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Oligodendrocyte progenitors are highly susceptible to various insults. Their limited antioxidant defenses and high levels of apoptotic factors, such as Bax and pro-caspase-3 contribute to their sensitivity. We previously showed that dopamine (DA) is toxic to oligodendrocyte progenitors by inducing superoxide generation, lowering glutathione levels and promoting apoptosis through caspase-3 activation. In contrast, factors that contribute to cell survival and defense against dopamine (DA) toxicity are less studied. Here, we explored the role of two molecules which play important roles in cell survival, namely the heat shock protein 90 (HSP-90) and the protein kinase Akt, using the selective inhibitors, 17-AAG and Akt inhibitor III, respectively. The HSP-90 inhibitor caused a decrease in P-Akt level, induced caspase-3 activation, increased nuclear condensation and caused a loss in cell viability. Furthermore, 17-AAG potentiated DA-induced apoptosis by enhancing caspase-3 activation. In addition, the Akt inhibitor alone exacerbated DA toxicity and in combination with 17-AAG caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. Together, these results indicate that HSP-90 is essential for oligodendrocyte progenitor survival. Both HSP-90 and Akt play important roles in concert in the defense against DA-induced apoptosis.
In the neonatal white matter, oligodendrocyte progenitors are highly sensitive to signals inducing cell death, both during development and in neurodegenerative diseases (Jelinski et al. 1999; Casaccia-Bonnefil 2000). The hypomyelinating disorder, periventricular leucomalacia (PVL) results from perinatal hypoxic/ischemic insults that produce oxidative stress leading to oligodendrocyte progenitor cell loss (Volpe 2001; Back et al. 2002; Rezaie and Dean 2002; Dewar et al. 2003). Regulating the survival of oligodendrocyte progenitors is critical for their proper development and differentiation into myelin producing cells. Various apoptotic and survival factors may contribute to a delicate balance regulating oligodendrocyte survival and defense against toxic insults. For example, caspase-3 is important for apoptosis of oligodendrocyte progenitors during development, however, contributes to aberrant cell loss in neurodegenerative diseases (De Louw et al. 2002; Knoblach et al. 2005). Furthermore, high levels of iron, a catalyst of oxidative reactions, and inadequate defenses, such as low levels of the antioxidant glutathione are among the factors contributing to the sensitivity of oligodendrocyte progenitors to oxidative stress (Thorburne and Juurlink 1996; Back et al. 1998).
In our previous studies, we showed that DA is toxic to oligodendrocyte progenitors. DA causes superoxide generation and reduces glutathione levels while its toxicity is enhanced in the presence of iron (Khorchid et al. 2002; Hemdan and Almazan 2006, 2007). Furthermore, DA induces an apoptotic mechanism of cell death by activation of caspases 9 and 3, and DNA fragmentation (Khorchid et al. 2002). In contrast, factors that contribute to defense of oligodendrocyte progenitors against oxidative stress have only been minimally studied. We recently demonstrated a role for intracellular glutathione in the cellular defense against DA toxicity, while deficient scavenging of peroxides by glutathione peroxidase is associated with the cells susceptibility to DA toxicity (Hemdan and Almazan 2007). Additionally, DA potently induces expression of heme-oxygenase-1 (HSP-32) (Khorchid et al. 2002), a member of a family of molecular chaperones known as heat shock proteins (HSPs), which are involved in cell survival, proliferation and differentiation (Helmbrecht et al. 2000; Richter-Landsberg and Goldbaum 2003; Beere 2005).
Heat shock proteins contribute to cell survival and protection by regulating the folding and stability of various cellular proteins. The family consists of several members including HSP-32, HSP-72, αB-crystallin, HSP-25 and HSP-90 (Sun and MacRae 2005), some of which are up-regulated in the brain following hypoxic/ischemic injury (Kato et al. 1994; Mariucci et al. 2007). Furthermore, heat shock preconditioning protected mature oligodendocytes from subsequent lethal heat shock treatment (Goldbaum and Richter-Landsberg 2001). However, the roles of HSPs in immature oligodendrocytes have not been addressed.
