HHS Public Access Author manuscript Author Manuscript
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14. Published in final edited form as: J Biochem Pharmacol Res. 2014 December ; 2(4): 185–190.
Expression of synaptophysin protein in different dopaminergic cell lines Xiang Baia,b,* and Randy Stronga,b,c,* aDepartment bBarshop
of Pharmacology, University of Texas Health Science Center
Institute for Longevity and Aging Studies, University of Texas Health Science Center
Author Manuscript
cGeriatric
Research, Education and Clinical Center, South Texas Veterans Health Care Networks, San Antonio, TX USA
Abstract
Author Manuscript
Synaptophysin is a specific presynaptic marker for neurons. Loss of synaptophysin occurs in Parkinson's disease, dementia with Lewy bodies and other neurodegenerative diseases. In vitro studies on synaptophysin are important to understand both the function of the protein itself and its implication in the pathogenesis of neurological diseases. In this study, we determined synaptophysin protein expression by Western analysis in 6 different dopaminergic cell lines including one human (SH-SY5Y), two rat (PC12 and N27) and 3 mouse (MN9D, Cath.a and CAD) cell lines. We found that synaptophysin protein is richly expressed in PC12 cells, but much less in other cells we studied. The order of synaptophysin expression from high to low for the other 5 cell lines was CAD> SH-SY5Y> MN9D> Cath.a = N27 cells, with Cath.a and N27 cells expressing almost undetectable content of synaptophysin protein. These data may be useful to other researchers in choosing a dopaminergic cell line as a model system to study the pathophysiology of neuron terminal loss.
Keywords synaptic degeneration; synaptophysin; Parkinson's Disease; dopaminergic cell line; SH-SY5Y; MN9D; PC12; Cath.a; CAD; N27
Introduction Author Manuscript
Synaptophysin, also known as the major synaptic vesicle protein p38, is expressed in neurons and has been regarded as a specific presynaptic marker [1]. Synapse loss has been found in the cortex of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced monkey model of Parkinson's disease (PD) [2]. Lower levels of synaptophysin (68%-78%) were found in cortex of Dementia with Lewy Bodies subjects versus similar aged control aged control [3]. Human wild type alpha-synuclein, which accumulates in PD, multiple
*
To whom correspondence should be addressed: [email protected];or [email protected]. Conflict of interests: The authors declare no conflict of interest.
Bai and Strong
Page 2
Author Manuscript
system atrophy and Dementia with Lewy Bodies, triggers synaptophysin loss in primary mouse cortical neurons and adult mouse hippocampal neurons [4]. Cell culture models of PD are important to study the role of synaptophysin in the pathogenesis of PD. However, we found that there was little information on expression of synaptophysin in different dopaminergic cell lines. In this study, we determined basal levels of synaptophysin protein expression in 6 different commonly used dopaminergic cell lines.
Author Manuscript Author Manuscript
We chose one human, two rat and three mouse dopaminergic cell lines for study. SH-SY5Y is a human cell line subclone of SK-N-SH cell, which was isolated from a bone marrow biopsy taken from a four year-old neuroblastoma patient. SH-SY5Y cells express dopaminergic markers [5]. The PC12 cell line was derived from a pheochromocytoma of the rat adrenal medulla [6]. The 1RB3AN27 (N27) cell line was derived from an immortalized clone of rat neurons by transfecting fetal mesencephalon cells with the plasmid vector pSV3neo carrying the LTa gene from SV40 virus [7]. Cath.a is derived from tyrosine hydroxylase positive tumors in the brain stem of a transgenic mouse carrying the SV40 T antigen oncogene under the transcriptional control of regulatory elements from the promoter region of the rat tyrosine hydroxylase gene [8]. It is reported that Cath.a cells express synaptophysin [8]. CAD cells are a variant of Cath.a cells in which the original immortalizing oncogene SV40 T antigen was lost [9]. CAD cells bear neurites while Cath.a cells do not [9]. Additionally, CAD cells are much bigger than Cath.a cells and CAD cells express synaptosomal proteins [9]. The MN9D dopaminergic cell line was generated by fusion of rostral mesencephalic neurons from embryonic C57BL/6J (embryonic day 14) mice with N18TG2 neuroblastoma cells [10]. MN9D is the cell line closest to primary mesencephalic dopamine neurons. It is reported that MN9D cells express synaptophysin [11]. All of these 6 dopaminergic cell lines have been reported to possess properties of dopaminergic neurons [5-10].
