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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0041fje. |
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-Synuclein regulates neuronal survival via Bcl-2 family expression and PI3/Akt kinase pathway1
Department of Pharmacology, National Creative Research Initiative Center for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, Seoul National University, Seoul 110–799, South Korea;
* Department of Neurosurgery, College of Medicine, Seoul National University, Boramae Hospital, Seoul 156–707, South Korea; and
Laboratory of Structural Proteomics, School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, South Korea
2Correspondence: Department of pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia, Seoul National University, Seoul, 110–799, South Korea. E-mail: yhsuh{at}plaza.snu.ac.kr
SPECIFIC AIMS
To provide the first direct evidence that 
-synuclein (-SN) has neuron-specific dual effects depending on its concentration or level of expression, we examined -SN-induced cytotoxic and neuroprotective effects by the extracellularly treatment of -SN or its transient transfection in primary neuronal cell and various neuronal cell lines. To investigate the mechanism mediated by -SN-induced cytotoxic or neuroprotective effects, we checked the expression level of the PI3/Akt signaling pathway and Bcl-2-related anti- or proapoptotic factors in NGF-differentiated PC12 cells with or without Bcl-2 stable transfection.
PRINCIPAL FINDINGS
1. Neurotoxic effects of -SN on the various neuronal cells
To study the effect of -SN on cell viability, rat cortical and hippocampal neuronal cells were treated with 0.1, 1, 10, or 20 µM -SN for 3 days. High concentrations of -SN (10 and 20 µM) significantly decreased cell viability in a time-dependent fashion whereas s nanomolar level of -SN did not. In nerve growth factor (NGF) -differentiated PC12 cells, SHSY-5Y and GT1–1 cells, -SN showed similar cytotoxicities with those in rat primary neuronal cells but -SN-induced cytotoxicity was decreased in Bcl-2 overexpressed PC12 cells (PC12-Bcl-2 cells). Overexpressed -SN decreased cell viability as significantly as treatment with 10 µM of -SN in PC12 cells; cytotoxicity induced by -SN was decreased in PC12-Bcl-2 compared with that in PC12 cells.
2. Possible neurotoxic mechanisms of -SN
The level of Bcl-xL was decreased but bax was increased in PC12 cells and PC12-Bcl-2 cells under treatment with 10 µM -SN for 2 days (Fig. 1
A). Similar results were observed in these cells transfected with -SN using a immunoblotting method (Fig. 1B
). As to the downstream factors of Bcl-2 families, cytochrome c released into the cytoplasm, activated caspase-3, and bax expression were time-dependently increased from the start of treatment until day 3 in the SHSY-5Y human neuroblastoma cell line (Fig. 1C
). -SN was detected as a 19 kDa band in whole-cell lysate and the media after transfection of -SN (Fig. 1B
, left 5th and 6th panels).
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-SN induced nitric oxide (NO) and inducible nitric oxide synthase time- and dose-dependently, and these increases were blocked by the pretreatment with NAME (NOS inhibitor), transcription factor NF kappa B (NF-
B) inhibitor, PD98059 (ERK inhibitor), or SB203580 (JNK inhibitor). As for -SN-induced NO production, I-B was degraded 6 h after -SN treatment; JNK and phospho-ERK also increased 12 h after -SN treatment. When neuronal cultures were treated with conditioned media from microglia incubated with 2.5 µM -SN, neuronal MTT reduction was significantly reduced, which was blocked by 2 h pretreatment of inhibitors as described above.
3. Protective effects of -SN on the various neuronal cells
-SN (0.1 µM) treatment showed a significant protective effect and 20 µM -SN increased neuronal death against cellular stress induced by serum deprivation in the rat cortical and hippocampal neurons. We consistently found similar protective effects of -SN in SHSY-5Y, GT1–1, and NGF-differentiated PC12 cells under serum deprivation and treatment with 200 µM hydrogen peroxide or 20 µM glutamate. In PC12-Bcl-2 cells, -SN revealed a more significant protective effect against serum deprivation or treatment with 200 µM hydrogen peroxide than in PC12 cells. In contrast, -SN did not affect the cell viability in U251 cells, a human glial cell line, indicating the cytotoxic effects of -SN may be neuroselective.
