Differential Modulation of Akt/Glycogen Synthase Kinase-3β Pathway Regulates Apoptotic and Cytoprotective Signaling Responses*

We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the pro-apoptotic factor glycogen synthase kinase-3β (GSK-3β). Ropinirole-induced protection was associated with phosphorylation of GSK-3β (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3β (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3β and reduced levels of β-catenin, a transcriptional activator that is regulated by GSK-3β. Knock-out of GSK-3β expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H2O2) dephosphorylates Akt and GSK-3β, increases GSK-3β activity, and promotes an interaction with β-catenin and its degradation. Inhibition of GSK-3β activity by inhibitor VIII protects cells from H2O2 similar to ropinirole. These results indicate that GSK-3β downstream of Akt plays a critical role in cell death and survival in these models.

The characteristic pathology of Parkinson disease (PD) 2 is degeneration of dopaminergic neurons coupled with Lewy body inclusions in the substantia nigra pars compacta (1). The mechanism underlying dopaminergic cell death in PD has not been elucidated. A variety of cellular and molecular changes indicative of mitochondrial dysfunction, oxidative stress, proteasomal dysfunction, and apoptosis have been identified in the parkinsonian brain (for review, see Refs. 2 and 3). Specifically, a large body of evidence suggests that oxidative stress or reactive oxygen species-mediated apoptosis may contribute to the progressive and selective neuronal degeneration observed in PD (4). Brains of PD patients have increased iron, which promotes free radical formation, decreased levels of reduced glutathione, which is the major anti-oxidant in the brain, and evidence of oxidative damage to DNA, lipids, and proteins (5). Furthermore, in the substantia nigra pars compacta of PD patients there are increased levels of cyclooxygenase, which contribute to formation of the oxidant species dopamine-quinone (5), and reduced mitochondrial complex I activity, which promotes free radical formation (6 -8).
Current therapies for PD are primarily based on a dopamine replacement strategy. Although they provide effective anti-parkinsonian effects, particularly in the early stages of the disease, PD patients eventually develop potentially disabling features such as falling, freezing, and dementia that are not satisfactorily controlled with available therapies (9). As a consequence, there has been an intensive search for therapies that might protect or restore function to neurons that would otherwise undergo degeneration in PD and thereby stop or slow the rate of disease progression.
Dopamine agonists that activate D 2 receptors are widely used to treat PD based on their capacity to provide short-term symptomatic improvements. Recent interest has also focused on the potential of dopamine agonists to provide neuroprotective effects and slow the rate of PD progression (18). Ropinirole and other dopamine agonists have been found to be capable of protecting dopamine neurons from a variety of toxins in both in vitro and in vivo models (10 -15). Furthermore, in clinical trials in PD patients, ropinirole delayed the rate of decline of a neuroimaging surrogate biomarker of nigrostriatal function in comparison to levodopa (16,17). These findings raise the possibility that ropinirole may be neuroprotective and slow the rate of PD progression. Although several mechanisms have been proposed to account for how these agents might provide neuroprotection (18), most interest has focused on the potential of their capacity to provide anti-apoptotic effects. However, the precise signaling mechanism whereby ropinirole induces antiapoptotic effects is not known. * This study was supported in part by a grant from GlaxoSmithKline Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Previously, we have demonstrated that some dopamine agonists protect PC12 cells from oxidative stress by activating a D 2 receptor-dependent PI-3K/Akt signaling pathway independent of their ability to activate GTP␥S binding (14,15). To elucidate the downstream effectors of the PI-3K/Akt signaling pathway that mediates the agonist-specific modulation of cell survival, we have investigated the anti-apoptotic signaling pathway activated by the dopamine agonist ropinirole. We now report that ropinirole-mediated protection against oxidative stress involves activation of PI-3K/Akt-mediated phosphorylation (deactivation) of GSK-3␤ and that oxidative stress has opposing effects on the modulation of this pathway.
