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Journal of the American College of Nutrition, Vol. 24, No. 6, 516-523 (2005)
Published by the American College of Nutrition

Selenomethionine Prevents Degeneration Induced by Overexpression of Wild-Type Human {alpha}-Synuclein during Differentiation of Neuroblastoma Cells

Bipin Kumar, MD, Piruz Nahreini, PhD, Amy J. Hanson, MS, Cynthia Andreatta, BS, Judith E. Prasad, BS and Kedar N. Prasad, PhD

Center for Vitamins and Cancer Research, Department of Radiology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262

Address reprint requests to: Kedar N. Prasad, Ph.D., Premier Micronutrient Corp., Antioxidant Res. Inst., 14 Galli Drive, Suite 200, Novato, CA 94949. E-mail: kprasad{at}premiermicronutrient.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: High levels of wild-type {alpha}-synuclein are found in autopsied brain samples of idiopathic Parkinson’s disease (PD), some familial PD, some Alzheimer’s disease (AD) and Down’s syndrome with dementia. Therefore, we have investigated whether overexpression of wild-type {alpha}-synuclein causes degeneration during adenosine, 3',5'-cyclic monophosphate (cAMP)-induced differentiation of murine neuroblastoma (NB) cells in culture. We have also studied whether selenomethionine can modify the effect of overexpression of {alpha}-synuclein during differentiation of NB cells.

Methods: To study these issues, we established a murine neuroblastoma (NB) clone (NBP2-PN54-C20) that expressed high levels of wild-type human {alpha}-synuclein as determined by real time PCR and Western blot. We have utilized RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and prostaglandin A1 (PGA1), a stimulator of adenylate cyclase, or RO20-1724 and dibutyryl cAMP to induce terminal differentiation in over 95% of the cell population by elevating the intracellular levels of cAMP in NB cells. The viability of cells was determined by MTT assay and LDH leakage assay, and the degeneration was documented by photomicrographs.

Results: The results showed that overexpression of human wild-type {alpha}-synuclein decreased viability and increased degenerative changes in comparison to those observed in vector control cells, when differentiation was induced by treatment with RO20-1724 and PGA1, but not with RO20-1724 and dibutyryl cAMP. When selenomethionine was added to NB cells overexpressing {alpha}-synuclein immediately after the addition of RO20-1724 and PGA1, the viability and degenerative changes were markedly reduced, suggesting the involvement of increased oxidative stress in the mechanism of action of {alpha}-synuclein. This protective effect was not observed after treatment with sodium selenite or methionine.

Conclusions: Data suggested that Overexpression of wild-type human {alpha}-synuclein-decreased viability and increased the levels of degenerative changes during differentiation of NB cells were reduced by selenomethionine treatment. This suggest that one of mechanisms of action {alpha}-synuclein may involve increased oxidative stress.

Key words: human wild-type {alpha}-synuclein, differentiated neuroblastoma cells, prevention, degeneration, selenomethionine

