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Human Molecular Genetics Advance Access originally published online on October 20, 2005
Human Molecular Genetics 2005 14(24):3801-3811; doi:10.1093/hmg/ddi396
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alpha-synuclein-induced toxicity

Wanli W. Smith1, Haibing Jiang1, Zhong Pei1, Yuji Tanaka7, Hokuto Morita1,3, Akira Sawa1,3,6, Valina L. Dawson2,3,4,5,6, Ted M. Dawson2,3,5,6 and Christopher A. Ross1,2,3,6,*

1Division of Neurobiology, Department of Psychiatry, 2Department of Neurology, 3Department of Neuroscience, 4Department of Physiology, 5Institute for Cell Engineering and 6Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA and 7Department of Neuropsychiatry, Okayama University School of Medicine, Okayama 7008558, Japan

* To whom correspondence should be addressed at: Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, CMSC 8-121, 600 North Wolfe Street, Baltimore, MD 21287, USA. Tel: +1 4106140010; Fax: +1 4106140013; Email: caross{at}jhu.edu

Received August 15, 2005; Accepted October 13, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to ~40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Parkinson's disease (PD) is a neurological disorder associated with tremor, muscle rigidity, disturbances of balance and bradykinesia. The two pathological hallmarks of PD are loss of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic eosinophilic inclusions termed Lewy bodies and Lewy neurites (1Go). The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors such as oxidative stress and proteasome inhibition (1Go–4Go). Mutations A53T, A30P and E46K in the alpha-synuclein gene cause rare familial forms of PD, suggesting that alpha-synuclein, a major protein component in Lewy bodies and Lewy neurites of both familial and sporadic PD, plays a role in the neurodegenerative process (5Go–7Go). Recent reports have demonstrated that alpha-synuclein over-expression results in the degeneration of dopaminergic neurons and motor deficits in Drosophila melanogaster and transgenic mice (8Go–12Go). These findings indicate that alpha-synuclein may play an important role in both familial and sporadic PD, but the molecular mechanisms underlying these pathological effects are unclear.

Alpha-synuclein is a highly conserved protein of 140 amino acids that is expressed predominantly in neurons and is abundant in pre-synaptic terminals. It may play a role in neuronal plasticity (13Go) and may regulate synaptic maturation and maintenance (14Go–16Go). Expression of mutant alpha-synuclein has been achieved in a number of cellular systems, with results ranging from a lack of an effect after over-expression (17Go), to adverse effects of varying severity (18Go–22Go). We and others have shown that the expression of mutant alpha-synuclein heightens the cytotoxic response of cells challenged with various stress agents (23Go–25Go). Recent biochemical studies and findings in transgenic mice suggest that A53T alpha-synuclein appears to exhibit more severe effects than A30P alpha-synuclein (8Go).

To investigate the effects of mutant A53T alpha-synuclein in a dopamine-containing cell environment, we generated mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. We previously showed that inducible expression of A30P, but not wild-type, alpha-synuclein in media containing 1% horse serum (HS) and 0.5% FBS is only toxic in combination with subtoxic doses of a proteasome inhibitor, lactacystin (23Go). Here, we found that using regular growth media (10% HS and 5% FBS), induction of either wild-type or mutant (A30P and A53T) alpha-synuclein expression was not toxic. However, in media without serum but containing the N2 supplement and nerve growth factor (NGF) (the induction and differentiation media), expression of A53T alpha-synuclein alone caused cell death even without additional cellular stressors. In contrast, induction of wild-type or A30P alpha-synuclein expression in similarly cultured cells with the same media was not toxic. Further investigation of the mechanisms underlying A53T alpha-synuclein-triggered toxicity revealed that expression of A53T alpha-synuclein decreased proteasome activity, increased intracellular ROS levels, and increased caspase-3, -9 and -12 activities. Interventions to block caspase activity and endoplasmic reticulum (ER) stress using inhibitors, and to knock down the expression of caspase-12 using siRNA, protected against A53T alpha-synuclein-induced cell death. Our results indicate that induction of A53T alpha-synuclein expression caused ER stress and mitochondrial dysfunction resulting in neuronal cell death.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of A53T alpha-synuclein expression causes cell death
To study the role of mutant A53T alpha-synuclein in PD pathogenesis, we employed the rat pheochromocytoma cell line PC12, which expresses dopamine and can be differentiated to a neuronal-like phenotype by NGF. We generated PC12 cell lines in which either wild-type alpha-synuclein or alpha-synuclein mutants A30P (23Go) or A53T were inducible using the Tet-off gene regulatory system. Cells expressed alpha-synuclein in the absence of Dox (induced condition) and did not express alpha-synuclein in the presence of Dox (repressed condition) (Fig. 1A and C). Inducible expression of either wild-type, A30P or A53T alpha-synuclein in media (without Dox) containing 10% HS and 5% FBS for 8 days and did not cause toxicity. Previously, we showed that the expression of A30P alpha-synuclein after induction and differentiation for 7 days, followed by lactacystin treatment for 24 h causes cell death (in media without Dox, containing 1% HS, 0.5% FBS and 100 ng/ml NGF), whereas the expression of wild-type alpha-synuclein in cells cultured similarly was indistinguishable from non-induced cells (23Go). Here, differentiation and induction of A53T alpha-synuclein expression alone for 6 days in media without Dox and serum, but containing N2 supplement and 100 ng/ml NGF, were found to increase cell death 3-fold, when compared with the non-induced condition (Fig. 1B and D). In contrast, cell death was unchanged following induction of wild-type or A30P alpha-synuclein for 6 days under similar culture conditions (Fig. 1B). We used four different clones with different expression levels of either wild-type or mutant alpha-synuclein and obtained similar results (data not shown).