Materials and methods Cell culture incubations All cells were cultured in a humidified, 5% CO2, 37 °C incubator in T75 flasks (Greiner Bio One). The T75 flasks for MN9D cells were coated with poly-l-lysine (Sigma) 5 hours before plating cells. The medium and supplements used for each cell line are listed in Table 1. The medium was changed every the other day and cells were splitat about 80% confluence. Cells used for experiments are from passage 6 to 9. Cells were harvested by 0.05% trypsin (Gibco) and seeded at the density of 10˄6 cells/flask.
Author Manuscript
Western blot analysis of protein expression Protein was extracted with 1 × RIPA buffer (CST #9806) with 1× Calbiochem Protease Inhibitor Cocktail Set I (Cat. No. 539131) and 1× Halt* Phosphatase Inhibitor Cocktail (Thermo Scientific 78240). The cell lysate was then centrifuged at 13k rpm for 10 min at 4°C. BCA (bicinchoninic acid) protein assay reagents were used to determine protein concentration in the supernatant using a kit from Pierce (#23225). The supernatant was denatured in 4× LDS buffer (Invitrogen) at 75 °C for 10 minutes. Protein was then subjected
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
Bai and Strong
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to 4-12% Criterion SDS-PAGE gel (Bio-Rad) and transferred onto PVDF membranes using the iBlot system ((Invitrogen) under program 4 for 7 minutes. Primary antibody against synaptophysin (EMD Millipore, 1:1000) and β-actin (CST, 1:1000) was incubated overnight at 4°C. These primary antibodies are reactive to human, rat and mouse antigen. The synaptophysin protein sequences are highly homologous among these three species. A secondary antibody from LiCor (IR800 for β-actin or IR680 for synaptophysin) was used accordingly for each primary antibody. Immunoreactive bands were scanned by the ODYSSEY® Quantitative Fluorescence Imaging System (LiCor).
Results
Author Manuscript
Different amounts of each cell lysate were assayed by Western analysis to detect synaptophysin signal. Figure 1 shows the synaptophysin signal after loading 10μg, 20μg, 40μg, 80μg, 100μg and 140μg of SH-SY5Y cell lysate. A weak synaptophysin signal became evident beginning with 40 μg of lysate, while at 10 μg and 20 μg of cell lysate loading, the protein signal is not detectable. Figure 2 shows the synaptophysin signal after loading 10μg, 20μg,40μg, 60μg, 80μg and 100μg of N27 cell lysate. No synaptophysin signal was detected after loading up to 100 μg of cell lysate. Figure 3 shows that synaptophysin is very robustly expressed in PC12 cells. The protein signal was very strong even with as little as 10 μg of cell lysate. In contrast, the synaptophysin signal was barely detected at 80 μg of MN9D cell lysate. Figure 4 shows that the synaptophysin signal is detectable in CAD cells after loading 20 μg of cell lysate. However, the synaptophysin signal was not detected at up to 120 μg of Catha cell lysate loading.
Discussion Author Manuscript
In this study, we determined synaptophysin protein expression by Western analysis in six different dopaminergic cell lines including one cell line from human (SH-SY5Y), two from rat (PC12 and N27) and three from mouse (MN9D, Cath.a and CAD). We found that synaptophysin protein is most richly expressed in PC12 cells, but much less in other cell lines we studied. The amount of synaptophysin expression ranks from high to low for the rest of the 5 cell lines is CAD, SH-SY5Y, MN9D, Cath.a and N27 cells, whereas Cath.a and N27 cells express almost no synaptophysin protein that can be detected by Western analysis.