Abbreviations: NB = neuroblastoma cells • cAMP = adenosine 3',5'-cyclic monophosphate • PGA1 = prostaglandin A1 • RO20-1724 = 4-3-butoxy-4-methoxybenzyl-2-imidazolidinone • AD = Alzheimer’s disease • PD = Parkinson’s disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The synuclein family consists of three homologous proteins, {alpha}-, ß- and {gamma}-synuclein. {alpha}-and ß-synuclein are located primarily in pre-synaptic regions of brain neurons, and {gamma}-synuclein is present in the peripheral nervous system and retina [1]. {alpha}-synuclein is an intrinsically unfolded protein that assembles into Lewy body (LB)-like filament, whereas ß- and {gamma}-synuclein do not [2]. {alpha}-synuclein appears to be associated with membrane compartments in neuronal cells in culture and brain tissue through interactions with acidic head groups of phospholipids [3,4]. In yeast cells, {alpha}-synuclein inhibits phospholipase D, induces lipid droplet accumulation, and affects vesicle trafficking [5]. Membrane-bound {alpha}-synuclein is in dynamic equilibrium with the cytoplasmic form, and it may play an important role in fibrillar formation [3,6]. {alpha}-synuclein may play a role in neuronal plasticity; however, the actual physiological function of this protein is not fully understood. Abnormal accumulation of {alpha}-synuclein [7] and abnormal proteolytic processing of {alpha}-synuclein [8] appear to be associated with neurodegeneration. Reports indicate that PD [9–11], AD [12,13] and 50% of Down’s syndrome patients with dementia [14] exhibit Lewy bodies (LBs) in the brain containing predominantly {alpha}-synuclein. A number of cellular and transgenic models have shown that overexpression of {alpha}-synuclein induces neurodegeneration [15–18]. A genomic triplication of wild-type {alpha}-synuclein leading to overexpression of wild-type {alpha}-synuclein was responsible for familial PD in the Iowa kindred [19]. In addition, extracellular senile plaques of AD brain contain {alpha}-synuclein [20–23]. In contrast to the above observation, some studies suggest that {alpha}-synuclein protects against oxidative stress in neuronal cells in culture [24,25] and might be of protective value in models of neuronal injuries [26]. However, it is unknown whether overexpression of wild type human {alpha}-synuclein in dividing nerve cells can influence the rate of proliferation and the level of differentiation or degeneration. To study this, we have developed a murine neuroblastoma (NB) cell clone that overexpresses wild-type human {alpha}-synuclein (NBP2-PN54-C20) under the control of cytomegalovirus (CMV) promoter, using a retroviral vector. We have utilized RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, and PGA1, a stimulator of adenylate cyclase, or RO20-1724 and dibutyryl cAMP to induce terminal differentiation in over 95% of the NB cell population [27,28].

We now report that overexpression of {alpha}-synuclein does not significantly affect the rate of proliferation or the level of differentiation in comparison to vector control cells. However, it decreases the viability and increases the levels of degenerative changes that are primarily located within the cell body during differentiation of NB cells induced by RO20-1724 and PGA1, but not by RO20-1724 and dibutyryl cAMP. Selenomethionine treatment protects against {alpha}-synuclein-induced degenerative changes, suggesting the involvement of increased oxidative stress in the mechanism of action of {alpha}-synuclein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell Culture
A murine neuroblastoma clone (NBP2), which has both tyrosine hydroxylase and choline acetyltransferase [29], was used in this study. Cells were grown in F12 medium (Gibco Products, Invitrogen Corp., Grand Island, NY) containing 3% Tet System Approved fetal bovine serum (Clontech, Palo Alto, CA), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco Products, Invitrogen Corp). All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.

Establishment of a NB Cell Line Overexpressing Wild Type Human {alpha}-Synuclein
NBP2 cells were used to generate a NB clone that expresses high levels of {alpha}-synuclein. The retroviral backbone plasmid, pLNCX, was obtained from Clontech, Inc. (originated from Dr. D. Miller’s laboratory at the Fred Hutchinson Institute, Seattle, Washington) [30]. A 293-derived amphotropic packaging cell line (Phoenix) was obtained from ATCC (originated from Dr. Gary Nolan’s laboratory at the University of California, San Francisco, CA) [31]. Plasmid pLNCX contains a neomycin resistance (Neor) gene driven by the viral long terminal repeat (LTR) promoter, followed by the cytomegalovirus immediate early (CMV) promoter [32]. Construction of PN-1 vector was described previously [32]. To construct PN-54, a full-length human {alpha}-synuclein cDNA (kindly provided by Dr. Makoto Hashimoto, UCSD, San Diego, CA) [33] was subcloned in a sense orientation into the NotI/HpaI sites downstream of the CMV promoter in PN-1 vector. The resulting expression vector for wild-type human {alpha}-synuclein was designated as PN-54. Retroviral production was carried out according to the manufacturer’s specifications (Clontech Laboratories, Inc., Palo Alto, CA). Briefly, PN-1 or PN54 vectors were co-transfected with an expression plasmid for vesicular stomatitis virus G glycoprotein (VSV-G) into Phoenix cells using a calcium phosphate transfection kit (Promega, Inc). Cultured NBP2 cells were transduced at a multiplicity of infection of one (moi:1) in the presence of 8 µg/ml Polybrene (Sigma Biochemicals). Transfected NBP2 cells were selected in the presence of 200 µg/ml of neomycin analog, G418 (Life Technologies). Neomycin resistant cells were cloned by standard techniques, and a clone (NBP2-PN54-C20) that expresses high levels of wild-type human {alpha}-synuclein was isolated for this study.