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  		Figure 1. Inducible expression of mutant A53T alpha-synuclein causes cell death. (A) Representative western blot analyses of alpha-synuclein expression levels in induced (Dox–) and non-induced (Dox+) conditions. (B) Expression of A53T caused cell death by 6 days after induction and differentiation using Trypan blue exclusion assay, whereas expression of wild-type and A30P alpha-synuclein exhibited no toxicity. *P<0.05 versus non-induced condition. (C) Representative western blot analyses of A53T alpha-synuclein expression levels in induced (Dox–) and non-induced (Dox+) conditions following induction and differentiation. (D) Cells expressing A53T alpha-synuclein exhibited toxicity in a time-dependent manner. Trypan blue exclusion was used to determine cell death. *P<0.05 versus non-induced conditions. (E) Cells expressed A53T alpha-synuclein for the indicated times (days) and were then cultured with Dox to shut down its expression. Dox+ and Dox– were induced and non-induced for 6 days. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus non-induced conditions.

 
Inducible expression of alpha-synuclein can be detected by western blotting 18 h after induction. The expression level of A53T alpha-synuclein (Fig. 1C) as well as wild-type or A30P alpha-synuclein (data not shown) reached peak levels at 4 days after induction. Expression of A53T alpha-synuclein caused cell death in a time-dependent manner. Six days after induction, cell death was up to ~40% (Fig. 1B and D). Whereas non-induced cells displayed a pyramidal shape with processes extending from each cell after differentiation with NGF and homogeneously labeled nuclei, induced cells exhibited a shrunken morphology with rounded cell bodies, retracted processes and highly condensed nuclei as measured by Hoechst 33342 and propidium iodide (PI) fluorescent labeling. On the basis of these observations, cell death was primarily apoptotic with ~3–5% necrotic cell death. By switching off the expression of A53T alpha-synuclein expression at various time points, we found that 1 day of expression of A53T alpha-synuclein was enough to cause cell toxicity, as measured by lactate dehydrogenase (LDH) analysis at day 6, and re-addition of Dox could not rescue the cells (Fig. 1E).

Expression of A53T mutant alpha-synuclein decreases proteasome activity and increases intracellular ROS in PC12 cells
Proteasome inhibition has been widely reported to cause apoptosis (26Go,27Go) and has been implicated in PD (2Go,28Go–31Go). Previously, we showed that proteasome activity in cells expressing A30P alpha-synuclein was lower than that in non-induced cells or cells expressing wild-type alpha-synuclein (23Go). Here, we found that the expression of A53T alpha-synuclein potently inhibited chymotryptic proteasome activity, when compared with the effect of expressing A30P alpha-synuclein (Fig. 2A). We also measured tryptic and post-acidic proteasome activities and obtained similar results (data not shown). This proteasome inhibition occurred as early as 1 day induction. Furthermore, by 1 day after inducing the expression of A53T alpha-synuclein, the intracellular level of ROS increased significantly relative to the levels seen in non-induced cultures or in cultures expressing inducible wild-type or A30P alpha-synuclein (Fig. 2B). These results indicate that the expression of A53T alpha-synuclein in PC12 cells causes early biochemical changes, leading to oxidative stress and proteasome inhibition.