Author Manuscript
Consistent with a previous report, in our study, MN9D cells displayed low expression levels of synaptophysin protein [11]. On the other hand, absence of an immunoblot signal in the Cath.a cells is not consistent with a previous report whereby synaptophysin signal was detected [8]. The difference may result from different methods used. Suri et al used antisynaptophysin rabbit serum and horseradish peroxidase linked to donkey anti-rabbit second antibody [8], while we used a commercial mouse synaptophysin antibody and IRDye 800CW Donkey anti-mouse IgG (H + L) as second antibody. Nevertheless, this discrepancy does not contradict the relative amount of synaptophysin expression in the 6 available dopaminergic cell lines we studied. In addition, the lack of immunoblot signal in the N27 cells is consistent with that of a previous report [12]. Although PC12 cells express synaptophysin richly, many labs including our own lab found that PC12 cells lack many characteristics of dopaminergic cells studied in this project in terms of electrophysiological
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
Bai and Strong
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Author Manuscript Author Manuscript
and biochemical function. We found that CAD cells express more synaptophysin compared to SHSY5Y cells. However, considering that SHSY5Y cells have long neurites, while CAD cells do not, we think that the SHSY5Y cell line may be a better choice for studies of synaptophysin. This study only provides information about the basal level of synaptophysin protein expression in these 6 dopaminergic cell lines. However, these cells are also often differentiated by different methods to induce neurite growth. For example, PC12 cells can be differentiated by nerve growth factor or basic fibroblast growth factor [13]; SH-SY5Y cells can be differentiated by retinoic acid [14]; Cath.a cells can be differentiated by CRF [15]; CAD cells can be differentiated by serum deprivation [9]; MN9D cells can be differentiated by sodium butyrate [16], and N27 cells can be differentiated with dibutyryl cAMP [17]. Some of these methods of differentiation might also affect synaptophysin expression. For instance, increased neurite length and synaptophysin protein level has been shown in retinoic acid differentiation of SH-SY5Y cells [14]. We did not test whether different differentiation methods alter synaptophysin protein level in these cell lines. We believe differentiation of these cells might be necessary and worth doing for some specific experiments depending on the aims of projects. We acknowledge that other different factors need to be considered including tyrosine hydroxylase expression and activity, and presence of other catecholamine synthesizing enzymes. In conclusion, this study provides information about expression of the presynaptic marker synaptophysin in 6 different dopaminergic cell lines. The data collected will be useful for other researchers when choosing which dopaminergic cell line to use when studying the role of synaptophysin in pathogenesis of PD in vitro.
Acknowledgments Author Manuscript
We are very grateful to Dr. Michael J. Zigmond at University of Pittsburgh for the generous provision of MN9D cells. We are very grateful to Ms. Juliann Jaumotte at University of Pittsburgh for all the help about MN9D cell culture. This study was supported by a grant from the VA Office of Research & Development VA 1 I01 BX001641 (RS), and PHS grant AG022307 (RS).
References
Author Manuscript
1. Calhoun ME, Jucker M, Martin LJ, Thinakaran G, Price DL, Mouton PR. Comparative evaluation of synaptophysin-based methods for quantification of synapses. J Neurocytol. 1996; 25:821–8. [PubMed: 9023727] 2. Raju DV, Ahern TH, Shah DJ, Wright TM, Standaert DG, Hall RA, et al. Differential synaptic plasticity of the corticostriatal and thalamostriatal systems in an MPTP-treated monkey model of parkinsonism. Eur J Neurosci. 2008; 27:1647–58. [PubMed: 18380666] 3. Mukaetova-Ladinska EB, Andras A, Milne J, Abdel-All Z, Borr I, Jaros E, et al. Synaptic proteins and choline acetyltransferase loss in visual cortex in dementia with Lewy bodies. J Neuropathol Exp Neurol. 2013; 72:53–60. [PubMed: 23242284] 4. Bate C, Gentleman S, Williams A. Alpha-Synuclein Induced Synapse Damage is Enhanced by Amyloid-β 1-42. Mol Neurodegener. 2010; 5(55) 1326-5-55. 5. Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 1978; 38:3751–7. [PubMed: 29704] 6. Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976; 73:2424–8. [PubMed: 1065897]