Solution Preparation
Solutions of prostaglandin A1 (PGA1, Sigma, St. Louis, MO), a stimulator of adenylate cyclase, 4-3-butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase (a gift from Hoffman La Roche, Nutley, NJ) and dibutyryl cAMP (Sigma) were prepared and stored as previously described [28].

Induction of Differentiation and Assay of Degeneration
Differentiation was induced by treatment of NB cells with PGA1 (2 µg/ml) and RO20-1724 (200 µg/ml) or by dibutyryl cAMP (2.0 mM) and RO20-1724 (200 µg/ml). The expression of differentiation functions was optimal and irreversible 3 days after treatment [27, 28]. Growth medium was changed and differentiating agents were replenished after 2 days of treatment. Degeneration was assayed by MTT assay, LDH leakage and propidium iodide staining 3 days after treatment with cAMP-elevating agents.

MTT Assay
This procedure has been used in our previous publication [34]. Cells (20,000 per well) were plated in a 24-well tissue culture plate and treated with cAMP-elevating agents. Viability of cells was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer’s instructions. The quantity of formazan product was determined by measuring absorbance at 490 nm in a GeniosTM microplate reader (Phenix Research, Hayward, CA). The absorbance is directly proportional to the number of living cells in culture. MTT assay may also be used for assessing mitochondrial damage. Each experiment was performed in triplicate.

Lactate Dehydrogenase (LDH) Assay
Cells were plated (20,000 cells per well) in a 24-well tissue culture plate. Damage to outer cellular membrane was determined 3 days after treatment with cAMP-elevating agents using the CytoTox-ONETM Homogeneous Membrane Integrity Assay (Promega, Madison, WI) according to the manufacturer’s instructions. Release of lactate dehydrogenase (LDH) into the culture media directly correlates with the number of non-viable cells present in the cell culture. The presence of LDH in the culture media was determined by a coupled enzymatic assay that results in the conversion of resazurin to fluorescent resorufin. The amount of fluorescence generated in each well was determined using a GeniosTM microplate reader (Phenix Research) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

RNA Isolation and RT-PCR
Total RNA was prepared from treated and untreated NB cells using Qiagen RNeasyTMMMini kit (Qiagen, Chatworth, CA) according to the manufacturer’s instructions. cDNA synthesis was carried out as previously described using 2 µg total RNA [35]. Real time PCR was performed using LightCycler FastStart DNA Master SYBR Green I reaction mix as previously described [36]. Primers used to detect {alpha}-synuclein and S29 (housekeeping gene) transcripts were as follows: {alpha}-synuclein 5'-TCCATAGAAGACACCGGG-'3; 5'-GCTGAGAAGACCAAAGAGCAA-'3; S29 5'-AAGGCAAGATGGGTCACCAGCAGCTCTA-'3 and 5'-TACAAAGACTAGCATGATCGGTTCCACT-'3. These primers were designed to detect vector derived human {alpha}-synuclein mRNA only.

Western Blot
Western blot analysis of wild-type {alpha}-synuclein was carried out on protein derived from NBP2-PN54-C20 cells after differentiation as described in our previous publication [37]. A rabbit anti-{alpha}-synuclein polyclonal antibody from BD Biosciences (San Diego, CA) and a rabbit anti-cyclophilin A polyclonal antibody from Upstate Cell Signaling Solutions (Lake Placid, NY) were used in this study. A secondary anti-IgG antibody conjugated to horseradish peroxidase was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Statistical Analysis
Significant differences between control and experimental groups were determined using a paired Student’s t-test with a p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
Selection and Characterization of a NB Clone Overexpressing Wild-Type Human {alpha}-Synuclein
A murine neuroblastoma (NB) cell line that overexpresses wild-type human {alpha}-synuclein (NBP2-PN54-C20) under the control of cytomegalous virus (CMV) promoter was developed using the retroviral vectors shown in Fig. 1. Clones were isolated by standard procedures and {alpha}-synuclein protein levels were determined in selected clones by Western blot. The human {alpha}-synuclein protein (19 kDa) was present at varying levels in NB cells overexpressing {alpha}-synuclein. A clone (NBP2-PN54-C20) showing high levels of wild-type human {alpha}-synuclein protein was selected for this study (Fig. 2A). Human {alpha}-synuclein protein was not present in vector control (NBP2-PN1) or parent controls (NBP2) cells. NBP2-PN54-C20 cells also expressed wild-type human {alpha}-synuclein mRNA as determined by real time PCR; however, as expected, vector control (NBP2-PN1) and parent control (NBP) cells did not produce vector derived {alpha}-synuclein mRNA (data not shown).



