Inhibition of vesicular monoamine transporter-2 activity in alpha-synuclein stably transfected SH-SY5Y cells

Abstract

alpha-Synuclein plays a key role in the pathological neurodegeneration in Parkinson's disease. Although its contribution to normal physiology remains elusive, the selective degeneration of alpha-synuclein-containing dopaminergic neurons in Parkinson's disease may be linked to abnormal alpha-synuclein induced toxicity. In the present study, a complex of alpha-synuclein and vesicular monoamine transporter-2 was identified by GST-Pull Down experiment. In wild-type alpha-synuclein stably transfected SH-SY5Y cell lines, the activity of vesicular monoamine transporter-2 decreased by 31% as determined by [(3)H] dopamine uptake, and its expression also decreased in both protein and mRNA levels using western and northern blot analysis. Overexpression of wild-type alpha-synuclein did not induce cell death or apoptosis, but significantly enhanced the intracellular reactive oxygen species level as assayed by flow cytometry. These data suggest that Up-regulated alpha-synuclein expression inhibits the activity of vesicular monoamine transporter-2, thereby interrupting dopamine homeostasis and resulting in dopaminergic neuron injury in Parkinson's disease.

Fig. 1

Interaction of α-synuclein with VMAT2 in human brain. (a) Western blot of GST-fused α-synuclein protein with anti-α-synuclein antibody. (b) Western blot of human brain homogenate after affinity purification with GST fusion protein encoding α-synuclein, but not by GST alone. A specific band at 62 kDa was detected in brain homogenates (lane 1), GST fused α-synuclein (lane 2), but not in GST alone (lane 3 )

Fig. 2

Overexpression of α-synuclein inhibits VMAT2 activity by [³H] dopamine uptake analysis. [³H] Dopamine uptake analysis was performed using 85 nM [³H]dopamine and radioactivity was assessed by liquid scintillation counting (CPM). All experiments were repeated at least three times. VMAT2-mediated [³H] DA uptakes were significantly decreased in α-synuclein transfected cell lines by 31% (2705 ± 150, n = 3, * P < 0.05) compared to that in control cells (3940 ± 341)

Fig. 3

Expression of α-synuclein and VMAT2 in stably transfected SH-SY5Y cells. (a) VMAT2 levels were decreased in cell lines stably expressing α-synuclein (Syn ,lane 2) compared to that in the control cell lines (LacZ, lane 1) determined by immunoblot with polyclonal anti-VMAT2 antibody. (b) β-Galactosidase-transfected SH-SY5Y cells (LacZ, lane1) express low endogenous α-synuclein levels compared with cells overexpressing α-synuclein (Syn, lane2) as determined by western blotting using a monoclonal antibody to α-synuclein

Fig. 4

Overexpression α-synuclein lowers mRNA level of VMAT2 by northern blot hybridization. Total RNA were electrophoresed, transferred onto nylon membranes, and hybridized with horseradish peroxidase labeled VMAT2 cDNA and GAPDH cDNA. Immunolabeled bands were detected using DAB. The mRNA level of VMAT2 was significantly decreased in the cell lines stably expressing α-synuclein ( Syn, lane 2) compared to that in the control cell lines (LacZ, lane1 )

Fig. 5

Effects of α-synuclein transfection on the cell viability of SH-SY5Y cells. The percentage of Trypan blue-positive cells is reported as the mean ± S.D. (n = 3). Stable overexpression of wild-type α-synuclein in SH-SY5Y cells did not show significant effect on the cell viability compared with the control cells (P > 0.05)

Fig. 6

Hoechst 33258 staining SH-SY5Y cells expressing α-synuclein and β-Galactosidase. Chromosomal condensation and DNA fragmentation were determined using the chromatin dye Hoechst 33258. Cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4°C, stained with 1 mM Hoechst 33258 for 5 min, and then the apoptotic features were analyzed under a fluorescence microscope ((IX-70, Olympus) with excitation at 352 nm and emission at 461 nm. The fluorescence was visualized using a 20 × objective

Fig. 7

Effects of α-synuclein expressing on intracellular ROS level. Cells were stained with 10 μM (DCFA)-DA for 20 min and then assayed on a FACScant flow cytometer. Data represent the mean dichlorofluorescein (DCF) signal and at least 10,000 events were collected per sample. The DCF signal in the cells expressing α-synuclein (503 ± 19) was significantly higher than that in the cells expressing β-Galactosidase (361 ± 30) (* P < 0.05)