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  		Figure 2. Decreased proteasome activity and increased intracellular ROS after induction of mutant alpha-synuclein. (A) Chymotryptic proteasome activities and intracellular ROS levels were measured at the indicated time periods after induction and also in non-induced cultures. Proteasome activities in induced conditions were normalized to those in non-induced cultures. (B) The intracellular ROS levels in non-induced conditions for all of the cell lines were comparable to those of induced pTet-off cells (data not shown); shown here are only the induced conditions of all cell lines. Data are shown as the mean±SE for three separate experiments performed in duplicate.

 
Expression of A53T alpha-synuclein decreases mitochondrial polarization and increases cytochrome c in cytosol
To study whether the expression of A53T alpha-synuclein alters mitochondrial depolarization, we used the fluorescentdye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Under non-induced conditions, there were no differences in mitochondrial membrane potentials in either cell line (Fig. 3A and B). In contrast, in induced conditions, cells expressing A53T alpha-synuclein showed a significant decrease (33%) in mitochondrial polarization, whereas cells expressing wild-type alpha-synuclein showed no difference (Fig. 3A).



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  		Figure 3. Inducible expression of A53T alpha-synuclein causes mitochondria-dependent cell death. (A) Cells expressing mutant A53T alpha-synuclein showed decreased mitochondrial polarization relative to cells expressing wild-type alpha-synuclein. Mitochondrial polarization was monitored by JC-1 red fluorescence. Data are shown as the mean±SE for three separate experiments performed in duplicate. *P<0.05 versus non-induced cells, #P<0.05 versus cells expressing wild-type alpha-synuclein. (B) CsA reversed the decrease of mitochondrial polarization triggered after inducing the expression of A53T alpha-synuclein. Mitochondrial polarization was monitored as described in (A). Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus non-induced cells, #P<0.05 versus untreated cells expressing A53T alpha-synuclein. (C) CsA inhibited cytochrome c release from mitochondria after inducing the expression of A53T alpha-synuclein. Cytochrome c was detected by ELISA. Data are shown as the mean±SE for three separate experiments performed in duplicate. #P<0.05 versus untreated cells expressing A53T alpha-synuclein. (D) CsA partially protected against A53T alpha-synuclein-induced cell death. Trypan blue exclusion was used to determine cell death. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus non-induced cells, #P<0.05 versus untreated cells expressing A53T alpha-synuclein.

 
Release of cytochrome c into the cytosol is a marker of mitochondrial dysfunction. We found that the induction of A53T expression increased the release of cytochrome c from the mitochondria and its accumulation in the cytosol, compared with the non-induced condition (Fig. 3C). To further assess the role of mitochondrial dysfunction in cell death induced by A53T alpha-synuclein, we used cyclosporin A (CsA), which inhibits the MPT process. MPT induces mitochondrial depolarization, is itself augmented by depolarization, and participates in the initiation of apoptosis (32Go–34Go). Treatment with CsA reversed mitochondrial polarization back to the non-induced condition and partially protected against cell death (Fig. 3B–D). Treatment with CsA reduced cytochrome c in the cytosol similar to the non-induced condition (Fig. 3C). These results indicate that the induction of A53T alpha-synuclein expression causes mitochondrial dysfunction.

Involvement of caspase-9/-3 activation in A53T alpha-synuclein-induced cell death
To determine whether A53T alpha-synuclein-induced cell death involved the activation of caspases, caspase-3, -8 and -9 activities were measured colorimetrically using DEVD-p-nitroanilide, IETD-p-nitroanilide or LEHD-p-nitroanilide as substrates, respectively. In lysates of cells after inducible expression of A53T alpha-synuclein, caspase-3 and -9 activities increased significantly over time, in marked contrast with the lack of increased activities of these caspases in the non-induced population (Fig. 4A and C). Moreover, we did not detect significant increases in caspase-8 activity (Fig. 4B). To further investigate caspase involvement in A53T alpha-synuclein-induced caspase-dependent cell death, specific inhibitors of caspase-3, -8 and -9 were used. Use of z-DEVD and z-LEHD, inhibitors of caspase-3 and -9, respectively, significantly protected against A53T alpha-synuclein-induced cell death, to an extent comparable to that achieved through use of the pan-caspase inhibitor z-VAD; in contrast, the caspase-8 inhibitor z-IETD did not alter cell death (Fig. 5). Caspase-8 and -9 are two key initiators of distinct caspase signaling pathways (35Go). Both of them can activate caspase-3, which is one of the key executioner caspases in apoptotic cell death (36Go). Our results suggest that the caspase-9/-3 cascade plays an important role in A53T alpha-synuclein-induced cell death, whereas the caspase-8/-3 cascade appears not to be involved.