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  		Fig. 1. Schematic representation of retroviral vectors used to establish an over-expressed {alpha}-synuclein (PN-54) NB cell line. PN-1 was used to generate a vector control cell line. For details see Materials and Methods. Abbreviations: LTR-long terminal repeat of murine leukemia virus; CMV-cytomegalovirus immediate early promoter; MCS-multiple cloning sites; Neo®–neomycin resistance gene; {alpha}-synuclein-human {alpha}-synuclein.

 


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  		Fig. 2. A. Western blot analysis of wild-type human {alpha}-synuclein holoprotein in undifferentiated (U) and differentiated (D) NBP2, NBP2-PN1 and NBP2-PN54 cells. 20 µg total cellular protein, anti-{alpha}-synuclein antibody, and goat anti-rabbit secondary antibody were used for the analysis. Human {alpha}-synuclein was detected as a 19 kDa protein. M-marker. B. Levels of cyclophilin A (Cyp-A) were determined and used as a loading control. Each experiment was repeated twice and similar results were obtained.

 
Effect of Overexpression of Wild-Type Human {alpha}-Synuclein on the Proliferation of NB Cells
The proliferation of each NB cell line was determined by MTT assay as a function of time after plating of undifferentiated NB cells. Results showed that NB cells overexpressing {alpha}-synuclein (NBP2-PN54-C20) grew at about same rate as that seen in vector control cells (NBP2-PN1) (Fig. 3). However, parent NB cells (NBP2) grew slightly faster than vector control NB cells.



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  		Fig. 3. Growth rates of undifferentiated NB cell lines. For each cell line, 10,000 cells were plated in each well of a 24 well tissue culture plate. Viability of cells was determined at the indicated time points by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.

 
Effect of Overexpression of Wild-Type Human {alpha}-Synuclein on Viability and Degeneration during Differentiation of NB Cells
The proliferation was determined by measuring the viability of cells by MTT assay. The results showed that NB cells overexpressing {alpha}-synuclein exhibited decreased proliferation during differentiation in comparison to vector control or parent control cells (Fig. 4). These cells did not divide after 1 day of treatment with RO20-1724 and PGA1 and remained quiescent for a period of 3 additional days. The rate of proliferation during differentiation was similar in parent and vector control NB cells.



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  		Fig. 4. Viability of NB cells differentiated with RO20-1724 and PGA1. Cells (20,000 cells/well) were plated in a 24 well tissue culture plate, and 200 µg/ml RO20-1724 and 2 µg/ml PGA1 were added 24 hours later. Viability of NB cells was determined at the indicated times by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.

 
Microscopic examination of differentiated cells 3 days after treatment revealed that cell bodies of NB cells overexpressing {alpha}-synuclein were degenerated as shown by the vacuolation and fragmentation of cytoplasmic materials without affecting the neurite or the nucleus (Fig. 5C) in comparison to parent control (Fig. 5A) or vector control (Fig. 5B). This was confirmed by decreased viability (Fig. 4). There was no evidence of LDH leakage in control or experimental groups. In contrast to the degenerative changes observed in {alpha}-synuclein overexpressing NB cells caused by treatment with RO20-1724 and PGA1, treatment of these cells RO20-1724 and dibutyryl cAMP failed to induce degenerative change (Fig. 5D).



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  		Fig. 5. Photomicrographs of differentiated NB cells. Cells (20,000 cells/well) were plated in a 24 well tissue culture plate and treated 24 hours later as described below. NBP2 (A), NBP2-PN1 (B), and NBP2-PN54-C20 (C) cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 µg/ml PGA1. D. NBP2-PN54-C20 cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 mM dibutyryl cAMP. E. NBP2-PN54-C20 cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 µg/ml PGA1 in the presence of 5 µg/ml selenomethionine.