Cell Cultures-PC12 and PC12-D 2 R cells were cultured as previously described in a humidified atmosphere containing 5% CO 2 at 37°C (14, 15, 19 -21). Medium was replaced with Opti-MEM1 3 h before various treatments of the cells. Dopaminergic neuronal cultures were prepared from embryonic day 14 rat fetuses (E14; Charles River Laboratories, Wilmington, MA) as previously described (22). Briefly, the ventral portion of the midbrain was removed in sterile ice-cold Ca 2ϩ and Mg 2ϩ -free HBSS/HEPES solution, cleaned free of meningeal tissue, and mechanically dissociated by passage through a flame-polished Pasteur pipette. Dissociated cells were plated at a density of ϳ1.2 ϫ 10 5 cells per cm 2 on poly-D-lysine-coated 24-well plates or glass coverslips. The neurons were maintained in a chemically defined medium consisting of Dulbecco's modified Eagle's medium/F-12 medium with N2 supplements, L-glutamine (0.5 mM), and penicillin/streptomycin (serum-free medium). Half of the culture medium was replaced every 2 days. Approximately 5-7-day-old cultures were used for experiments.
CellTiter-Blue Cytotoxicity Assays-For analysis of cell survival, cells were plated at a density of 10 4 cells/well on 96-microwell cell culture plates (in 100 l of medium) and grown for 24 h. Thereafter, 200 M H 2 O 2 was added either with or without ropinirole at the indicated concentrations, and cells were incubated for another 24 h. After treatments, 20 l of the Cell-Titer-Blue reagent was added to each well, and the plates were incubated for 2-3 h. The conversion of non-fluorescent Cell-Titer-Blue reagent to highly fluorescent substrate by living cells was quantified using a spectrofluorometer (Spectra Max Gemini XS, Molecular Devices, Sunnyvale, CA) as described (19,20). The data are expressed as percentages of the vehicle-treated controls, and the values represent the means Ϯ S.E. from eight microwells from each of three independent experiments (n ϭ 24). Uptake of [ 3 H]Dopamine-[ 3 H]Dopamine uptake by primary mesencephalic neurons was carried out as described previously (19). The results were expressed as percentages of vehicletreated control culture response.
For immunoprecipitation, the protein extract was incubated sequentially (2 h for each incubation at 4°C) with anti-GSK-3␤ antibody and protein A/G-agarose (Santa Cruz Biotechnology) with gentle agitation. Immunoprecipitates were washed 3 times with lysis buffer, boiled for 5 min in 3ϫ Laemmli sample buffer, and processed for Western blotting using GSK-3␤ or ␤-catenin antibody. The blots were stripped and reprobed with anti-␤actin antibodies.
Statistical Analysis-Data were analyzed by either two-tailed t test or analysis of variance followed by the Tukey's test to correct for multiple comparisons.

The Dopamine Agonist Ropinirole Protects PC12-D 2 R Cells and Primary Mesencephalic Neurons from Cell Death Induced by Oxidative Stress via Activation of the D 2 Receptors-We
studied the potential of the dopamine agonist ropinirole to protect against apoptosis induced by H 2 O 2 in PC12 cells that express D 2 receptors (PC12-D 2 R). Cells were preincubated (1 h) with varying concentrations of ropinirole (10 Ϫ11 to 10 Ϫ3 M) before the addition of H 2 O 2 (200 M). After 24 h of incubation, cell viability was assessed using the CellTiter-Blue cell death assay. The administration of 200 M of H 2 O 2 induced a 52.9 Ϯ 5.0% reduction in cell survival in comparison to controls. Ropinirole protected PC12-D 2 R cells from H 2 O 2 -induced apoptosis in a robust and concentration-dependent manner (Fig. 1A). In contrast, ropinirole did not protect PC12 cells that lacked D 2 receptors from exposure to H 2 O 2 . Ropinirole also did not protect against H 2 O 2 when PC12-D 2 R cells were pretreated with the dopamine antagonist haloperidol (10 M) before the addition of ropinirole (Fig. 1B).