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  		Figure 4. Selective activation of caspases after induction of A53T alpha-synuclein. (A) Caspase-3 activity increased in a time-dependent manner in induced cultures but remained unchanged in non-induced cultures. (B) Lack of significant differences in caspase-8 activation when comparing induced and non-induced cultures. We used treatment of Fas ligand as positive control in the caspase-8 activity assay.(C) Caspase-9 activity increased in a time-dependent manner in induced cultures, remaining unchanged in non-induced cultures. Data are shown as the mean±SE for three separate experiments performed in duplicate.

 


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  		Figure 5. Effects of caspase inhibitors on A53T alpha-synuclein-triggered toxicity. (A and B) Cells expressing A53T alpha-synuclein were left untreated or treated with either 50 µM z-VAD, 50 µM z-LEHD, 50 µM IETD or 50 µM DEVD. Trypan blue exclusion (A) and LDH (B) assays were used to determine cell death. Cas, caspase. Data represent the mean±SE for three separate experiments performed in duplicate. *P<0.05 versus non-induced conditions. #P<0.05 versus untreated cells expressing A53T alpha-synuclein.

 
Involvement of caspase-12 activation and ER stress in A53T alpha-synuclein-induced cell death
To determine whether the accumulation of A53T alpha-synuclein elicits ER stress, we investigated downstream effectors of ER stress, including eIF2{alpha}, chaperone GRP78, GADD153 and caspase-12, in cells that were non-induced or induced to express A53T alpha-synuclein. As shown in Figure 6, the level of phosphorylated eIF2{alpha} (an indicator of PERK activation) in induced cells increased significantly by 5 and 6 days after induction, as did GADD153. The levels of expression of the chaperone GRP78 were also slightly increased in induced cells relative to non-induced cells (Fig. 6A). A hallmark of ER stress-induced apoptosis is the cleavage and activation of the ER membrane-associated procaspase-12 (37Go–39Go). By 6 days after induction, the activity of caspase-12 was significantly increased in induced cells relative to non-induced cells (Fig. 6B). These results suggest that the expression of A53T alpha-synuclein causes ER stress.



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  		Figure 6. Expression of A53T alpha-synuclein induces ER stress and caspase-12 activation. (A) Lysates prepared from A53T alpha-synuclein cells in induced (Dox–) and non-induced (Dox+) conditions at the times indicated following induction and differentiation were subjected to western blot analysis using anti-phospho-eIF2-{alpha}, anti-eIF2-{alpha}, anti-GRP78 or anti-GADD153 antibodies. The experiment was repeated three times with similar results. Lysates prepared from PC12 cells treated with Tunicamycin (a well-known ER stressor) as positive control. (B) Caspase-12 activity was significantly increased after 5 and 6 days inducible expression of A53T alpha-synuclein. Data are shown as the mean±SE for three separate experiments performed in duplicate.

 
The broad spectrum inhibitor z-VAD preferentially prevents cell death through the caspase-9 or -3 pathway at lower doses, but can inhibit caspase-12 activity at high doses (38Go). To study the participation of caspase-12 in A53T-induced cell death, z-VAD and CsA were used to determine the relationship between the activation of caspase-12 and the mitochondrial/caspase-9/caspase-3 cascade. Treatment of cells with z-VAD partially prevented cell death induced by A53T alpha-synuclein induction. Fifty micromolar z-VAD exhibited maximal protection, as increasing the dosage to 100 µM did not protect further (Fig. 7A). Indeed, under non-induced conditions, treatment with concentrations of z-VAD >100 µM caused toxicity. Treatment with z-VAD completely blocked A53T alpha-synuclein-induced increase of caspase-9 and -3 activities but only partially blocked caspase-12 activity (Fig. 7B–D). However, treatment with CsA did not alter caspase-12 activity (Fig. 7D), suggesting that the caspase-12 activation and mitochondrial depolarization were parallel early events in the A53T alpha-synuclein-induced cell death pathways.



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  		Figure 7. Treatment with z-VAD partially protects against A53T alpha-synuclein-induced toxicity and caspase-12 activation. (A) Cells expressing A53T alpha-synuclein were either left untreated or treated with z-VAD at the concentrations indicated. Cell death was measured by LDH assay. Data are shown as the mean±SE for three separate experiments performed in duplicate. (B and C) Cells expressing A53T alpha-synuclein were either left untreated or treated with 50 µM z-VAD. Caspase-3 (B) and caspase-9 (C) activities were monitored. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus untreated cells expressing A53T alpha-synuclein.(D) Cells expressing A53T alpha-synuclein were either left untreated or treated with z-VAD or CsA. Caspase-12 activity was monitored. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus untreated cells expressing A53T alpha-synuclein.