4. Possible neurotrophic mechanisms of -SN
-SN (0.1 µM) increased the Bcl-xL expression level more significantly than in the control group but decreased bax expression in PC12 cells and PC12-Bcl-2 cells per immunoblotting (Fig. 2
A) and mRNA analysis (Fig. 2B
). To examine the protective mechanism of -SN, we checked phosphorylated serine/threonine kinase (pAkt) levels and the effect of PI3 kinase inhibitor on serine/threonine kinase (Akt) levels. pAkt expression was increased by treatment with 0.1 µM -SN, which was blocked by pretreatment with LY294002, a PI3 kinase inhibitor (Fig. 2C
).
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CONCLUSIONS AND SIGNIFICANCE
Although -SN is a major component of intracellular inclusions in Parkinson’s disease (PD), dementia with Lewy bodies, and glialcytoplasmic inclusion of multiple system atrophy, the primary physiological function of -SN remains to be elucidated. In the present study, we provide direct evidence that -SN at nanomolar concentrations protects neurons against cellular stress conditions such as serum deprivation, glutamate toxicity, or oxidative stress caused by hydrogen peroxide. Its protective effect against neurotoxicity is observed at a physiological range and is mediated by activation of the pro-survival PI3K/Akt pathway, followed by Bcl-2 family antiapoptotic expression. The survival effect by -SN was increased more by Bcl-2 stable transfection; PI3 inhibitor pretreatment decreased this antiapoptotic effect (Fig. 2D
). Thus, it is likely that activation of the PI3K/Akt and Bcl-2 family expression pathway is responsible for its protective effect. PI3K and its downstream effector, the Akt, have been reported to play a role in neuronal survival: overexpression of activated PI3K and Akt promoted the survival of superior cervical neurons in culture, and in several different cell types, including hippocampal neurons and PC12 cells, activated Akt stimulated changes in Bcl-2 and bax expression and showed an antiapoptotic effect. In support, activation of Akt has been shown to lead to phosphorylation of Bad, decreasing interaction of bad with Bcl-xL and increasing the Bcl-xL/bad ratio, and to phosphorylation of caspase-9, leading to its inactivation and eventually promoting cell survival.
We have demonstrated that treatment with high concentrations or overexpression of -SN leads to up-regulation of bax expression, an increase in caspase activity, and subsequent release of cytochrome c from mitochondria even though we cannot precisely control the expression level of -SN. It has been demonstrated recently that -SN in the defining lesions of synucleinopathy is specifically nitrated. The selective nitration of -SN provided direct evidence that nitrative and oxidative stress play highly significant roles in the onset and progression of various synucleinopathies. However, there have been no reports on the origin of this nitration. In this study, our data strongly suggest that nitration is caused by NO from the microglia situated near the neurons. Once the extracellular concentration of -SN increases to greater than a few micromolar under pathological conditions, it may activate neighboring microglia to produce NO via the mitogen-activated protein kinase pathway and NF-B translocation. Increased -SN in dying neurons damaged by microglia activation could diffuse out after neuronal rupture and exert a direct neurotoxic effect through an increase in the ratio of pro- to antiapoptotic signaling molecules, which in turn would build a positive feedback loop. Translocation of cytosolic bax into mitochondria induces cytochrome c release to activate caspase-3, leading to cell death. In contrast, Bcl-2 and Bcl-xL expression could block apoptosis by inhibiting bax expression and its conformational changes. It has recently been reported that -SN interacts with dephospho-bad. Dephospho-bad is known to stimulate bax to undergo allosteric activation leading to facilitate mitochondrial membrane permeabilization, which is inhibited by antiapoptotic members of the Bcl-2 family. This interaction could provide another underlying molecular basis for the proapoptotic effect of -SN expression in cultured neurons.
Taken together, our results provide the first evidence that low or physiological levels of -SN can protect neurons against various toxic insults such as hypoxia, oxidative stress, and glutamate toxicity via the PI3/Akt pathway. In contrast, our results also demonstrate that high concentrations or overexpression of -SN exert a cytotoxic effect on neurons that might be mediated through the Bcl-2 family-related caspase pathway and inflammatory events to produce NO, suggesting another role of -SN in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and PD.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0041fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0041fje 
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) and PC12-Bcl2 (
) cells (D). *P < 0.05, significantly different from control by Student’s t test, n = 8; 0.1 µM, 0.1 µM 