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
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7. Prasad KN, Carvalho E, Kentroti S, Edwards-Prasad J, Freed C, Vernadakis A. Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue. In Vitro Cell Dev Biol Anim. 1994; 30A:596–603. [PubMed: 7820310] 8. Suri C, Fung BP, Tischler AS, Chikaraishi DM. Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J Neurosci. 1993; 13:1280–91. [PubMed: 7680068] 9. Qi Y, Wang JK, McMillian M, Chikaraishi DM. Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. J Neurosci. 1997; 17:1217–25. [PubMed: 9006967] 10. Choi HK, Won LA, Kontur PJ, Hammond DN, Fox AP, Wainer BH, et al. Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res. 1991; 552:67– 76. [PubMed: 1913182] 11. Chen CX, Huang SY, Zhang L, Liu YJ. Synaptophysin enhances the neuroprotection of VMAT2 in MPP+-induced toxicity in MN9D cells. Neurobiol Dis. 2005; 19:419–26. [PubMed: 16023584] 12. Thomas MG, Saldanha M, Mistry RJ, Dexter DT, Ramsden DB, Parsons RB. Nicotinamide Nmethyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling. Cell Death Dis. 2013; 4:e669. [PubMed: 23764850] 13. Chen JH, Lee DC, Chiu IM. Cytotoxic effects of acrylamide in nerve growth factor or fibroblast growth factor 1-induced neurite outgrowth in PC12 cells. Arch Toxicol. 2013 14. Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, et al. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology. 2009; 30:127–35. [PubMed: 19056420] 15. Cibelli G, Corsi P, Diana G, Vitiello F, Thiel G. Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways. Eur J Neurosci. 2001; 13:1339–48. [PubMed: 11298794] 16. Rick CE, Ebert A, Virag T, Bohn MC, Surmeier DJ. Differentiated dopaminergic MN9D cells only partially recapitulate the electrophysiological properties of midbrain dopaminergic neurons. Dev Neurosci. 2006; 28:528–37. [PubMed: 17028430] 17. Zhang D, Kanthasamy A, Anantharam V, Kanthasamy A. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line. Toxicol Appl Pharmacol. 2011; 254:65–71. [PubMed: 21310168]
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Figure 1.
Synaptophysin expression in SH-SY5Y cells. The image shows synaptophysin protein immunoblot detected by synaptophysin antibody using Western analysis. Loading amount are shown at the top of each lane. β-actin was used as a loading control. Mouse brain lysate was used as a positive control.
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Author Manuscript Author Manuscript Figure 2.
Synaptophysin expression in N27 cells. The experimental conditions were the same as described in Figure 1 legend.
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Figure 3.
Synaptophysin expression in PC12 and MN9D cells. The experimental conditions were the same as described in Figure 1 legend.
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Figure 4.
Synaptophysin expression in CAD and Cath.a cells. The experimental conditions were the same as described in the Figure 1 legend.
Author Manuscript Author Manuscript J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
Author Manuscript
Author Manuscript 2 mM n/a n/a n/a
Glutamax (Invitrogen)
Donor horse serum (Invitrogen)
Bovine calf serum (Invitrogen)
NaBicarbonate
n/a
5%
5%
2 mM
50 unit/ml
5%
5%
Fetal Bovine Serum (Hyclone) 50 unit/ml
advanced DMEM (Gibco)
Advanced DMEM:F12 1:1 (Gibco)
medium
Penicillin/Streptomycin (Gibco)
PC12
SH-SY5Y
Cell line
n/a
n/a
n/a
2 mM
50 unit/ml
10%
RPMI1640 (Gibco)
N27
n/a
n/a
n/a
2 mM
50 unit/ml
10%
RPMI1640 (Gibco)
Cath.a
Author Manuscript Table 1
n/a
n/a
n/a
2 mM
50 unit/ml
5%
advanced DMEM (Gibco)
CAD
3.7g/L, pH7.2
n/a
n/a
2 mM
50 unit/ml
10%
DMEM (Sigma)
MN9D
Author Manuscript
Culture medium and supplement of all the cell lines
Bai and Strong Page 10
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14. Published in final edited form as: J Biochem Pharmacol Res. 2014 December ; 2(4): 185–190.
