An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage

Abstract

Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson's disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitin- and alpha-synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nm rotenone for 1-4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-synuclein and ubiquitin. After a latency of >2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.

Fig. 1.

Chronic rotenone exposure increased α-synuclein protein levels. A, One week of rotenone treatment increased soluble α-synuclein levels, whereas 4 weeks of rotenone treatment elevated α-synuclein levels in both soluble and insoluble protein fractions. Results are expressed as percent increase from control and represent mean ± SEM of three or four independent experiments. *p < 0.05 compared with control cells. Compared with control cells (B), chronic rotenone exposure (C) caused cytoplasmic α-synuclein accumulation, as determined by immunocytochemistry. Scale bar, 10 μm. Similar results were observed in four independent experiments.

Fig. 2.

Chronic rotenone exposure increased levels of ubiquitin immunoreactivity. A, B, Chronic rotenone exposure resulted in elevated cytoplasmic ubiquitin levels, as determined by immunocytochemistry. A, Control cells.B, Cells treated with rotenone for 4 weeks. Scale bar, 10 μm. Similar results were observed in four independent experiments.

Fig. 3.

Chronic rotenone treatment caused delayed oxidative damage. A, Chronic rotenone exposure (5 nm) decreased cellular GSH. Control values at 1 week were 4.97 ± 1.2 nmol/mg of protein. Results are expressed as percentages of levels in control cells at each time point and represent mean ± SEM of four independent experiments at each time point.B, Delayed oxidative protein damage in rotenone-treated cells. Protein carbonyl levels are expressed as percentages of levels in control cells at each time point. Results show mean ± SEM of four independent experiments at each time point. *p< 0.05 compared with control.

Fig. 4.

Chronic rotenone treatment caused oxidative DNA damage. Control cells (A, C) and cells treated with rotenone for 4 weeks (B, D) were stained with antibodies against 8-oxo-dG, a marker of oxidative DNA damage (top panels). The same cells were also labeled with bisbenzimide for nuclear morphology (bottom panels). Rotenone-treated cells showed increased 8-oxo-dG immunoreactivity before H₂O₂ exposure (B). H₂O₂ increased 8-oxo-dG staining to a greater extent in rotenone-treated cells (D) than in control cells (C). Many cells with oxidative DNA damage showed fragmented nuclear morphology characteristic of apoptosis (B, D). Similar results were observed in four replicates.

Fig. 5.

Chronic rotenone treatment sensitized cells to H₂O₂-induced death and oxidative protein damage. Cells were grown in medium supplemented with 5 nmrotenone for 1–4 weeks before exposure to 300 μmH₂O₂. Cell death was then monitored over 24 hr. Cells treated with rotenone for 1 week (A) showed responses to H₂O₂ similar to those of control cells. However, after exposure to rotenone for 4 weeks (B), cells were markedly sensitized to H₂O₂. Results show mean ± SEM of three independent experiments at each time point. *p < 0.05. A.U., Arbitrary units.

Fig. 6.

Chronic rotenone treatment increased apoptotic cell death before and after H₂O₂ exposure. Cells were analyzed for DNA fragmentation using TUNEL staining before and 24 hr after H₂O₂ exposure. Control cells showed little evidence of apoptosis (A), whereas cells treated with rotenone for 4 weeks showed a small but significant increase in apoptosis before H₂O₂ exposure (C, E). Cultures treated with rotenone for 4 weeks showed more TUNEL-positive cells after H₂O₂exposure (D) compared with control cultures (B). Arrows indicate some TUNEL-positive cells. E, Quantification of apoptotic cells. Cultures treated with rotenone for 4 weeks showed elevated apoptosis before and 24 hr after H₂O₂ exposure. Results are expressed as mean ± SEM of four experiments. *p < 0.05 compared with control.

Fig. 7.

Time course of cytochromec redistribution and caspase-3 activation in control and rotenone-treated cells after H₂O₂ exposure. Control cells (A–C) and cells treated with rotenone for 4 weeks (D–F) were triple-stained for nuclear morphology (bisbenzimide; blue), cytochromec (green), and activated caspase-3 (red) before (A, D) and 2 hr (B, E) and 4 hr (C, F) after H₂O₂ exposure. Under basal conditions, both control and rotenone-treated cells showed punctate cytochromec staining, consistent with its mitochondrial localization (A, D). After H₂O₂exposure (B, C, E, F), there was release of cytochrome c from mitochondria to cytosol, resulting in loss of punctate staining and a more diffuse, less intense staining pattern. A, B, insets, Cytochromec redistribution at higher power. D, In occasional rotenone-treated cells under basal conditions, there was redistribution of cytochrome c such that staining was less punctate and less intense, consistent with cytosolic distribution. Increased caspase-3 activation was evident 2 and 4 hr after H₂O₂ exposure but occurred to a greater extent in rotenone-treated cells (E, F) than in control cells (B, C). Many of the rotenone-treated cells expressing activated caspase-3 contained fragmented nuclei characteristic of apoptosis, indicating that rotenone treatment was associated with more advanced stages of apoptosis. Similar results were observed in four independent experiments. G, Quantification of cells with caspase-3 activation after H₂O₂ treatment. More rotenone-treated cells expressed activated caspase-3 before and 2–4 hr after H₂O₂ exposure. *p < 0.05 compared with controls. H, Caspase activation in live cells after H₂O₂ exposure. Cells treated with rotenone for 4 weeks were exposed to 300 μmH₂O₂ for 6 hr and then imaged simultaneously for cell morphology using phase-contrast microscopy (gray), nuclear morphology using bisbenzimide (blue), and caspase activation using rhodamine 110 bis-l-aspartic acid amide (green). H₂O₂ caused some cells to retract their processes, round up, and undergo nuclear fragmentation (arrows). Some cells showed caspase activation before nuclear fragmentation (arrowhead). Similar results were observed in four independent experiments.

Fig. 8.

Caspase inhibition delayed and reduced H₂O₂-induced death. Cells treated with rotenone for 4 weeks were incubated with vehicle or caspase inhibitor (Z-DEVD-FMK) for 2 hr before and throughout the exposure to H₂O₂. Cell death was analyzed as described. Results are expressed as mean ± SEM. Similar results were obtained in three independent experiments. *p < 0.05 compared with cells treated with rotenone and H₂O₂ alone.