 
Further evidence in support of a role for caspase-12 in the toxicity engendered by A53T was obtained through experiments aimed at inhibiting caspase-12 expression by short interfering RNA (siRNA). After transfection of caspase-12-directed siRNA into A53T-PC12 cells, western blot analysis showed that the expression of caspase-12 in A53T-PC12 cells decreased by ~ 80%, when compared with transfection groups in which a control ‘scrambled’ siRNA were transfected (Fig. 8A). Knockdown of caspase-12 expression also further protected against cell death after treatment of z-VAD (Fig. 8B and C). To confirm these findings, we used salubrinal, an ER stress inhibitor, which can regulate eIF2{alpha} phosphorylation (40Go). Like z-VAD, treatment of cells with 5 µM salubrinal alone partially prevented cell death induced by A53T alpha-synuclein induction (Fig. 8D). Increase of the dosage of salubrinal did not further increase the protective effect. Interestingly, treatment of cells with 5 µM salubrinal plus 50 µM z-VAD almost completely prevented cell death induced by A53T alpha-synuclein induction (Fig. 8D). Taking together, these results indicated that both ER and mitochondrial dysfunction contributed to A53T alpha-synuclein-induced cell death.



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  		Figure 8. Effects of z-VAD and ER stress inhibition on A53T alpha-synuclein toxicity. (A) Representative western blot analysis shows the expression level of caspase-12 in non-induced (Dox+) conditions at the times indicated following differentiation and transfection of siRNA-targeting caspase-12 in A53T-PC 12 cells, using an anti-caspase-12 antibody (top). NS, the non-specific bands show loading control. 737-siRNA was targeted rat caspase-12 (5'-CCGACAGCACATTCCTGGTCTTTAT-3'), complementary to the caspase-12 mRNA starting from nucleotide 737. (B and C)A53T-PC 12 cells were left untransfected or transfected with either caspase-12 siRNA or scrambled RNA. Five hour after transfection, media were removed and changed with differentiation and induction media. At the same time, cells were either left untreated or treated with 50 µM z-VAD. Cell death was measured 6 days after transfection and induction. (B) LDH assay; (C) Trypan blue exclusion assay. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus untransfected induced condition cells. (D) Cells expressing A53T alpha-synuclein were either left untreated or treated with 50 µM z-VAD or 5 µM salubrinal or 50 µM z-VAD plus 5 µM salubrinal. Cell death was measured 6 days after transfection and induction by LDH assay. Data are mean±SE for three separate experiments performed in duplicate. *P<0.05 versus untreated induced condition cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we found that A53T alpha-synuclein, but neither wild-type nor A30P alpha-synuclein, directly induced cell death in cultures following a differentiation program that induces a dopamine-containing neuronal-like phenotype. Unlike our previous results with A30P alpha-synuclein (23Go), the cell toxicity was directly caused by the expression of A53T mutant alpha-synuclein without additional stressor stimuli (in media without serum but containing N2 supplement and NGF). Expression of A53T caused early biochemical changes including decreased proteasome activity and increased intracellular ROS levels. Furthermore, we have identified two major cell death pathways, ER stress and mitochondrial dysfunction, likely acting in concert that contribute to A53T alpha-synuclein-induced cell death.

