Study On The Aggregation Of α-synuclein Induced By Rotenone And The Mechanisms Under This Phenomenon

Part I The Neurotoxic effect of Rotenone on Rat Phechromocytoma PC12 CellsObjective To investigate the neurotoxicity of rotenone on dopaminergic cells and possible mechanisms under this.Methods PC12 cells were treated with rotenone at different concentrations for 24 hours. MTT assay was used to evaluate the cell viability. Apoptosis was determined by nuclear condensation and/or fragmentation after Hoechst 33258 staining and apoptotic cell counting with flow cytometry after Annexin V- FITC staining. Levels of intracellular reactive oxygen species (ROS) and mitochondrial membrane potentials were both determined by flow cytometry technique. Glutathione assay kit was used to assess the level of intracellular glutathione. Caspase 3 activity was measured by fluorescence assay using the probe Ac-DEVD-AMC. In addition, PC 12 cells was pretreated with the antioxidant, N-acetylcysteine(500μmol/Lor 2.5mmol/L), for 30 minutes before the incubation of rotenone.Results Decrease of cell viability, increase of apoptotic cell counting and apoptosis rate of PC12 cells were induced with incubation of rotenone(10nmol/L,100nmol/L,1μmol/L or 10μmol/L). Rotenone treatment also caused elevated intracellular ROS, decreased mitochondrial membrane potentials and increase of caspase 3 activity. Moreover, the above effect of rotenone was concentration-dependent. When compared with control group, level of glutathione increased to 144% and 117% after 10nmol/L and 100nmol/L of rotenone treatment for 24 hours, respectively. However, higher concentrations of rotenone (1μmol/L or 10μmol/L) caused loss of intracellular glutathione. Pretreatment of N-acetylcysteine (500μmol/Lor 2.5mmol/L) could partially inhibit the toxic effect of 1μmol/L of rotenone.Conclusion Rotenone induces the apoptosis of dopaminergic cells by enhancing oxidative stress and destroying mitochondrial membrane potentials with a concentration-dependent manner. The antioxidant, N-acetylcysteine, can protect PC 12 cells against the neurotoxicity of rotenone. Part II Rotenone Induces Up-regulation and Aggregation ofα-Synuclein in Dopaminergic CellsObjective To evaluate the effect of rotenone on the expression and aggregation ofα-synuclein in dopaminergic cells.Methods PC12 cells were treated with rotenone at different concentrations for 24 hours. Western blot was used to assess the intracellular expression level ofα-synuclein. The formation ofα-synuclein aggregates was observed with laser scaning confocal technique andβ-amyloid structure was evaluated with thioflavin S(TS) staining. The effect of N-acetylcysteine on the expression and aggreagation ofα-synuclein was also measured.Results Rotenone treatment for 24 hours up-regulated the expression of cytoplasmicα-synuclein with a concentration-dependent manner. Pretreatment of N-acetylcysteine (500μmol/L) could partially inhibit the above effect of 1μmol/L of rotenone. In normal state,α-synuclein diffusely located in the cytoplasma. However, 1μmol/L of rotenone induced formation ofα-synuclein-positive and TS-negative aggregsomes. Pretreatment with N-acetylcysteine decreased the number of aggregsomes and the size also minished.Conclusion Rotenone can promote the up-regulation and aggregation ofα-synuclein by enhancing oxidative stress, and the aggregates do not bear the structure ofβ-amyloid.Part III Study on the Up-regulation ofα-Synuclein Induced by Rotenone in Dopaminergic CellsObjective To investigate the possible mechanisms for the up-regulation ofα-synuclein induced by rotenone in dopaminergic cells.Methods PC 12 cells were incubated with rotenone for 24 hours at different concentrations, with or without pretreatment of N-acetylcysteine for 30 minutes. The levels ofα-synuclein mRNA were analyzed with real-time fluorescence PCR. Cell lysate was prepared at different time points (0h, 2h, 6h, 12h, 18h and 24h) when cells were treated with 1μmol/L of rotenone. After this, three enzymatic activities of 20S proteasome (trypsin-like, chymotrypsin-like and peptide-glutamacylhydrolase) were measured by fluorescence assay using the corresponding probes. Meanwhile, the relationship between α-synuclein expression and the time course of rotenone treatment was observed using Western blot .Results When compared with control group,α-synuclein mRNA in PC12 cells was up-regulated after 24h-treatment with rotenone with a concentration-dependent manner. Pretreatment with N-acetylcysteine dramatically alleviated the up-regulation ofα-synuclein mRNA induced by 1μmol/L of rotenone. PC12 cells treated with 1μmol/L of rotenone, the enzymatic activities started a decrease at the time point of 2 hour and the up-regulation ofα-synuclein expression 6 hour both with a time-dependent manner. Pretreatment with N-acetylcysteine partially inhibited the damage in enzymatic activity of 20S proteasome induced by rotenone treatment.Conclusion Rotenone induces the up-regulation ofα-synuclein mRNA and enzymatic activity damage of 20S proteasome by the mechanism of oxidative stress, which promote the up-regulation ofαα-synuclein protein.PART IV Dopamine Mediates the Neurotoxicity of Rotenone and Promotesα-Synuclein Aggregation in PC12 CellsObjective To explore the role of dopamine in the neurotoxicity of rotenone andα-synuclein aggregation in dopaminergic cells.Methods PC12 cells were treated with 1μmol/L of rotenone for 24 hours, with or without pretreatment of reserpine(1μmol/L or 5μmol/L) for 3 hours. MTT assay was used to determine the cell viability. Intracellular reactive oxygen species were measured by the fluorescence probe of dihydrorhodamine 123 (DHR123) with flow cytometry. By Western blot, the expression ofα-synuclein protein was detected. The formation ofα-synuclein aggregates was observed with laser scaning confocal technique.Results 24h-treatment with rotenone dramatically decreased the cell viability and reserpine pretreatment prevented the damage of PC12 cells induced by rotenone. When compared with control group, rotenone caused an increase of 181% of intracellular reactive oxygen species and up-regulation ofα-synuclein protein. However, reserpine partially inhibited the above effects of rotenone. The formation ofα-synuclein immunoreactive aggregsomes was observed in rotenone group and pretreatment with reserpine decreased the number of these aggregsomes.Conclusion Dopamine mediates the toxicity of rotenone and promotes the formation ofα-synuclein aggregates.