HSP-90 is a ubiquitous and highly abundant molecule that plays key roles in the regulation of the cell cycle and cell survival in biological systems by interacting with a multitude of client proteins including survival as well as apoptotic factors (Terasawa et al. 2005). HSP-90 can bind to the pro-apoptotic protein Apaf-1, thus preventing apoptosome formation and apoptosis (Pandey et al. 2000). HSP-90 can also inhibit apoptosis by binding and stabilizing the survival factor, Akt (Basso et al. 2002; Georgakis et al. 2006). Akt is a serine-threonine kinase activated by IGF-1 receptor signaling through phosphoinositol-3-kinase (PI3-k) (Datta et al. 1999). IGF-1 receptor signaling has been shown to promote normal oligodendrocyte development in the CNS (Zeger et al. 2007), and Akt protects oligodendrocyte progenitors from growth factor deprivation (Cui et al. 2005), glutamate (Ness et al. 2004) and tumor necrosis factor-α damage (Ye et al. 2007). However, a role in protection against oxidative stress has not been shown.
In this study, we assessed the role of HSP-90 and Akt in oligodendrocyte progenitor survival and defense against DA toxicity using the specific pharmacological inhibitors, 17-AAG (Schulte and Neckers 1998) and AI(III), respectively. Our results indicate that HSP-90 is vital for the survival of oligodendrocyte progenitors, and plays an important role in the defense against DA-mediated apoptosis, by interfering with caspase-3 activation. Furthermore, Akt is involved in the defense of oligodendrocyte progenitors against DA, and the cooperative actions of HSP-90 and Akt account for some of their cytoprotective effects.
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- Materials and methods
Mechanisms that counter apoptosis operate in normal nervous system development and in pathologic states. HSPs are a large group of proteins that regulate cell development and survival, through their actions as molecular chaperones, mediating protein folding (Beere 2005). Various HSPs are up-regulated following cerebral ischemia (Kato et al. 1994; Mariucci et al. 2007), in neurodegenerative diseases (Gordon 2003) and following trauma (Hayes et al. 1995). HSP-90, a constitutive member of this group, is a major survival factor that is over-expressed in tumors (Georgakis et al. 2006). Reducing its levels has been consistently associated with reduced tumor growth (Nimmanapalli et al. 2003; Rahmani et al. 2003; Georgakis et al. 2006). However, its role in the survival of normal cells during development, and in neurodegenerative conditions is less understood.
In this study, we observed that various HSPs (HSP-32, HSP-25 and αB-crystallin) were not detected in oligodendrocyte progenitors, but were expressed in differentiating oligodendrocytes. In contrast, HSP-90 was constitutively expressed at similar levels throughout development. This suggested that the high sensitivity of oligodendrocyte progenitors to toxic stimuli may be partly due to their deficient HSPs expression, and suggested HSP-90 may be vital for their survival. Thus, we assessed the role of HSP-90 in oligodendrocyte progenitor survival and protection against DA toxicity, using a specific inhibitor of HSP-90. Similar experiments were carried out with mature oligodendrocytes to determine whether HSP-90 has the same function throughout differentiation. The induction of HSP-72 by 17-AAG, a competitive inhibitor of ATP-induced HSP-90 client protein folding was used as an indicator of 17-AAG pharmacological effectiveness in our system. An induction of HSP-72 was observed following 17-AAG treatment in both progenitors and mature oligodendrocytes. In addition, combination of 17-AAG and DA further induced HSP-72 in oligodendrocyte progenitors. These results indicate that 17-AAG provided effective HSP-90 inhibition. It may be argued that induction of HSP-72 is expected to protect cells as HSP-72 itself is another HSP that plays an important role in survival of many cells (Yenari et al. 2005). However, although the role of HSP-72 in our system is not clarified, the lack of protection suggests that other, survival effects of HSP-90 are being hampered by its inhibition. HSP-90 mediates its protective effects by binding, folding and stabilizing numerous client proteins involved in the cell cycle, cell survival and apoptosis (Terasawa et al. 2005). Indeed, HSP-90 inhibition induced massive oligodendrocyte progenitor death by apoptosis and enhanced DA-induced toxicity as assessed by several criteria, namely mitochondrial activity, nuclear condensation and caspase-3 activation.