Although PC12 cells are used as a good model to study dopaminergic function, they are non-neuronal cells derived from adrenal pheochromocytomas. Therefore, to further determine whether ropinirole protects dopamine neurons, we used primary rat mesencephalic neuronal cultures treated with 6-hydroxydopamine (6-OHDA). As shown in Fig. 1, C and D, ropinirole (1 M) offered significant neuroprotection against 6-OHDA-induced neuronal loss. Our results are consistent with the report that ropinirole protects the primary mesencephalic neurons from 1-methyl-4-phenylpyridinium, the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine toxicity (10). Ropinirole did not protect against 6-OHDA toxicity when primary mesencephalic neurons were co-incubated with ropinirole plus the dopamine antagonist eticlopride (10 M) (Fig. 1B). These results indicate that ropinirole protects dopamine neurons against cell death induced by H 2 O 2 and 6-OHDA and that this protection occurs by way of functional D 2 receptors.

Ropinirole Induced Protection in PC12-D 2 R Cells, and Primary Mesencephalic Neurons Involves the PI-3K/Akt
Signaling Pathway-We have previously reported that the increase in cell survival mediated by D 2 receptor activation is abolished by inhibitors of PI-3K (14). We, therefore, studied whether PI-3K signaling is modulated by the D 2 receptor when complexed with ropinirole. To determine whether PI-3K signaling is involved in ropinirole-mediated protection, we tested the effect of the PI-3K inhibitor LY294002 (10 M). Inhibition of PI-3K completely abolished the capacity of ropinirole to protect against cell death induced by oxidative stress in PC12 cells and by 6-OHDA in primary mesencephalic neuronal cultures (Fig.  1, B-D). However, LY294002 by itself had no effect on cell survival (Fig. 1B). These data suggest that activation of the PI-3K pathway contributes to the protective effects of ropinirole against apoptosis induced by oxidative stress in PC12-D 2 R cells and by 6-OHDA in primary mesencephalic neuronal cultures.
To examine if Akt, the principle downstream target of PI-3K (25), is implicated in ropinirole-mediated neuroprotection, we measured the translocation and phosphorylation of Akt after ropinirole administration. Akt phosphorylation and its protective effects occur after it translocates to the plasma membrane through an interaction of its N-terminal PH domain with phosphatidylinositol 3,4,5-triphosphate (PIP3) (26), thereby bringing the enzyme into the proximity of additional PIP3-dependent and -independent protein kinases (27). The distribution of Akt was assessed using a PH-Akt-GFP (15). In unstimulated cells, phosphorylated Akt was mainly localized in the perikarya (supplemental Fig. 1). In contrast, D 2 receptor activation by ropinirole caused a rapid (15 min) translocation of PH-Akt-GFP to peripheral membrane regions ( Fig.  2A, right panel). Ropinirole-induced translocation was similarly demonstrated using antibodies to endogenous phospho-Akt (Fig. 2, B and C). The addition of ropinirole to normal PC12-D 2 R cells was also associated with a significant increase in phosphorylated Akt, with phosphorylation occurring at the serine 473 (Ser 473 ) site. These results were also observed 15 min after the addition of the drug and returned to basal levels at 60 min (Fig. 2, D and E). Levels of total Akt protein remained unchanged. Thus, D 2 receptor stimulation by ropinirole in PC12-D 2 R cells causes rapid translocation and phosphorylation of Akt. These changes in Akt translocation and phosphorylation were prevented by co-administration of the PI-3K inhibitor LY294002 (data not shown), indicating that ropinirole induces Akt activation through a PI-3K signaling pathway.
Effects of Ropinirole on Downstream Effectors of PI-3K/Akt Signaling Pathway-The PI-3K/Akt pathway is known to promote cell survival by inactivating pro-apoptotic factors and activating anti-apoptotic factors by phosphorylation-dependent mechanisms (28 -30). To determine whether ropinirole induces the activation of any specific effectors of the PI-3K/Akt pathway, we examined the effect of ropinirole on the phosphorylation at Ser/Thr of Akt substrate proteins (28) when admin- istered to untreated PC12-D 2 R cells. Western blot analysis demonstrated that ropinirole treatment induced a transient accumulation of phosphorylated Akt substrates coincident with the timing of the phosphorylation of Akt (Fig. 3A). One of the phosphorylated downstream substrates of Akt was established to be GSK-3␤ (Fig. 3, B and C), which in its activated (dephosphorylated) state promotes cell death in response to oxidative stress (31). Ropinirole did not induce phosphorylation of p70S6 kinase or FKHR (Fig. 3B), two other downstream substrates of Akt. Blockade of PI-3K by LY294002 blocked the ropinirole-induced phosphorylation of Akt substrates including GSK-3␤ (Fig. 4).