Expression of synaptophysin protein in different dopaminergic cell lines Xiang Baia,b,* and Randy Stronga,b,c,* aDepartment bBarshop
of Pharmacology, University of Texas Health Science Center
Institute for Longevity and Aging Studies, University of Texas Health Science Center
Author Manuscript
cGeriatric
Research, Education and Clinical Center, South Texas Veterans Health Care Networks, San Antonio, TX USA
Abstract
Author Manuscript
Synaptophysin is a specific presynaptic marker for neurons. Loss of synaptophysin occurs in Parkinson's disease, dementia with Lewy bodies and other neurodegenerative diseases. In vitro studies on synaptophysin are important to understand both the function of the protein itself and its implication in the pathogenesis of neurological diseases. In this study, we determined synaptophysin protein expression by Western analysis in 6 different dopaminergic cell lines including one human (SH-SY5Y), two rat (PC12 and N27) and 3 mouse (MN9D, Cath.a and CAD) cell lines. We found that synaptophysin protein is richly expressed in PC12 cells, but much less in other cells we studied. The order of synaptophysin expression from high to low for the other 5 cell lines was CAD> SH-SY5Y> MN9D> Cath.a = N27 cells, with Cath.a and N27 cells expressing almost undetectable content of synaptophysin protein. These data may be useful to other researchers in choosing a dopaminergic cell line as a model system to study the pathophysiology of neuron terminal loss.
Keywords synaptic degeneration; synaptophysin; Parkinson's Disease; dopaminergic cell line; SH-SY5Y; MN9D; PC12; Cath.a; CAD; N27
Introduction Author Manuscript
Synaptophysin, also known as the major synaptic vesicle protein p38, is expressed in neurons and has been regarded as a specific presynaptic marker [1]. Synapse loss has been found in the cortex of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced monkey model of Parkinson's disease (PD) [2]. Lower levels of synaptophysin (68%-78%) were found in cortex of Dementia with Lewy Bodies subjects versus similar aged control aged control [3]. Human wild type alpha-synuclein, which accumulates in PD, multiple
*
To whom correspondence should be addressed: [email protected];or [email protected]. Conflict of interests: The authors declare no conflict of interest.
Bai and Strong
Page 2
Author Manuscript
system atrophy and Dementia with Lewy Bodies, triggers synaptophysin loss in primary mouse cortical neurons and adult mouse hippocampal neurons [4]. Cell culture models of PD are important to study the role of synaptophysin in the pathogenesis of PD. However, we found that there was little information on expression of synaptophysin in different dopaminergic cell lines. In this study, we determined basal levels of synaptophysin protein expression in 6 different commonly used dopaminergic cell lines.
Author Manuscript Author Manuscript
We chose one human, two rat and three mouse dopaminergic cell lines for study. SH-SY5Y is a human cell line subclone of SK-N-SH cell, which was isolated from a bone marrow biopsy taken from a four year-old neuroblastoma patient. SH-SY5Y cells express dopaminergic markers [5]. The PC12 cell line was derived from a pheochromocytoma of the rat adrenal medulla [6]. The 1RB3AN27 (N27) cell line was derived from an immortalized clone of rat neurons by transfecting fetal mesencephalon cells with the plasmid vector pSV3neo carrying the LTa gene from SV40 virus [7]. Cath.a is derived from tyrosine hydroxylase positive tumors in the brain stem of a transgenic mouse carrying the SV40 T antigen oncogene under the transcriptional control of regulatory elements from the promoter region of the rat tyrosine hydroxylase gene [8]. It is reported that Cath.a cells express synaptophysin [8]. CAD cells are a variant of Cath.a cells in which the original immortalizing oncogene SV40 T antigen was lost [9]. CAD cells bear neurites while Cath.a cells do not [9]. Additionally, CAD cells are much bigger than Cath.a cells and CAD cells express synaptosomal proteins [9]. The MN9D dopaminergic cell line was generated by fusion of rostral mesencephalic neurons from embryonic C57BL/6J (embryonic day 14) mice with N18TG2 neuroblastoma cells [10]. MN9D is the cell line closest to primary mesencephalic dopamine neurons. It is reported that MN9D cells express synaptophysin [11]. All of these 6 dopaminergic cell lines have been reported to possess properties of dopaminergic neurons [5-10].