Oxidative stress and proteasome inhibition have been implicated in PD and other neurodegenerative disorders (3Go,4Go,28Go,41Go,42Go). Proteasome inhibition can cause apoptosis (26Go,27Go) and increased intracellular ROS levels can cause DNA and protein damage leading to cell death (43Go,44Go). A number of reports have shown that both proteasome inhibition and ROS can trigger mitochondrial and ER stress cell death pathways (45Go–48Go). Here, we found that the expression of mutant alpha-synuclein decreased proteasome activity and increased intracellular ROS levels as early as 1 day following induction. One day of A53T alpha-synuclein expression is enough to cause cell toxicity and cannot be rescued by re-addition of Dox, indicating that proteasome inhibition and increased ROS levels were critical early events in A53T-induced cell death. Inducible expression of alpha-synuclein can be detected by western blot 18 h after induction and reached peak levels by 4 days after induction. Increases in caspase-9 and -3 activities were detected by 3 days following induction, whereas caspase-12 activity was found elevated by 4 days after induction. By day 5, caspase-12, phosphorylated eIF2-{alpha} and GADD153 expressions increased significantly. These data suggest the following order of events of A53T alpha-synuclein-caused cell death in this PC12 model: first, expression of A53T alpha-synuclein decreased proteasome activity and increased intracellular ROS levels. Secondly, mitochondrial cell death pathways are activated by day 3 and remain active till cell death. Thirdly, the ER stress cell death pathway is activated by day 4, as this pathway seems to response to high levels of alpha-synuclein expression and greater proteasome inhibition to trigger an accumulation of unfolded proteins to initiate cell death. In addition, treatment with z-VAD completely blocked A53T alpha-synuclein-induced increase of caspase-9 and -3 activities but only partially blocked caspase-12 activity. Treatment with CsA did not alter caspase-12 activity and only partially protected against cell death. These results indicate that caspase-12 activation and mitochondrial depolarization were parallel events in the A53T alpha-synuclein-induced cell death pathways.

How A53T alpha-synuclein decrease of proteasome activity in our PC12 cell model remains to be identified. It is possible that mutant alpha-synuclein directly affects the proteasome complex because it has been reported that alpha-synuclein interacts with a subunit of proteasome regulatory complexes (49Go). In our cell system, we found a small amount of aggregation of A53T alpha-synuclein, consistent with recent data indicating a complex relation between aggregation and toxicity (50Go). PD patients show increased levels of oxidative damage to DNA, lipids and proteins (43Go,44Go,51Go,52Go). In vitro studies show that oxidative stress augments alpha-synuclein aggregation (53Go,54Go). Oxidatively modified alpha-synuclein is more prone to aggregation than native protein (55Go,56Go). Alpha-synuclein can enhance the formation of hydrogen peroxide in vitro (57Go,58Go), whereas dopamine metabolism or oxidative phosphorylation can produce ROS (59Go–62Go). Given these findings, the expression of A53T alpha-synuclein in PC12 cells, which contain dopamine, may increase ROS by changing dopamine metabolism or affecting the formation of H2O2.

Previously, we and others found that mitochondrial dysfunction may have a role in the pathogenesis of PD (23Go,63Go,64Go). Here, we report that cells expressing mutant A53T alpha-synuclein alone exhibited a selective increase in mitochondrial depolarization and cytochrome c accumulated in cytosol resulting in cell death. The ensuing mitochondrial dysfunction and cell death were reduced by treatment with CsA, suggesting that mitochondrial dysfunction is involved in A53T alpha-synuclein-induced cell death. We further showed that the activities of caspase-9 and -3, two downstream effectors of mitochondrial dysfunction (32Go), increased after A53T alpha-synuclein induction, whereas caspase-8 activity remained unchanged. Use of inhibitors of caspase-3 and -9, as well as use of the CsA and pan-caspase inhibitor z-VAD, partially protected against A53T alpha-synuclein-induced cell death, whereas a caspase-8 inhibitor had no effect. These findings indicate that A53T alpha-synuclein-induced cell death was partially dependent on mitochondrial dysfunction via a caspase-9 or -3 pathway, but was not dependent on a caspase-8-mediated pathway. Consistent with our findings, the recent studies have shown that caspase 9 is activated, and mitochondrial function is impaired by over-expressing alpha-synuclein in the rat substantia nigra (65Go).

Recent studies point to the ER as an additional subcellular compartment implicated in the execution of cell death. ER stress stimuli activate the unfolded protein response (UPR) signal transduction pathways, which initiate cell death (66Go). The UPR activates the ER transmembrane kinase PERK, activates ER resident caspase-12 and up-regulates the expression of ER stress response genes, including GRP78 (a chaperone residing in the ER), and gadd153 (a growth arrest and DNA damage-inducible gene, also known as CHOP). PERK activation induces the phosphorylation of the translation initiation factor eIF2{alpha}, which causes global inhibition of protein synthesis (67Go). In the cell model described here, induction of the expression of A53T alpha-synuclein caused ER stress, as evidenced by the elevation in the expression of GADD153/CHOP and GRP78, the increased phosphorylation of eIF2{alpha} and the activation of caspase-12. We also found that RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our findings here in PC12 cells may be relevant to human cell systems. To our knowledge, the sequence of human caspase-12 has not yet been reported; however, earlier observations in HeLa cells (38Go) and 293T cells (39Go) indicated that there exists a ‘human caspase-12-like protein’ that is recognized by mouse caspase-12 antibodies. The human caspase-12-like protein has a similar molecular mass as the mouse caspase-12 and is a phosphoprotein (68Go). Our results suggest that activated caspase-12 may directly process downstream caspases in cytosol such as caspase-9, in keeping with previous reports (69Go). The cell death triggered by activated caspase-9 was prevented by z-VAD, as the inhibitor was capable of totally blocking the A53T alpha-synuclein-induced caspase-9 and -3 activation. Alternatively, activated caspase-12 may target other, as yet unidentified substrates that influence or cause the progression towards cell death. Cell death could not be prevented by z-VAD because z-VAD only partially inhibited caspase-12 activation. Our results strongly suggest that mitochondrial dysfunction and ER stress both contribute to A53T alpha-synuclein-induced cell death but do not exclude the potential involvement of other cell death pathways.