Akt and GSK-3␤ Are Essential for Cell Survival-To investigate the role of Akt/GSK-3␤ signaling in cell survival, shRNA specific to Akt1 and GSK-3␤ were used (23). We used a PH-Akt-GFP construct to determine the transfection efficiency of PC12-D 2 R cells. Transient expression of PH-Akt-GFP yielded ϳ50 -60% GFP-positive cells after 48 h of transfection (data not shown). When normal PC12-D 2 R cells were transfected with either Akt shRNA or GSK-3␤ shRNA, markedly reduced cell viability was observed 48 h after transfection (Fig. 5, A and B). When compared with transfection of the U6-XASH3 HP control vector, transfection of hairpin small interfering RNA expression vectors against Akt and GSK-3␤ reduced the expression of Akt and GSK-3␤, respectively (Fig. 5, A and B). Because GSK-3␤ is a downstream effector of Akt, we examined the phosphorylation of GSK-3␤ in Akt knockdown cells. As shown in Fig. 5C, Akt suppression reduced phosphorylation of GSK-3␤ and the levels of the GSK-3␤ target ␤-catenin (32). To further confirm the role of Akt in cell survival, we have assessed the levels of Akt in cells transfected with Akt1 shRNA. We find that Akt levels are preserved in cells showing normal nuclear morphology and markedly reduced in cells showing condensed nuclei, indicating a strong correlation between cell death and low levels of Akt (Fig. 5D). These results suggest that Akt acting through GSK-3␤ signaling is necessary for the survival of PC12-D 2 R cells. Previously we have demonstrated that p53 and extracellular-regulated kinase (ERK) signaling plays an important role in mediating cell death and survival, respectively, in these cells (19 -21). A significant increase in the levels of phosphorylated p53 and decreased ERK was observed in Akt knockdown cells (Fig. 5C), suggesting a cross-talk between Akt and pro-and anti-apoptotic signaling pathways.
Oxidative Stress Causes, and Ropinirole Prevents, Dephosphorylation of Akt/GSK-3␤-It has previously been reported that activated (dephosphorylated) GSK-3␤ promotes cell death, whereas N-terminal serine phosphorylation deactivates GSK-3␤ and promotes cell survival (33)(34)(35)(36). We examined the phosphorylation states of Akt and GSK-3␤ after exposure to H 2 O 2 . We found that H 2 O 2 caused dephosphorylation of Ser 473 of Akt (which causes it to become inactive) and Ser 9 of GSK-3␤ (which causes it to become active) (Fig. 6, A and B). However, H 2 O 2 treatment had no effect on the total levels of Akt or GSK-3␤ proteins (Fig. 6A).
To determine the effect of oxidative stress on the activation state of GSK-3␤, we examined the levels of ␤-catenin, a transcriptional activator that is regulated by GSK-3␤. Phosphorylation of ␤-catenin by activated GSK-3␤ leads to its rapid degradation (for review, see Ref. 32). H 2 O 2 treatment of PC12-D 2 R  cells leads to dephosphorylation of Ser 9 of GSK-3␤ (activation) (Fig. 6, A and B) and increased degradation of ␤-catenin (Fig.  6C). Immunocytochemical staining of phospho-Ser 9 of GSK-3␤ and total ␤-catenin further confirm that treatment of PC12-D 2 R cells with H 2 O 2 dephosphorylates GSK-3␤ (Fig. 6D,   top left panel) and promotes degradation of ␤-catenin (Fig. 6D, bottom  left panel).
