDA-induced apoptosis of PC12 cells and its protection promoted by cAMP and antioxidants.#

In this report, we described that 6-OHDA increased the intracellular superoxide production and induced caspase activation, Bid cleavage, mitochondrial membrane depolarization and chromatin condensation, which were independent of MPT in PC12 cells, and that cAMP suppressed the apoptosis through the restoration of the phospho-Akt levels and the inhibition of p38 phosphorylation without the inhibition of superoxide generation and mitochondrial membrane depolarization.#

In the present work, we demonstrated that 6-OHDA-induced apoptosis was dependent on superoxide production, and was inhibited by pCPT-cAMP in PC12 cells. The decrease in mitochondrial membrane potential was not inhibited by pCPT-cAMP and was not likely to be involved in the apoptosis machinery in this model.#

It has been reported that 6-OHDA induces MPT in isolated brain mitochondria (Kim et al., 2001). In isolated rat liver mitochondria, we also detected that 6-OHDA induces cytochrome c release through a CMPT mechanism, which showed mitochondrial swelling and membrane depolarization with a CsA sensitive mechanism (data not shown). In the whole PC12 cells, however, 6-OHDA-induced mitochondrial membrane depolarization and chromatin condensation were not inhibited by CsA (Fig. 4). These results indicate that CMPT, which characterized by depolarization and swelling in a CsA-sensitive mechanism, is not involved in the mechanism of apoptosis (Di Paola et al., 2006). Presumably, the decrease in mitochondrial membrane potential was rather a result of cell death. In this context, we observed that tiron, which is a superoxide scavenger, but not pCPT-cAMP, suppressed the 6-OHDA-induced mitochondrial membrane depolarization and superoxide generation (Figs. 10B and 11B and D). Furthermore, it has been reported that 6-OHDA induced lipid peroxidation, which induces the depolarization of the mitochondrial membrane in a CsA-insensitive mechanism (Chaloupka et al. 1999; Nobre et al. 2003; Ogawa et al. 1994). These results may indicate that the 6-OHDA-induced superoxide and/or products of its chain reaction, such as lipid peroxide, trigger mitochondrial membrane depolarization in a CsA insensitive mechanism. Thus, we presented a possible mechanism of the 6-OHDA-induced apoptosis in Fig. 12.#

Caspase-8 activation and tBid appear to be early events in our apoptosis model. It is generally accepted that Bax and tBid trigger the release of cytochrome c independently of the CMPT mechanism. The activation of caspase-8 leads to Bid cleavage and facilitates mitochondria-mediated downstream apoptotic events (Li et al., 1998). In the present experiments, we demonstrated that 6-OHDA activated caspase-8 in a time-dependent manner (Fig. 2), and that tBid was detected after the addition of 6-OHDA (Fig. 8A). Furthermore, we demonstrated that Ac-IETD-CHO, which was an inhibitor of caspase-8, suppressed caspase-9 activity (Fig. 8B). These results indicate that the cleavage of Bid by activated caspase-8 triggers the activation of the caspase cascade in 6-OHDA-treated PC12 cells.#

Cyclic AMP protected neuronal cells (Neame et al., 1998) and PC12 cells (Rideout et al. 2001; Yamada et al. 1997) from apoptosis induced by various stimulations. Cyclic AMP induced the transactivation of the receptors for nerve growth factor, thereby the modulating activation of Akt in PC12 cells (Piiper et al., 2002) and regulated the cellular level of p-Akt through a PI3-kinase-dependent pathway (Tsygankova et al., 2001). In this experiment, we found that 6-OHDA induced the downregulation/dephosphorylation of Akt (Fig. 9) and that pCPT-cAMP induced Akt phosphorylation and suppressed the 6-OHDA-induced caspase activation and chromatin condensation (Figs. 5 and 6). Moreover, we found that LY294002, which was an inhibitor of PI3-kinase/Akt pathway, promoted 6-OHDA-induced chromatin condensation (Fig. 5). These results indicated that the PI3-kinase/Akt pathway promoted cell survival against 6-OHDA-induced apoptosis, and that pCPT-cAMP suppressed the apoptosis of PC12 cells through this pathway (Fig. 12).#

Akt is localized upstream of caspase-8 activation and is activated by phosphorylation and protects cells from apoptosis (Suhara et al., 2001). Recent studies indicated that p-Akt increases the expression of FLICE inhibitory protein (FLIP), which inhibits caspase-8 activation (Panka et al. 2001; Suhara et al. 2001). In this experiment, we found that pCPT-cAMP suppressed the 6-OHDA-induced caspase-8 activation and chromatin condensation (Figs. 5 and 6), but not mitochondrial membrane depolarization (Fig. 7). These results indicate that pCPT-cAMP acts at upstream of caspase-8 activation.#

