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Glycogen synthase kinase 3ß (GSK3ß) mediates 6-hydroxydopamine-induced neuronal death

GANG CHEN^*, KIMBERLY A. BOWER^*, CUILING MA^*, SHENGYUN FANG, CAROL J. THIELE and JIA LUO^*,,1

^* Department of Microbiology, Immunology & Cell Biology, West Virginia University School of Medicine, Robert C. Byrd Health Sciences Center, Morgantown, West Virginia, USA;
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland, USA;
Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland, USA; and
Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai, P.R. China

¹Correspondence: Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Morgantown, WV 26506, USA. E-mail: jluo{at}hsc.wvu.edu

SPECIFIC AIMS

The causes of sporadic Parkinson’s disease (PD) are poorly understood. 6-Hydroxydopamine (6-OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. The purpose of this study is to investigate mechanisms of 6-OHDA-induced neuronal apoptosis.

PRINCIPAL FINDINGS

1. 6-OHDA induces endoplasmic reticulum (ER) stress
ER stress, which is triggered by the loss of calcium homeostasis or an accumulation of unfolded/misfolded proteins in the ER lumen, is generally characterized by up-regulation of GRP78, GADD153, phosphorylation of eukaryotic initiation factor-2 (eIF2), and cleavage of ER-specific procaspase-12. We showed that 6-OHDA exposure caused a significant increase in the expression of GRP78, GADD153 and phosphorylation of eIF2 at Ser51 in a human dopaminergic neuronal cell line (SH-SY5Y), indicating the occurrence of ER stress. Similarly, we observed significant up-regulation of GRP78, GADD153, phosphorylation of eIF2, and cleavage of procaspase-12 after 6-OHDA exposure in primary cultures of cerebellar granule neurons (CGNs).

2. 6-OHDA activates glycogen synthase kinase-3 ß (GSK3ß)
Glycogen synthase kinase-3 ß (GSK3ß) is shown to respond to ER stress, and its activity is regulated by phosphorylation. GSK3ß activity is regulated negatively by the phosphorylation of serine 9 (Ser9) and positively by phosphorylation of tyrosine 216 (Tyr216). 6-OHDA significantly inhibited phosphorylation of GSK3ß at Ser9 but induced hyperphosphorylation of Tyr216 with little effect on GSK3ß expression in SH-SY5Y (Fig. 1 ). Similarly, 6-OHDA decreased phosphorylation of GSK3ß at Ser9 in PC12 cells and CGNs. Akt is one of the most important upstream signaling components that regulate GSK3ß phosphorylation at Ser9. 6-OHDA also induced a transient inhibition of Akt phosphorylation at Ser473 in SH-SY5Y cells, PC12 cells, and CGNs. Cyclin D1 is a substrate of GSK3ß; phosphorylation of cyclin D1 by GSK3ß facilitates a ubiquitin-dependent degradation of cyclin D1. 6-OHDA down-regulated cyclin D1 expression in SH-SY5Y cells and PC12 cells, indicating activation of GSK3ß. For a comparison, two ER stress inducers (thapsigargin and tunicamycin) dephosphorylated GSK3ß at Ser9 in a similar pattern. It has been demonstrated that ER stress-induced GSK3ß dephosphorylation (Ser9) is mediated by protein phosphatase 2A (PP2A) in SH-SY5Y cells. We sought to determine whether PP2A was involved in 6-OHDA-mediated GSK3ß dephosphorylation. Blocking PP2A activity by okadaic acid (1 nM) eliminated 6-OHDA-mediated GSK3ß dephosporylation (Ser9). At concentrations of <5 nM, okadaic acid specifically blocks PP2A activity without affecting PP1.

View larger version (34K): [in this window] [in a new window]   Figure 1. Effect of 6-OHDA on GSK3ß, Akt, and cyclin D1. SH-SY5Y cells were cultured in serum-free medium for 24 h and exposed to 6-OHDA (50 µM). Phosphorylation and expression of GSK3ß and Akt were determined by immunoblots. The same blots were stripped and reprobed with an anti-actin antibody (left). The relative amount of phosphorylated proteins imaged on the films was measured and quantified microdensitometrically (right panel). Each data point (±SEM; bars) is the mean of 3 replicates.