Because ropinirole protected primary mesencephalic neurons, we hypothesized that the increase in phospho-GSK-3␤ Ser 9 levels, indicative of decreased GSK-3␤ activity, might contribute to neuroprotection against 6-OHDA toxicity. We, thus, investigated the effects of ropinirole and 6-OHDA on the phosphorylation of GSK-3␤ in primary mesencephalic neurons. Ropinirole caused an increase in Ser 9 phosphorylation of GSK-3␤, specifically in tyrosine hydroxylase-positive dopaminergic neurons (Fig. 7 and supplemental Fig. 2). In contrast, 6-OHDA caused small but significant decrease in the levels of Ser 9 of GSK-3␤ in primary mesencephalic neurons ( Fig. 7 and supplemental Fig. 2). These results are compatible with our hypothesis that the activation state of GSK-3␤ contributes to the cell survival state and that increased serine phosphorylation of GSK-3␤ protein induced by ropinirole contributes to dopaminergic neuronal survival against oxidative stress.
To further examine the role of GSK-3␤ on cell death induced by oxidative stress, we examined the effects of the GSK-3␤ inhibitor VIII. Pretreatment (1 h) of PC12-D 2 R cells with the GSK-3␤ inhibitor VIII induced a significant increase in cell survival after H 2 O 2 treatment (Fig.  8). Significant neuroprotection was observed at a concentration of 0.5 M, and the maximal response was observed at a concentration of 1 M.
We next investigated the mechanism by which GSK-3␤ promotes cell survival and cell death. It is known that phosphorylation of ␤-catenin by GSK-3␤ causes its degradation by the ubiquitin proteasome system; therefore, we examined the activity of GSK-3␤ in cells treated with H 2 O 2 with or without ropinirole or GSK-3␤ inhibitor VIII pretreatment. GSK-3␤ has been shown to phosphorylate at Ser 33 , Ser 37 , and Thr 41 of ␤-catenin in vivo and in vitro (37). Thus, we used GST-tagged ␤-catenin protein as a substrate to measure the kinase activity of GSK-3␤. The kinase assay indicated that the ability of  A and B, p Ͻ 0.001 compared with XASH3 HP3. C, cells were transfected with Akt1 HP3 shRNA construct. After 48 h, total cell lysates were prepared, and Akt, GSK-3␤, phospho (p)-GSK-3␤, ␤-catenin, p53, and ERK levels were determined by Western immunoblotting. A shRNA construct XASH3 HP was used as a control. ␤-Actin was a loading control. Note that knockdown of Akt induces down-regulation of phosphorylated (deactivation) GSK-3␤, ␤-catenin, with increased levels of the p-p53 and reduced levels of ERK. D, immunocytochemical analysis of Akt levels in Akt knockdown cells. PC12-D 2 R cells were transfected with XASH3 or Akt1 shRNA and 44 h later labeled with anti-Akt antibody (red). In all, nuclei were stained with DAPI (blue). Note that Akt1 shRNA causes a reduction in the levels of Akt and that co-localizes in cells with condensed nuclei (arrows).
GSK-3␤ to phosphorylate ␤-catenin was significantly higher in H 2 O 2 -treated cells as compared with vehicle-treated cells or in H 2 O 2 -treated cells that were pretreated with ropinirole or GSK-3␤ inhibitor (Fig. 9A).

DISCUSSION
We have determined that GSK-3␤ is a downstream effector of the D 2 receptor-activated PI-3K/Akt signaling pathway that mediates neuroprotection against oxidative stress by the D 2 agonist ropinirole in dopaminergic PC12 cells and in primary mesencephalic cultures. We show that oxidative stress activates GSK-3␤ and inactivates Akt with resultant cell death. In contrast, ropinirole activates Akt and deactivates GSK-3␤ through activation of the D 2 receptor/PI-3K signaling pathway to mediate cell survival. Our results demonstrate that the differential modulation of the Akt/GSK-3␤ signaling pathway leads to cytoprotective or pro-apoptotic signaling responses.