Materials and methods Cell culture incubations All cells were cultured in a humidified, 5% CO2, 37 °C incubator in T75 flasks (Greiner Bio One). The T75 flasks for MN9D cells were coated with poly-l-lysine (Sigma) 5 hours before plating cells. The medium and supplements used for each cell line are listed in Table 1. The medium was changed every the other day and cells were splitat about 80% confluence. Cells used for experiments are from passage 6 to 9. Cells were harvested by 0.05% trypsin (Gibco) and seeded at the density of 10˄6 cells/flask.
Author Manuscript
Western blot analysis of protein expression Protein was extracted with 1 × RIPA buffer (CST #9806) with 1× Calbiochem Protease Inhibitor Cocktail Set I (Cat. No. 539131) and 1× Halt* Phosphatase Inhibitor Cocktail (Thermo Scientific 78240). The cell lysate was then centrifuged at 13k rpm for 10 min at 4°C. BCA (bicinchoninic acid) protein assay reagents were used to determine protein concentration in the supernatant using a kit from Pierce (#23225). The supernatant was denatured in 4× LDS buffer (Invitrogen) at 75 °C for 10 minutes. Protein was then subjected
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
Bai and Strong
Page 3
Author Manuscript
to 4-12% Criterion SDS-PAGE gel (Bio-Rad) and transferred onto PVDF membranes using the iBlot system ((Invitrogen) under program 4 for 7 minutes. Primary antibody against synaptophysin (EMD Millipore, 1:1000) and β-actin (CST, 1:1000) was incubated overnight at 4°C. These primary antibodies are reactive to human, rat and mouse antigen. The synaptophysin protein sequences are highly homologous among these three species. A secondary antibody from LiCor (IR800 for β-actin or IR680 for synaptophysin) was used accordingly for each primary antibody. Immunoreactive bands were scanned by the ODYSSEY® Quantitative Fluorescence Imaging System (LiCor).
Results
Author Manuscript
Different amounts of each cell lysate were assayed by Western analysis to detect synaptophysin signal. Figure 1 shows the synaptophysin signal after loading 10μg, 20μg, 40μg, 80μg, 100μg and 140μg of SH-SY5Y cell lysate. A weak synaptophysin signal became evident beginning with 40 μg of lysate, while at 10 μg and 20 μg of cell lysate loading, the protein signal is not detectable. Figure 2 shows the synaptophysin signal after loading 10μg, 20μg,40μg, 60μg, 80μg and 100μg of N27 cell lysate. No synaptophysin signal was detected after loading up to 100 μg of cell lysate. Figure 3 shows that synaptophysin is very robustly expressed in PC12 cells. The protein signal was very strong even with as little as 10 μg of cell lysate. In contrast, the synaptophysin signal was barely detected at 80 μg of MN9D cell lysate. Figure 4 shows that the synaptophysin signal is detectable in CAD cells after loading 20 μg of cell lysate. However, the synaptophysin signal was not detected at up to 120 μg of Catha cell lysate loading.
Discussion Author Manuscript
In this study, we determined synaptophysin protein expression by Western analysis in six different dopaminergic cell lines including one cell line from human (SH-SY5Y), two from rat (PC12 and N27) and three from mouse (MN9D, Cath.a and CAD). We found that synaptophysin protein is most richly expressed in PC12 cells, but much less in other cell lines we studied. The amount of synaptophysin expression ranks from high to low for the rest of the 5 cell lines is CAD, SH-SY5Y, MN9D, Cath.a and N27 cells, whereas Cath.a and N27 cells express almost no synaptophysin protein that can be detected by Western analysis.