3. Blocking GSK3ß activity by selective inhibitors prevented 6-OHDA-induced neuronal apoptosis
It has recently been recognized that GSK3ß is an important modulator of apoptosis. To determine whether 6-OHDA-induced neuronal apoptosis was mediated by GSK3ß, we blocked GSK3ß activity by preincubating SH-SY5Y cells with lithium, a widely used selective GSK3ß inhibitor, or a synthetic chemical GSK3ß inhibitor (TDZD-8). TDZD-8 has been shown to specifically inhibit GSK3ß activity with little effect on other kinases. Pretreatment with TDZD-8 and lithium eliminated 6-OHDA-induced cell death as measured by cell viability (MTT assay) and apoptosis (DNA fragmentation ELISA) (Fig. 2 A, B). TDZD-8 and lithium blocked 6-OHDA-mediated caspase-3 activation and cleavage of PARP, which confirmed the neuroprotective effect of GSK3ß inhibitors. Similarly, neuroprotection of TDZD-8 and lithium against 6-OHDA-induced damage was observed in cultured CGNs. Involvement of GSK3ß in 6-OHDA-induced cell death was further validated using a novel cell-permeable peptide inhibitor of GSK3ß (L803-mts). L803-mts is a substrate-specific, competitive phosphorylated peptide inhibitor of GSK3ß, shown to selectively block GSK3ß activity without affecting the activity of other kinases. L803-mts protected SH-SY5Y and PC12 cells against 6-OHDA-induced cell death in a concentration-dependent manner (Fig. 2C ). L803-mts also blocked 6-OHDA-induced down-regulation of cyclin D1 (Fig. 2D ), indicating that this peptide effectively inhibited GSK3ß activity.

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of inhibitors of GSK3ß on 6-OHDA-mediated cell death. A) SH-SY5Y cells were cultured in serum-free medium and pretreated with inhibitors of GSK3ß, LiCl (Li, 20 mM), or TDZD-8 (10 µM) for 1 h. After that, cells were exposed to 6-OHDA (50 µM) and the numbers of viable cells were determined by MTT assay. The amount of cells relative to the untreated control was calculated. B) Apoptosis was determined with DNA fragmentation ELISA. Each data point (±SEM; bars) is the mean of 4 independent trials. *P < 0.05, statistically significant difference from untreated controls. C) SH-SY5Y and PC12 cells were cultured in serum-free medium and pretreated with a cell-permeable peptide inhibitor of GSK3ß (L803-mts, 0–20 µM) for 1 h. Cells were exposed to 6-OHDA (50 µM) and the number of viable cells was determined by MTT assay. Each data point (±SEM; bars) is the mean of 4 independent trials. *P <0.05, statistically significant difference from 6-OHDA-only groups. D) SH-SY5Y cells were pretreated with L803-mts (0 or 15 µM) for 1 h and exposed to 6-OHDA (50 µM). Expression of cyclin D1 was determined by immunoblots (top panel). The same blots were stripped and reprobed with an anti-actin antibody. The relative amount of cyclin D1 was measured microdensitometrically and normalized to the expression of actin (bottom panel). The experiment was replicated 3 times.

CONCLUSIONS

We demonstrate here that 6-OHDA, a PD mimetic, causes ER stress characterized by an increase in ER chaperones, cleavage of ER-specific caspase, and phosphorylation of eIF2. These findings are supported by recent studies showing that PD mimetics could induce unfolded protein response (UPR) and the expression of genes involved in ER stress response. The ER is an intracellular compartment that plays an important role in maintenance of Ca²⁺ homeostasis and proper folding of newly synthesized membranous and secretory proteins. Conditions associated with ER dysfunction induce highly conserved stress responses; these include the unfolded protein response (UPR), ER overloaded response (EOR), and ER-associated degradation (ERAD). Accumulating evidence indicates that ER stress may be a common denominator for acute brain injury (e.g., cerebral ischemia) and chronic degenerative diseases (Alzheimer’s disease and Parkinson’s disease). GSK3ß, a serine/threonine kinase originally identified as a regulator of glycogen metabolism, is now recognized as an important modulator of apoptosis. Recent studies indicate that cellular stress such as ER stress, oxidative stress, heat shock, and hyperosmotic stress can modulate GSK3ß activity. Our study shows that 6-OHDA induces GSK3ß dephosphorylation (Ser9). ER stress can activate PP2A, which dephosphorylates Akt and GSK3ß (Ser9). We show that blocking PP2A activity eliminates 6-OHDA-induced GSK3ß dephosphorylation (Ser9). More important, our study reveals that GSK3ß is a key intermediate for 6-OHDA-triggered apoptotic signaling; blocking GSK3ß activity prevents 6-OHDA-mediated caspase-3 activation, cleavage of PARP, and DNA fragmentation. Neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are promising agents for neuroprotective therapies. GDNF and BDNF can protect dopaminergic neurons against 6-OHDA-induced damage in an Akt-dependent manner. Akt is upstream of GSK3ß, and its activation results in an inhibition of GSK3ß. It is likely, therefore, that GSK3ß is involved in growth factor-mediated neuroprotection. These results suggest that neuroprotection provided by neurotrophic factors and GSK3ß inhibitors may share the same mechanism: modulation of a common intermediate, GSK3ß. Our study links PD mimetic-evoked ER stress to GSK3ß activation, providing important insight into the relationship between ER stress and degenerative diseases. The potential mechanism underlying 6-OHDA-induced neuronal death is summarized in Fig. 3 .

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic diagram of mechanisms of 6-OHDA-induced neuronal apoptosis. 6-OHDA triggers ER stress and activates PP2A, which dephopshophorylates Akt and GSK3ß (Ser9). This process blocks the anti-apoptotic signal of PI3K/Akt while it activates proapoptotic GSK3ß, which results in neuronal apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1551fje; doi: 10.1096/fj.04-1551fje

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