We have previously reported that activation of D 2 receptors by some (but not all) experimental dopamine agonists can induce a PI-3K/Akt pro-survival signaling pathway in PC12-D 2 R cells that is linked to transactivation of the epidermal growth factor receptor. This effect is independent of activation of G proteins and varies in degree with different dopamine agonists (14,15). In the present study, we examined the signaling mechanisms whereby ropinirole might mediate a neuroprotective effect. We find no evidence for receptor-independent cytoprotective activities of ropinirole against oxidative stress in PC12 cells and 6-OHDA toxicity in primary mesencephalic neurons. Rather, we find that the activation of dopamine D 2 receptors is required for the prevention of apoptosis induced by H 2 O 2 in this model. This conclusion is based on the observations that similar protective effects could not be obtained in PC12 cells that lacked D 2 receptors or that were pretreated with haloperidol. Loss of neuroprotection observed with the D 2 /D 3 antagonist eticlopride in primary cultures of mesencephalic neurons treated with ropinirole similarly suggests that D 2 receptors are involved in mediating the neuroprotective effect of ropinirole. Furthermore, we show that these protective effects are dependent on the phosphorylation and deactivation of the Akt substrate GSK-3␤. Protective effects of ropinirole were lost with PI-3K inhibition in both PC12 cells and primary mesencephalic neurons. The phosphorylation of Akt and its translocation to the cell membrane in both its native and GFP-linked forms are also lost with PI-3K inhibition (which prevents phosphorylation and activation of Akt). Functional readout of Akt activation was demonstrated by an increase in the phosphorylation of downstream Akt substrates at an Akt-specific phosphorylation site. Activated Akt is known to promote cell survival by inactivating its pro-apoptotic substrates, such as FKHR, Bcl-2-associated death protein (BAD), caspase-9, and GSK-3␤ (30,32). Specifically, in our study we found that treatment with ropinirole and cell protection were associated with Akt-mediated phosphorylation at Ser 9 of GSK-3␤, which down-regulates its pro-apoptotic activity (39). The capacity of ropinirole to phosphorylate Ser 9 of GSK-3␤ (deactivation) and the loss of GSK-3␤ phosphorylation (activation) in the presence of a chemical inhibitor of PI-3K suggest that GSK-3␤ is a downstream effector of the D 2 receptor-mediated PI-3K/Akt signaling pathway. Akt suppression by shRNA resulted in cell death, indicating that Akt is necessary for cell survival. Down-regulation of GSK-3␤ Ser 9 phosphorylation and its target ␤-catenin in Akt knockdown cells further confirm that GSK-3␤ is downstream of Akt in PC12 cells. Activated Akt is known to promote cell survival by inactivating the pro-apoptotic protein p53 and activating anti-apoptotic ERK (30,32). The level of p53 is regulated by proteasomal degradation after its ubiquitination, which is mediated by the E3 ubiquitin ligase Mdm2. Akt plays a critical role in controlling Mdm2 activity (40). We have previously demonstrated the existence of a negative signaling cross-talk pathway from p53 to ERK in these cells (21). This ERK suppression pathway most likely contributes to the low levels of ERK observed in Akt knockdown cells.