Author Manuscript
Consistent with a previous report, in our study, MN9D cells displayed low expression levels of synaptophysin protein [11]. On the other hand, absence of an immunoblot signal in the Cath.a cells is not consistent with a previous report whereby synaptophysin signal was detected [8]. The difference may result from different methods used. Suri et al used antisynaptophysin rabbit serum and horseradish peroxidase linked to donkey anti-rabbit second antibody [8], while we used a commercial mouse synaptophysin antibody and IRDye 800CW Donkey anti-mouse IgG (H + L) as second antibody. Nevertheless, this discrepancy does not contradict the relative amount of synaptophysin expression in the 6 available dopaminergic cell lines we studied. In addition, the lack of immunoblot signal in the N27 cells is consistent with that of a previous report [12]. Although PC12 cells express synaptophysin richly, many labs including our own lab found that PC12 cells lack many characteristics of dopaminergic cells studied in this project in terms of electrophysiological
J Biochem Pharmacol Res. Author manuscript; available in PMC 2015 August 14.
Bai and Strong
Page 4
Author Manuscript Author Manuscript
and biochemical function. We found that CAD cells express more synaptophysin compared to SHSY5Y cells. However, considering that SHSY5Y cells have long neurites, while CAD cells do not, we think that the SHSY5Y cell line may be a better choice for studies of synaptophysin. This study only provides information about the basal level of synaptophysin protein expression in these 6 dopaminergic cell lines. However, these cells are also often differentiated by different methods to induce neurite growth. For example, PC12 cells can be differentiated by nerve growth factor or basic fibroblast growth factor [13]; SH-SY5Y cells can be differentiated by retinoic acid [14]; Cath.a cells can be differentiated by CRF [15]; CAD cells can be differentiated by serum deprivation [9]; MN9D cells can be differentiated by sodium butyrate [16], and N27 cells can be differentiated with dibutyryl cAMP [17]. Some of these methods of differentiation might also affect synaptophysin expression. For instance, increased neurite length and synaptophysin protein level has been shown in retinoic acid differentiation of SH-SY5Y cells [14]. We did not test whether different differentiation methods alter synaptophysin protein level in these cell lines. We believe differentiation of these cells might be necessary and worth doing for some specific experiments depending on the aims of projects. We acknowledge that other different factors need to be considered including tyrosine hydroxylase expression and activity, and presence of other catecholamine synthesizing enzymes. In conclusion, this study provides information about expression of the presynaptic marker synaptophysin in 6 different dopaminergic cell lines. The data collected will be useful for other researchers when choosing which dopaminergic cell line to use when studying the role of synaptophysin in pathogenesis of PD in vitro.
Acknowledgments Author Manuscript
We are very grateful to Dr. Michael J. Zigmond at University of Pittsburgh for the generous provision of MN9D cells. We are very grateful to Ms. Juliann Jaumotte at University of Pittsburgh for all the help about MN9D cell culture. This study was supported by a grant from the VA Office of Research & Development VA 1 I01 BX001641 (RS), and PHS grant AG022307 (RS).
References
Author Manuscript
1. Calhoun ME, Jucker M, Martin LJ, Thinakaran G, Price DL, Mouton PR. Comparative evaluation of synaptophysin-based methods for quantification of synapses. J Neurocytol. 1996; 25:821–8. [PubMed: 9023727] 2. Raju DV, Ahern TH, Shah DJ, Wright TM, Standaert DG, Hall RA, et al. Differential synaptic plasticity of the corticostriatal and thalamostriatal systems in an MPTP-treated monkey model of parkinsonism. Eur J Neurosci. 2008; 27:1647–58. [PubMed: 18380666] 3. Mukaetova-Ladinska EB, Andras A, Milne J, Abdel-All Z, Borr I, Jaros E, et al. Synaptic proteins and choline acetyltransferase loss in visual cortex in dementia with Lewy bodies. J Neuropathol Exp Neurol. 2013; 72:53–60. [PubMed: 23242284] 4. Bate C, Gentleman S, Williams A. Alpha-Synuclein Induced Synapse Damage is Enhanced by Amyloid-β 1-42. Mol Neurodegener. 2010; 5(55) 1326-5-55. 5. Biedler JL, Roffler-Tarlov S, Schachner M, Freedman LS. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 1978; 38:3751–7. [PubMed: 29704] 6. Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976; 73:2424–8. [PubMed: 1065897]