Enhancement of DA-induced cell loss and caspase-3 activation by the HSP-90 inhibitor was detected only using the 75 μM DA concentration. At the lower concentration (50 μM), 17-AAG had no effect, suggesting other antioxidant defenses (Hemdan and Almazan 2007) may be operating or that the level of HSP-90 inhibition is not sufficient to potentiate toxicity under mild oxidative stress. Additionally, at a higher concentration of DA (150 μM), which induces a maximal level of activated caspase-3 as well as cell death by necrosis (Hemdan and Almazan 2006, 2007), 17-AAG was not effective. Collectively, these results indicate that HSP-90 is vital for survival, and is involved in the cellular defense against DA-induced apoptosis but not necrosis. Similar to our results, HSP-90 inhibition increased lipopolysaccharide-induced reactive oxygen species generation and caspase-3 activation in macrophages (Hsu et al. 2007), and enhanced hydrogen peroxide-induced cytochrome c release and nuclear condensation in endothelial cells (Zhang et al. 2005). With the exception of a few cell types, in most other systems including cancer and neural cells, HSP-90 inhibition by 17-AAG or compounds of its class required at least (0.5–3 μM) concentrations to produce effects on cell survival. On the other hand, the massive loss of oligodendrocyte progenitors we observed by a concentration of only 0.1 μM (i.e., ∼ 10-fold less than literature values) suggested HSP-90 plays a central role in their survival. In addition, in 12d differentiated oligodendrocytes, concentrations of 17-AAG up to 25 μM did not induce cell death, suggesting that as oligodendrocyte progenitors differentiate, they may acquire other survival factors or mechanisms, such that HSP-90 is no longer indispensable. The greatly increased expression of αB-crystallin and HSP-25 in mature oligodendrocytes are among the possible contributors to their survival in the face of HSP-90 inhibition. HSPs have been shown in various systems to play redundant roles, and thus may compensate for the absence of each other.
Several studies have revealed that HSP-90 may be involved in inhibition of apoptosis by suppressing cytochrome c-mediated Apaf-1 oligomerization (Pandey et al. 2000), or by stabilizing the survival factor, Akt (Basso et al. 2002). Akt promotes its anti-apoptotic effects by a number of mechanisms including phosphorylating the pro-apoptotic proteins, Bad, forkhead transcription factors and caspase-9, and inhibiting cytochrome c release (Datta et al. 1999; Kennedy et al. 1999; Hirai et al. 2004). Conversely, Akt inactivation by its dephosphorylation and/or degradation has been associated with apoptosis in some systems (Luo et al. 2003). We found that while highly toxic concentrations of 17-AAG and DA caused a decrease in P-Akt level, lower toxic concentrations, alone and when combined did not significantly alter P-Akt. Hence, exacerbation of DA toxicity by HSP-90 inhibition may involve a mechanism other than decreased Akt level. Thus, HSP-90 may interfere with DA toxicity by interactions with other proteins upstream of caspase-3, such as Apaf-1 and cytochrome c. Alternatively, small functional changes in Akt activity which are not detected by western blotting may be occurring with the lower drug concentrations.