GSK-3␤ activity is negatively regulated by Akt-mediated phosphorylation at Ser 9 in the pseudosubstrate domain (32). This pathway is activated in response to growth factors and neurotrophins (41,42) as well as ropinirole as shown in our study. Our data demonstrate that oxidative stress induced by H 2 O 2 leads to dephosphorylation of Akt (deactivation) and Ser 9 of GSK-3␤, indicative of activation of this pro-apoptotic molecule. The activation of GSK-3␤ by H 2 O 2 was confirmed by our finding of increased phosphorylation of GST-␤-catenin in H 2 O 2 -treated cells and of an interaction between GSK-3␤ and ␤-catenin with evidence of its degradation (Fig. 9). Chemical inhibition of GSK-3␤ activity by GSK-3␤ inhibitor VIII also prevented cell death caused by H 2 O 2 , further suggesting that Akt/GSK-3␤ signaling plays a critical role in cell death induced by oxidative stress. The critical role of GSK-3␤ in cell survival was further illustrated by evidence that a reduction of GSK-3␤ expression using a specific shRNA leads to cell death. Our results are consistent with the reports that GSK-3␤ plays a crit-  ) and an anti-phospho (p)-GSK-3␤ (red) antibodies as described under "Experimental Procedures." Nuclei were stained with DAPI (blue). Cultures then were examined by using fluorescent microscope. Experiments were repeated three times, and representative images are shown. Bottom panel, quantification of phospho-GSK-3␤ immunofluorescence signal in dopamine neurons. To measure the relative increase in cytoplasmic phospho-Ser 6 of GSK-3␤, line profiles that transected the neuron but avoided the nucleus were used to assess fluorescence intensity (see supplemental Fig. 2). Six to eight-line cells per group were assessed using ImageJ (NIH) to obtain an average profile of fluorescence intensity for each of the treatment groups. Note the marked increase (p Ͻ 0.001) in phospho-GSK-3␤ in ropinirole-treated dopamine neurons and a small but significant decrease (p Ͻ 0.05) in 6-OHDA-treated cells. ical role in a variety of neuronal cell death mechanisms including PD (33,35,(43)(44)(45)(46)(47)(48). GSK-3␤ is thought to induce apoptosis by promoting the release of cytochrome c from mitochondria and activating caspase-3 (43,49). Inhibition of GSK-3␤ activity by ropinirole, as indicated by phosphorylation at Ser 9 and reduced GSK-3␤ activity in ropinirole pretreated cells incubated with H 2 O 2 , suggest that interference with this molecule plays an important role in protecting dopaminergic cells in this model system as well.
It is noteworthy that deactivation of GSK-3␤ protects cells from H 2 O 2 toxicity but that complete knockdown of GSK-3␤ is not compatible with cell survival. In fact, it has been shown that inhibition of GSK-3␤ activity by either overexpression of the GSK-3␤-binding protein, FRAT-1, the use of a kinase-dead dominant negative mutant of GSK-3␤, or pharmacological inhibitors such as lithium, each, protects cortical neurons from trophic withdrawal (50). It has also been reported that marked chemical inhibition of GSK-3␤ prevents apoptosis induced by H 2 O 2 in neuronally differentiated PC12 cells (33). There is, thus, strong evidence indicating that activation of GSK-3␤ promotes apoptosis, but normal GSK-3␤ activity appears to be essential for cell survival. It is possible that GSK-3␤ acts through multiple pathways and that RNA inhibition interferes with its activity in a different way than the inhibitor. It appears that although GSK-3␤ in its inactive form can induce cell death, some level of GSK-3␤ activity is necessary for cell survival.
Taken together, our findings suggest that intracellular GSK-3␤ is a downstream target of the PI-3K/Akt signaling pathway and plays a major role in mediating the cell death induced by oxidative stress and the protection mediated by the dopamine agonist ropinirole. There are multiple downstream targets for GSK-3␤, some of which are involved in controlling cell survival against oxidative stress. To maintain redox homeostasis, aerobic cells have developed an antioxidant mechanism that includes a group of antixenobiotic genes termed phase II detoxification genes such as NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, glutamate-cysteine ligase, glutathione peroxidases, and heme oxygenase (51)(52)(53)(54). The transcription factor nuclear factor E2-related factor 2 regulates the expression of antioxidant phase II genes and contributes to preserve redox homeostasis and cell viability in response to oxidant insults (55,56). Recent reports suggest that a survival signal elicited by PI-3K/Akt acting through GSK-3␤ is the key mediator of the antioxidant phase II cell response (57,58). It is widely accepted that in PD, dopaminergic neurons have compromised antioxidant mechanisms. It will be interesting to investigate the role of antioxidant phase II genes in mediating the cell survival and death in experimental models of PD. It has been shown that there is a loss of oxidative stress tolerance with aging that is linked to a parallel reduction in Akt activity (59) and to an increase in GSK-3␤ activity (60).
It is not yet known if the Akt/GSK-3␤ signaling pathway is involved in the cell death process that occurs in PD. However, ropinirole has been shown to have protective effects in experimental models of PD, and a clinical trial shows positive effects of the drug on a biomarker of nigrostriatal function consistent with a protective effect (10,13,16,17). The molecular mechanisms described here could, thus, explain how ropinirole might provide neuroprotective effects in PD.