The findings presented here provide mechanistic insight into the pathogenesis of cell death caused by the expression of the A53T mutant alpha-synuclein, which may be relevant to neuronal loss in PD pathogenesis. This is the first report of ER stress and caspase-12 activation mediating A53T mutant alpha-synuclein-induced toxicity. Our findings should be extended to studies in transgenic mice and human tissues. In summary, our results demonstrate that mitochondrial dysfunction and ER stress both contribute to A53T alpha-synuclein-induced cell death. This study may help clarify the pathogenesis of mutant alpha-synuclein cellular toxicity in PD and may provide a cell model for screening PD therapeutics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Media and N2 supplements for cell culture were from Invitrogen (Carlsbad, CA, USA). NGF was from Roche (Indianapolis, IN, USA), Hoechst 33342, PI and DCFDA from Molecular Probes (Eugene, OR, USA) and CsA from BioMol (Plymouth Meeting, PA, USA). Anti-alpha-synuclein monoclonal antibody (directed against amino acids 15–123 of rat alpha-synuclein but exhibiting good reaction with human alpha-synuclein) was from BD Biosciences (Palo Alto, CA, USA). Anti-caspase-12 antibody was from Sigma (Saint Louis, MO, USA). Anti-eIF2{alpha} and anti-phospho-eIF2{alpha} antibody were from Cell Signaling Technology (Beverly, MA, USA). Anti-GADD153 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Salubrinal was from Calbiochem (San Diego, CA, USA).The caspase inhibitors z-VAD-fmk, DEVD-fmk, IETD-fmk and LEHD-fmk were purchased from Enzyme Systems Products(Livermore, CA, USA).

Cell culture and inducible cell lines
Cells were grown in DMEM containing 10% HS and 5% FBS in a 5% CO2 atmosphere. PC12 Tet-off cells (Clontech) were used to create PC12 cell lines expressing inducible A53T alpha-synuclein. Tet-responsive alpha-synuclein expression constructs were engineered by cloning full-length cDNA of A53T mutant alpha-synuclein into pTRE (Clontech) vector. A53T alpha-synuclein construct was co-transfected into PC12 Tet-off cells with pTK-Hyg (Clontech) at a 10:1 molar ratio. Single colonies were obtained by limiting dilution of cells cultured in the presence of 100 µg/ml G418, 200 µg/ml hygromycin B (Clontech) and 200 ng/ml Dox for 2–3 weeks. Clones were analyzed for the expression of alpha-synuclein by western blotting. Wild-type and A30P alpha-synuclein PC12 cell lines were generated as previously described (23Go). Differentiation was initiated by the addition of 100 ng/ml NGF to the culture medium (DMEM with N2 supplement). NGF was replenished every day after differentiation.

Measurement of cell death and viability
Trypan blue exclusion was used to measure cell death by counting the number of dead (blue) and live cells in the cultures after induction of expression. Hoechst/PI labeling of cells to detect apoptotic and necrotic cell death were performed as described previously (70Go). LDH cytotoxicity assay was performed according to the manufacturer's protocol (Roche); the colorimetric assay quantifies LDH activity released from the cytosol of damaged cells into the supernatant and thus serves to quantify cell death.

Measurements of intracellular ROS
The levels of cytosolic ROS were measured by DCFDA (Molecular Probes) as previously described (71Go). Briefly, cells were induced or non-induced at indicated time period, washed with PBS, then incubated for 45 min with DCFDA, which is initially non-fluorescent and is converted by oxidation to the fluorescent molecular DCF. DCF was then quantified using a CytoFluor Multi-well Plate Reader, Series 400 (Perseptive Biosystems) with 485 nm excitation and 538 nm emission filters.