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7. Prasad KN, Carvalho E, Kentroti S, Edwards-Prasad J, Freed C, Vernadakis A. Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue. In Vitro Cell Dev Biol Anim. 1994; 30A:596–603. [PubMed: 7820310] 8. Suri C, Fung BP, Tischler AS, Chikaraishi DM. Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J Neurosci. 1993; 13:1280–91. [PubMed: 7680068] 9. Qi Y, Wang JK, McMillian M, Chikaraishi DM. Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. J Neurosci. 1997; 17:1217–25. [PubMed: 9006967] 10. Choi HK, Won LA, Kontur PJ, Hammond DN, Fox AP, Wainer BH, et al. Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res. 1991; 552:67– 76. [PubMed: 1913182] 11. Chen CX, Huang SY, Zhang L, Liu YJ. Synaptophysin enhances the neuroprotection of VMAT2 in MPP+-induced toxicity in MN9D cells. Neurobiol Dis. 2005; 19:419–26. [PubMed: 16023584] 12. Thomas MG, Saldanha M, Mistry RJ, Dexter DT, Ramsden DB, Parsons RB. Nicotinamide Nmethyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling. Cell Death Dis. 2013; 4:e669. [PubMed: 23764850] 13. Chen JH, Lee DC, Chiu IM. Cytotoxic effects of acrylamide in nerve growth factor or fibroblast growth factor 1-induced neurite outgrowth in PC12 cells. Arch Toxicol. 2013 14. Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, et al. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology. 2009; 30:127–35. [PubMed: 19056420] 15. Cibelli G, Corsi P, Diana G, Vitiello F, Thiel G. Corticotropin-releasing factor triggers neurite outgrowth of a catecholaminergic immortalized neuron via cAMP and MAP kinase signalling pathways. Eur J Neurosci. 2001; 13:1339–48. [PubMed: 11298794] 16. Rick CE, Ebert A, Virag T, Bohn MC, Surmeier DJ. Differentiated dopaminergic MN9D cells only partially recapitulate the electrophysiological properties of midbrain dopaminergic neurons. Dev Neurosci. 2006; 28:528–37. [PubMed: 17028430] 17. Zhang D, Kanthasamy A, Anantharam V, Kanthasamy A. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line. Toxicol Appl Pharmacol. 2011; 254:65–71. [PubMed: 21310168]
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Figure 1.
Synaptophysin expression in SH-SY5Y cells. The image shows synaptophysin protein immunoblot detected by synaptophysin antibody using Western analysis. Loading amount are shown at the top of each lane. β-actin was used as a loading control. Mouse brain lysate was used as a positive control.
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Author Manuscript Author Manuscript Figure 2.
Synaptophysin expression in N27 cells. The experimental conditions were the same as described in Figure 1 legend.
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Figure 3.
Synaptophysin expression in PC12 and MN9D cells. The experimental conditions were the same as described in Figure 1 legend.
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Figure 4.
Synaptophysin expression in CAD and Cath.a cells. The experimental conditions were the same as described in the Figure 1 legend.
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Author Manuscript 2 mM n/a n/a n/a
Glutamax (Invitrogen)
Donor horse serum (Invitrogen)
Bovine calf serum (Invitrogen)
NaBicarbonate
n/a
5%
5%
2 mM
50 unit/ml
5%
5%
Fetal Bovine Serum (Hyclone) 50 unit/ml
advanced DMEM (Gibco)
Advanced DMEM:F12 1:1 (Gibco)
medium
Penicillin/Streptomycin (Gibco)
PC12
SH-SY5Y
Cell line
n/a
n/a
n/a
2 mM
50 unit/ml
10%
RPMI1640 (Gibco)
N27
n/a
n/a
n/a
2 mM
50 unit/ml
10%
RPMI1640 (Gibco)
Cath.a
Author Manuscript Table 1
n/a
n/a
n/a
2 mM
50 unit/ml
5%
advanced DMEM (Gibco)
CAD
3.7g/L, pH7.2
n/a
n/a
2 mM
50 unit/ml
10%
DMEM (Sigma)
MN9D
Author Manuscript
Culture medium and supplement of all the cell lines
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