Surprisingly, time course analysis showed an early rise in P-Akt level (at 3 h) by higher concentrations of 17-AAG (0.5 μM), DA (300 μM) and their combination (0.2 μM 17-AAG ± 300 μMDA), after which it declined. This may be due to several processes as observed in other studies, such as DA action on DA receptors (Brami-Cherrier et al. 2002), oxidant-induced inactivation of serine/threonine phosphatases involved in Akt dephosphorylation, for example PTEN, by hydrogen peroxide (Leslie et al. 2003) or various effects related to HSP-90 inhibition, and the resulting dysregulation of its client proteins, ex. HSP-25. HSP-25 can be up-regulated by 17-AAG, displayed protection against it (McCollum et al. 2006), and can directly phosphorylate Akt. As well, short term (1 h) treatment by Geldanamycin, and another HSP-90 inhibitor disrupted the ability of the phosphatase, PP2A to dephosphorylate Akt, causing increased Akt phosphorylation (Yun and Matts 2005). Finally, by activating another HSP-90 client protein, Src kinase, Geldanamycin caused transient phosphorylation (15 min to 1 h) of Akt which decreased back to control levels by 2 h (Koga et al. 2006). In our time-dependent experiments, decreases in P-Akt and increases in caspase-3 activity occurred concurrently, and not sequentially, at 8, 12, and 24 h following treatment with high concentrations of 17-AAG, DA and their combination, thus further suggesting that these two events may be related.
However, a clear protective role for Akt against DA toxicity was demonstrated by the observations that Akt inhibitor III alone increased DA-induced cell loss, and in combination with the HSP-90 inhibitor prior to DA treatment caused synergistic cell loss and caspase-3 activation. This suggests that HSP-90 and Akt cooperate in protecting cells from DA. Others have reported synergy in cell killing caused by combinations of HSP-90 inhibitors and inhibitors of other HSP-90 client proteins in various systems which was linked with the enhanced loss of client protein activity (George et al. 2004; Barker et al. 2006; Premkumar et al. 2006; Wang et al. 2006). Although we cannot conclude if a direct or indirect interaction occurs between HSP-90 and Akt, their simultaneous inhibition reduces the threshold of DA toxicity. In line with these results, 17-AAG and the PI3-kinase inhibitor, LY294002 synergistically induced cell death by caspase-3 activation in glioma cells. On the contrary, non-neoplastic astrocytes did not exhibit caspase-3 activation by this treatment (Premkumar et al. 2006).
In contrast to the death-promoting effects of HSP-90 inhibition in oligodendrocyte progenitors, in other neural systems, protection occurred. For example, 17-AAG reduced lipopolysaccharide-induced nitrite and IL-1β production in astrocytes and microglia, and reduced pathology in the multiple sclerosis animal model, experimental allergic encephalomyelitis. These results may be explained by HSP-90 regulation of various genes including NFκB. Thus, disruption of HSP-90 binding to the inhibitor IκK causes IκB up-regulation and results in inhibition of NFκB-induced cytokine production (Dello Russo et al. 2006). In addition, geldanamycin protected against neuronal damage induced by ischemia (Ouyang et al. 2005), huntingtin aggregates (Sittler et al. 2001) and 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity (Shen et al. 2005), through induction of HSP-72.
In summary, our results indicate that HSP-90 is vital for oligodendrocyte progenitor survival and defense against DA toxicity. HSP-90 mediates its effects by counteracting the apoptotic process, and caspase-3 activation. The exact mechanism linking HSP-90 inhibition to cell death remains to be identified. However, synergistic enhancement of DA-induced cell death mediated by simultaneous inhibition of Akt and HSP-90 points to an interaction between the HSP-90 and Akt survival pathways. This may involve dysregulation of various anti-apoptotic effects common to the two proteins. Our results have important implications for PVL, since HSPs, including HSP-90 are up-regulated in neural cells (Kawagoe et al. 1993; Kato et al. 1994; Mariucci et al. 2007) and in oligodendrocytes (Jelinski et al. 1999) following hypoxia/ischemia. In addition, IGF-1, the growth factor responsible for Akt activation protects oligodendrocyte progenitors and suppresses caspase-3 activation following cerebral ischemia (Cao et al. 2003; Lin et al. 2005).