In the 6-OHDA-induced apoptosis pathway, the oxidative stress-induced phosphorylation of p38 was linked to the activation of caspase-8 and -9 in MN9D cell and primary cultures of mesencephalic neurons (Choi et al., 2004). The protein kinase activity of p38 was required for the apoptosis of PC12 cells in some models (Jenkins and Barone, 2004). In addition, PI3-kinase/Akt signaling promotes cell survival by inhibiting the p38 mitogen-activated protein kinase-dependent apoptosis (Gratton et al., 2001). In the present experiment, we found that pCPT-cAMP worked as an Akt activator, and suppressed the 6-OHDA-induced p38 phosphorylation (Fig. 9), but not superoxide generation (Fig. 10). These results suggest that p38 phosphorylation is involved in 6-OHDA-induced apoptosis, and that pCPT-cAMP acts upstream of the activation of p38 as well as caspase-8, and downstream of superoxide generation in PC12 cells (Fig. 12).#

Accumulated evidence indicates that 6-OHDA induces neuronal cell apoptosis through ROS generation from oxidation of 6-OHDA and this ROS acts as a second messenger in cellular signaling (Berman and Hastings 1999; Choi et al. 1999; Graham 1978; He et al. 2000; Kumar et al. 1995). We studied the intracellular superoxide production by 6-OHDA in the PC12 cells using hydroethidine (Fig. 10) (Budd et al. 1997; Zuo et al. 2000). Hydroethidine is a noncharged, membrane-permeable fluorescence probe for the superoxide anion, and the oxidized product emits a strong red fluorescence in the presence of DNA when hydroethidine reacts with superoxide (Yamada et al., 2003b). 6-OHDA increased the red fluorescence in a time and concentration-dependent manner, and this was attenuated by tiron, which is a membrane permeable superoxide scavenger (Fig. 10) (Zuo et al., 2000). Tiron also attenuated the 6-OHDA-induced p38 phosphorylation, mitochondrial membrane depolarization and chromatin condensation (Fig. 11). In this case, it is noteworthy that the attenuation depended on the time of preincubation with tiron. Pretreatment with tiron attenuated the 6-OHDA-induced mitochondrial depolarization and apoptosis, probably through ROS scavenging. These results indicate that 6-OHDA generated intracellular ROS, especially superoxide, at an earlier step of the apoptosis pathway. Moreover, the ROS might be generated through 6-OHDA quinone, a product of 6-OHDA auto-oxidation (Padiglia et al., 1997).#

A previous study shows that 6-OHDA does not cause apoptosis in PC12 cells, but rather mainly necrosis is induced (Woodgate et al., 1999). However, our results showed typical chromatin condensation and caspase activation (Figs. 1 and 2). In addition, the chromatin condensation was inhibited by a caspase inhibitor (Fig. 1). In other reports, 6-OHDA-induced PC12 cell death was almost totally dependent on caspase-3 activation, which also showed that the 6-OHDA-induced PC12 cell death was mainly apoptosis (Ha et al., 2003). The reason for this discrepancy is not clear at this time, but it may be due to the different experimental conditions, such as the cell culture conditions.#

In a recent study, GSK-3β, a downstream molecule of the PI3-kinase/Akt pathway, plays a critical role in the 6-OHDA-induced apoptosis of PC12 cells, and the PI3-kinase/Akt pathway protects through the inhibition of the GSK-3β activity (Chen et al., 2004). We studied the phosphorylation of GSK-3β after 12h of 6-OHDA treatment (data not shown). Contrary to the previous report, GSK-3β (Ser9) phosphorylation did not decrease despite the decrease in Akt phosphorylation. The discrepancy may be due to the difference in culture conditions (i.e., we did not keep the PC12 cells under serum-free condition before 6-OHDA treatment. Some kinases other than Akt may have participated in GSK-3β (Ser9) phosphorylation under our conditions), or the experiment not being performed at the optimal time point.#

Taken together, we propose the following causal sequence of 6-OHDA-induced apoptosis of PC12 cells: the intracellular generation of ROS by 6-OHDA is an initial event and the ROS suppresses the Akt activity and activating phosphorylation of p38, thereby activating caspase-8, which stimulates the cleavage of Bid, and induces the activation of caspase-9 and -3 independently from mitochondrial depolarization (Fig. 12).#