 
Effect of Overexpression of Wild-Type Human {alpha}-Synuclein on Proteasome Activity during Differentiation of NB Cells
Since inhibition of proteasome activity appears to be associated with neurodegeneration [38–40], it was thought that {alpha}-synuclein-induced neurodegeneration during differentiation may be due to an inhibition of proteasome activity. Therefore, we measured the proteasome activity in the extract of NB cells differentiated for 3 days with RO20-1724 and PGA1. The results showed that the activity of proteasome was similar in differentiated NB cell lines (NBP2-PN54-C20, NBP2-PN1 and NBP2) (Table 1).


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  		Table 1. Effect of Overexpression of {alpha}-Synuclein on Proteasome Activity in Neuroblastoma Cells

 
Effect of Selenomethionine, Sodium Selenite or Methionine on {alpha}-Synuclein-Induced Degeneration during Differentiation of NB Cells
Microscopic examination of cells after 3 days of treatment showed that addition of selenomethionine immediately after RO20-1724 and PGA1 markedly reduced {alpha}-synuclein-induced degeneration during differentiation of NB cells (Fig. 5E). This was also confirmed by increase in viability (Fig. 6). Selenomethionine also reduced spontaneous degenerative changes seen in vector control cells during differentiation. However, sodium selenite (1–5 µg/ml) or methionine 1–5 µg/ml) did not protect NB cells against {alpha}-synuclein-induced degenerative changes during differentiation.



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  		Fig. 6. Effects of selenomethionine on the viability of NB cells differentiated with RO20-1724 and PGA1. Cells (20,000/well) were plated in a 24 well tissue culture plate and were treated with differentiating agents 24 hours after plating. Various concentrations of selenomethionine were added concomitantly with the differentiating agents. Cell viability was measured 3 days after treatment by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.

 

   DISCUSSION
 
This study shows that overexpression of {alpha}-synuclein in NB cells does not alter the rate of proliferation in comparison to vector control cells, suggesting that insertion of plasmid vector or vector with wild-type {alpha}-synuclein did not affect the proliferation of NB cells. However, vector control NB cells grow slightly slower than parent control cells. This could be due to a difference in sub-clones or insertion of plasmid vector into the cell.

This study also reveals that the overexpression of {alpha}-synuclein in NB cells does not affect the levels of cAMP-induced differentiation as measured by neurite formation, since they are similar (over 95% of cell population) in all cell lines. However, NB cells overexpressing {alpha}-synuclein exhibit an increased level of degenerative changes during differentiation-induced by RO20-1724 and PGA1 in comparison to vector control cells. Degenerative changes are primarily located in the cell bodies, while nuclei and neurites remain intact. The degenerative changes are confirmed by decreased viability. It is interesting to observe that {alpha}-synuclein-induced degeneration is observed only when differentiation is induced by RO20-1724 and PGA1, but not by RO20-1724 and dibutyryl cAMP. This suggests that {alpha}-synuclein may interact with PGA1 rather than with cAMP to induce degenerative changes during differentiation. The mechanism of this interaction remains to be defined. This result may be of particular significance, because PGE2, one of the secretory products of inflammatory reactions, has been implicated in neurodegeneration associated with AD [30, 41, 42]. PGA1 is formed during extraction of PGE2, and is very stable. Therefore, we have used PGA1 in this study.

It has been postulated that {alpha}-synuclein-induced neurotoxicity is mediated via increased oxidative stress [43]. Indeed, pretreatment of neurons with vitamin E reduced {alpha}-synuclein- induced degeneration in experimental neurons [43]. Our results show that selenomethionine reduces the levels of {alpha}-synuclein-induced degeneration during differentiation of NB cells, suggesting that one of the mechanisms of action of {alpha}-synuclein may involve increased oxidative stress. The fact that sodium selenite or methionine treatment does not reduce the level of {alpha}-synuclein-induced degenerative changes suggests that this organic form of selenium is necessary for its protective effect. The effect of other antioxidants on {alpha}-synuclein-induced degeneration during differentiation of NB cells remains to be determined. Data obtained from this study cannot be directly extrapolated to degeneration associated with AD or PD. However, evidence of increased oxidative damage [44–55] has been observed in autopsied samples of brains of both AD and PD patients. Thus, we suggest that reducing oxidative stress by an appropriate mixture of antioxidants that includes selenomethionine may be one of the best strategies to reduce the incidence of AD and PD and/or the rate of progression of these neurological diseases.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
This study was supported by a grant from the NIH AG0118285.

Received April 12, 2004. Accepted December 1, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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