Effects of chronic low dose rotenone treatment on human microglial cells

Abstract

Background: Exposure to toxins/chemicals is considered to be a significant risk factor in the pathogenesis of Parkinson's disease (PD); one putative chemical is the naturally occurring herbicide rotenone that is now used widely in establishing PD models. We, and others, have shown that chronic low dose rotenone treatment induces excessive accumulation of Reactive Oxygen Species (ROS), inclusion body formation and apoptosis in dopaminergic neurons of animal and human origin. Some studies have also suggested that microglia enhance the rotenone induced neurotoxicity. While the effects of rotenone on neurons are well established, there is little or no information available on the effect of rotenone on microglial cells, and especially cells of human origin. The aim of the present study was to investigate the effects of chronic low dose rotenone treatment on human microglial CHME-5 cells.

Methods: We have shown previously that rotenone induced inclusion body formation in human dopaminergic SH-SY5Y cells and therefore used these cells as a control for inclusion body formation in this study. SH-SY5Y and CHME-5 cells were treated with 5 nM rotenone for four weeks. At the end of week 4, both cell types were analysed for the presence of inclusion bodies, superoxide dismutases and cell activation (only in CHME-5 cells) using Haematoxylin and Eosin staining, immunocytochemical and western blotting methods. Levels of active caspases and ROS (both extra and intra cellular) were measured using biochemical methods.

Conclusion: The results suggest that chronic low dose rotenone treatment activates human microglia (cell line) in a manner similar to microglia of animal origin as shown by others. However human microglia release excessive amounts of ROS extracellularly, do not show excessive amounts of intracellular ROS and active caspases and most importantly do not show any protein aggregation or inclusion body formation. Human microglia appear to be resistant to rotenone (chronic, low dose) induced damage.

Figure 1

Detection of inclusion bodies. Representative photomicrographs showing H & E staining (A and B), α-synuclein staining (C & D) and ubiquitin staining (E and F) in the two cell types (SH-SY5Y and CHME-5) collected at the end of 4 week's of rotenone (5 nm) treatment. SH-SY5Y cells showed eosinophilic (A, arrow head), α-synuclein positive (C, arrow head) and ubiquitin positive (E, arrow head) inclusion bodies. No inclusion bodies were seen in human microglial CHME-5 cells (B, D and F). Results shown are representative of three independent experiments. Scale bar: 50 μm.

Figure 2

Glut-5 levels in microglial CHME-5 cells. Figure 2A: Representative immunoblot showing the levels of Glut-5 protein (55 kDa, first row) in the cell lysates collected at the end of week 4 from the control and rotenone (5 nm) treated CHME-5 cells. β-actin (42 kDa, second row) was used as an internal loading control. Figure 2B: Histogram showing relative semi-quantitation of Glut-5 levels. Glut-5 levels were normalised against the β-actin levels in each well and expressed in densitometric units. P (p = 0.039) value indicates a significant difference in Glut-5 levels between control and rotenone treated groups. Each column represents the mean and standard deviation from three independent experiments. Figure 2C: Representative photomicrographs showing immunostaining of CR3/43 (HLA DR/DP/DQ) in control and rotenone treated CHME-5 cells at the end of week 4 of the treatment period. CR3/43 staining is dramatically increased in rotenone treated cells as visualised by the green fluorescence. Scale bar: 50 μm.

Figure 3

Detection of Reactive Oxygen Species (ROS). Figure 3A: Detection of ROS using DHE fluorescent dye in CHME-5 and SH-SY5Y cells from the two treatment groups (DMSO control and 5 nm rotenone) at the end of week 4. Hoechst 33258 stain (blue) was used to identify the nucleus of cells. The fluorescent intensity of the red dots in the nucleus indicates the relative levels of ROS present in the cell. Scale bar: 20 μm. Figure 3B: Histogram showing the levels of ROS in each of the treatment groups in both cell types (CHME-5 and SH-SY5Y) at the end of week 4. The levels are expressed in densitometric units. P (*p > 0.05) value indicates that there was no significant difference in ROS levels between the control and rotenone treated CHME-5 cells. However rotenone treated SH-SY5Y cells showed a significant (**P < 0.001) two fold increase in the intracellular ROS levels compared to the untreated controls. Each column represents the mean and standard deviation from three independent experiments Figure 3C: Histogram representing the extracellular levels of ROS using the Acridan Lumigen PS-3 assay in the medium collected from both treatment groups (DMSO control and 5 nm rotenone) and both cell types (CHME-5 and SH-SY5Y) at the end of week 4. The levels are expressed in light intensity units. CHME-5 cells released significantly higher (*p < 0.001) levels of ROS in the medium compared to controls, while the extracellular release of ROS in the medium from rotenone treated and untreated SH-SY5Y cells was not significantly different (**P > 0.05). Each column represents the mean and standard deviation from three independent experiments. Figure 3D: Image showing the effects of antioxidants on chemiluminescence in the Acridan Lumigen PS-3 assay. Chemiluminescent signals were captured using Fujifilm Luminescent Image Analyzer (Dots) and light intensity units were recorded using the BioTek Synergy 2 plate reader (bar graph). a: culture medium from CHME-5 cells treated with 5 nm rotenone for 4 weeks, b: culture medium (as in a) incubated with 100 μM ascorbic acid (vitamin C) for 5 minutes, c: culture medium (as in a) incubated with NuPAGE antioxidant (1:500 diluted). The dramatic reduction of chemiluminescence in the media incubated with antioxidants indicates that the chemiluminescence in Acridan PS-3 assay is ROS specific.

Figure 4

Detection of active caspases using FLICA assay. Histogram showing the levels of active caspases in each of the treatment groups from the two cell types (CHME-5 and SH-SY5Y) at the end of week 4. The levels are expressed in fluorescence units/10⁶ cells. P (*P > 0.05) value indicates that there was no significant difference in the level of active caspases between the rotenone treated and untreated CHME-5 cells. Rotenone treated SH-SY5Y cells showed significantly increased (**P < 0.001) levels of active caspases (fluorescence units) compared to untreated control cells. Each column represents the mean and standard deviation from three independent experiments.

Figure 5

Superoxide dismutase (SOD) levels in both CHME-5 and SH-SY5Y cells. Figure 5A: Representative immunoblot and histogram showing levels of SOD1 in the two types of cells (CHME-5 and SH-SY5Y) collected from two treatment groups (DMSO control and rotenone treated). CHME-5 cells showed a significant (p < 0.05) two fold increase in SOD1 levels, whereas SH-SY5Y cells showed a more significant 2.4 fold increase (p < 0.01) in SOD1 levels after 4 weeks of rotenone treatment. The base levels of SOD1 appeared to be higher in SH-SY5Y cells compared to CHME-5 cells. Figure 5B: Representative immunoblot and histogram showing levels of SOD2 in the two types of cells (CHME-5 and SH-SY5Y) collected from two treatment groups (DMSO control and rotenone treated). CHME-5 cells did not show any change in SOD2 levels (p > 0.05), whereas SH-SY5Y cells showed a 1.5 fold increase in SOD2 levels after 4 weeks of rotenone treatment, although this increase was not significant (p > 0.05). The base levels of SOD2 appeared to be higher in SH-SY5Y cells compared to CHME-5 cells. 1: CHME-5 control; 2: CHME-5 rotenone; 3: SH-SY5Y control; 4: SH-SY5Y rotenone. Each column represents the mean and standard deviation from three independent experiments.