Background/Aims Hypercholesterolemia is recently considered a risk factor for Parkinson’s disease

Background/Aims Hypercholesterolemia is recently considered a risk factor for Parkinson’s disease (PD), the most consistent neurodegenerative movement disorder. (TH)-positive neuronal loss were determined by HPLC-electrochemical detection and TH immunocytochemistry respectively. Mitochondrial membrane potential in cells stained by tetramethylrhodamine methyl ester dye INNO-406 inhibition was analysed by flow cytometry. Results Cholesterol treatment potentiated a reduction of neuronal viability with loss of TH-positive neurons in cultures. MPP+-induced depletion of dopamine level in the post-mitotic MAP-2 immunoreactive neurons and loss of mitochondrial membrane potential were also heightened by cholesterol. Conclusion Apparently, changes in neuronal cholesterol content significantly influenced the neurotoxicity and the direct mitochondrial mechanisms involved in MPP+-induced cell death. Our observations demonstrate that high cholesterol incorporated into the differentiated human neuroblastoma cells worsened dopaminergic neuronal survivability through increased depolarization of mitochondrial membrane potential, which is a known mechanism of dopaminergic cell death by MPP+. The present findings support the hypothesis that hypercholesterolemia could be a risk factor for PD. = 4; d.f. = 31). Analysis of Biogenic Amines by HPLC Electrochemistry Cells were scrapped into 50 L ice-cold 0.4 M HClO4 containing ethylenediaminetetraacetic acid disodium salt, sonicated in cold under low energy, kept on ice in the dark for 30 min, and centrifuged at 12,500 rpm for 5 min, and 10 L of the supernatant was injected into the HPLC-ECD system for assaying biogenic amines. Before and after sample injections, a standard INNO-406 inhibition solution of biogenic amines containing 4 pmol of each biogenic amine, were assayed to confirm the retention time of the analytes. The flow rate was 0.7 mL/min and the detection was performed at 0.74 V with sensitivity set at 20 nA [16]. TH-Immunocytochemistry SH-SY5Y cells (1 105 cells/mL) were seeded on confocal dishes and differentiated by retinoic acid treatment for 6 days. After completion of the treatment period, cells were treated with MPP+ and/or cholesterol (50 M) for 24 h, fixed in 4% paraformaldehyde and processed for immunofluorescence staining of TH as described [13]. The cells were then incubated overnight at 4C with goat anti-MAP 2 or chicken anti-TH primary antibodies (1: 250 dilution) in 0.05% Triton-X 100. The cells were washed with phosphate buffered saline (PBS) and incubated with fluorescence labelled Alexa Fluor? 488 donkey anti-goat IgG and Alexa Fluor? 568 goat anti-chicken IgG secondary antibodies (1: 500 dilution) and stained with DAPI nuclear stain. Cells were examined using a confocal microscope (Andor Technology, Ireland). Fluorescence intensity of cells stained for TH-immunoreactivity (red) were measured using ImageJ software. Mitochondrial Membrane Potential Assay by FACS For flow cytometry, 2 105 cells were plated for TMRM staining. Cells were trypsinized and incubated in fresh medium, containing 50 nM of TMRM, at 37C in CO2 incubator for 30 min. The cells were then washed with D-PBS to Rabbit Polyclonal to OR2AG1/2 remove excess of stain and resuspended in 500 L of D-PBS. The cells were then analyzed employing flow cytometry (BD LSR Fortessa FACS), and the software-FACS Diva 6.2 (BD Bioscience, USA). Phycoerythrin (PE-A) filter was used with an excitation and emission of 540 and 570 nm. Ten thousand of events from each sample were used for measuring the mean fluorescence intensity. The experiments were repeated 3 or more times, performed on different days. Statistical Analysis One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was employed for finding significant differences among different treatment groups. Values of 0.05 were considered statistically significant. Results are given as mean SEM. Results Cholesterol Enhanced MPP+-Mediated Neurotoxicity of Differentiated SH-SY5Y Neurons The data from the -cyclodextrin-treated control did not differ significantly when compared to type 1 Milli-Q water control in cell viability experiments. A cholesterol concentration of 50 M was selected to study the impact of cholesterol on MPP+-mediated neurotoxicity (Fig. 1a, b), and to assess the mechanisms underlying the exacerbated cell toxicity of INNO-406 inhibition MPP+ in cholesterol-treated, differentiated SH-SY5Y neurons. Differentiated SH-SY5Y cells were treated with cholesterol 50 M alone, MPP+, and both cholesterol 50 M and MPP+ for 24 h and was further subjected to cell viability assay. In the cholesterol-alone treated cells, 82% was the observed cell viability and it was observed that there was 67% cell viability in only neurotoxin-treated cells and this.