Proteasome activity
Proteasome activity was measured at indicated time period after induction and differentiation. The fluorogenic substrates, Suc-LLVY-AMC, Z-LLE-AMC and Boc-LRR-AMC, were used to measure the chymotryptic-like, tryptic-like and post-acidic activities, respectively, as previously described (23Go). Cells were harvested using proteolysis buffer (10 mM Tris–HCl (pH 7.8), 0.5 mM dithiothreitol, 5 mM ATP and 5 mM MgCl2). The fluorogenic substrate was incubated with different lysates for 60 min at 37°C. Cleavage products were measured using a CytoFluor Multi-well Plate Reader, Series 400 (Perseptive Biosystems) with 380 nm excitation and 460 nm emission filters. Background fluorescence was determined by incubating lysates with the proteasome inhibitor lactacystin (50 µM) for 30 min prior to the addition of proteasome substrate.

Western blot analysis
Cells were harvested in lysis buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM ß-glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Na3VO4 and 5 mM NaF). Lysates were resolved on 4–12% NuPAGE Bis-Tris gels (30 µg/lane) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked in TBST (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat milk and then probed with different antibodies. Proteins were detected by using enhanced chemiluminescence reagents (NEN Life Science, Boston, MA, USA).

Measurement of cellular caspase activity
Cells were harvested in cell lysis buffer (50 mM HEPES, pH 7.4, 1 mM DTT, 0.1 mM EDTA, 0.1% CHAPS and 0.1% Triton X-100). DEVD-p-nitroanilide, IETD-p-nitroanilide and LEHD-p-nitroanilide were the substrates for caspase-3, -8 and -9, respectively. The experiments were performed according to the manufacturer's protocol (Biosource International). For caspase-12 activity assay, ATAD-AFC was used as a substrate. Cell lysate aliquots were assayed in a CytoFluor Multi-well Plate Reader, Series 400 (Perseptive Biosystems) with 400 nm excitation and 505 nm emission filters according to the manufacturer's protocol (BioVision).

Mitochondrial membrane potential and cytochrome c assay
Loss of mitochondrial membrane potential, indicative of apoptosis, was detected using JC-1 MitoPTTM detection kit (B-bridge International Inc.) according to the manufacturer's protocol. Briefly, cells were incubated with MitoPT dye (JC-1) at 37°C for 15 min in a CO2 incubator. Fifty thousand cells from each experiments group were dispensed in 100 µl assay buffer in a black flat-bottom 96-well plate. The red JC-1 fluorescence was measured using a CytoFluor Multi-well Plate Reader, Series 400 (Perseptive Biosystems) with 485 nm excitation and 590 nm emission filters. By comparing the average of red fluorescent signal in induced versus non-induced control samples, the loss of mitochondrial membrane potential can be monitored. The apoptotic cells generate a lower reading of red fluorescence. For cytochrome c ELISA, mitochondrial and cytoplasmic fractions were prepared using the Mitochondrial Fractionation Kit (Active Motif, Carisbad, CA, USA). Cytochrome c levels were measured using a commercially available ELISA kit (R&D Systems).

Caspase-12 siRNA knockdown
The siRNA targeting rat caspase-12 (5'-CCGACAGCACATTCCTGGTCTTTAT-3', complementary to the caspase-12 mRNA starting from nucleotide 737) was designed and ordered from Invitrogen. The corresponding scrambled RNA oligonucleotide sequence was used as a negative control (5'-CCGACGATTACGTCCTCTGTCATAT-3'). The transfection of siRNA into PC12 cells was performed with lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. In brief, the siRNA was transfected into A53T-PC 12 cells using 0.5 µg lipofectamine 2000 for cells in each well of 24-well plates (toxicity assay) and 2.5 µg lipofectamine 2000 for cells in each well of six-well plates (immunoblotting analysis). Five hour after transfection, cells were washed and replaced with differentiation and induction medium. The expression level of caspase-12 was detected by western blot analysis. Cell toxicity was measured by LDH and Trypan blue exclusion assay after 6 days of transfection.

Data analysis
Quantitative data are expressed as arithmetic mean±SE based on at least three separate experiments performed in duplicate. The difference between two groups was statistically analyzed by Student's t-test or an analysis of variance (one-way-ANOVA). A P-value <0.05 was considered significant.


   ACKNOWLEDGEMENTS
 
This research was funded by NINDS NS38377, Udall PD Research Center, National Institutes of Health. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases. We thank Dr Myriam Gorospe and Dr Yusen Liu for helpful discussions.

Conflict of Interest statement. None declared.


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