Study On The Toxic Mechanism Of Dopamine On Cellular Energy Metabolism Pathway

Parkinson’s disease（PD）is a common neurodegenerative disease whose etiology and pathogenesis remain unclear.However,the pathology of PD is mainly damages or death of dopaminergic neurons（DN）of substantia nigra（SN）in midbrain.It is believed that this damage is closely related to the oxidation of dopamine（DA）which generates free radicals and reactive quinones（DAQ）.DAQ actively modify the thiol group of proteins,and these modified proteins aggregate aberrantly without proper functions,resulting in neurotoxicity.Whether this is the cause of persistent death of DN in PD patients is currently mixed.We previously reported that the DAQ modification of lactate dehydrogenase B（LDHB）,α-enolase（Enolase 1,ENO1）,and malate dehydrogenase（MDH1）was significantly increased in the SN of aged rats.As all these enzymes play key roles in energy production to maintain cell viability,the study in whether DAQ modify these enzymes involved in the death of DN would aid in understanding of the pathogenesis of PD.First,the human neuroblastoma cell line（SH-SY5Y）was used as a model of dopaminergic neurons in this study.We incubated the cells with a serial concentration of exogenous DA,and then measured the survival rate of the cells by MTT method,quantified the changes of total content of quinone proteins in the cells by NBT method,and then detected the amount of LDHB,ENO1,and MDH1 by Western-blotting.We found that the survival rate of the DA treated cells decreased significantly than that of DA non-treated cells and that the amount of total quinone proteins increased with the elevated levels of DA.After incubation of 100 mol/L DA with the cells for 24 hours,the content of LDHB,ENO1,and MDH1 decreased obviously in the experiment.These results suggested that high levels of exogenous DA may cause DAQ modification of these critical enzymes for energy production and decrease their content.Second,using the RNAi technology,we generated three cell lines LDHB^Ri,ENO1^Ri,and MDH1^Rii with a lower expression level of LDHB,ENO1,and MDH1,respectively,which separately mimics the functional loss of these enzymes in the DA treated cells to find the correlation between decreased enzyme activity and cell survival rate and possible ways to affect survival rate..We then measured the survival rate,ATP content,mitochondrial membrane potential,and the NADPH/NADP⁺ratio of these cells.Results showed that the survival rate,ATP content,mitochondrial membrane potential,and NADPH/NADP⁺ratio were lower in the enzyme knockdown cell lines than in control cell line.Moreover,we treated the knockdown cells with DA and L-3,4-dihydroxyphenylalanine（L-DOPA,the DA precursor）and found that these cells were more susceptible to the toxic effects of DA and L-DOPA because the survival rate of these cells was significantly lower than that of non-treated cells.It suggested that once the cells were damaged,they would be more susceptible to DA toxicity.In summary,we showed that DA was oxidized to form DAQ,which modified the LDHB,ENO1,and MDH1,resulting in the enzyme inactivation and cytotoxicity.We also showed that the decreased expression of these enzymes is correlated positively to the decrease of intracellular mitochondrial membrane potential,ATP content,and the NADPH/NADP⁺ratio of the cells,suggesting that the oxidative stress and blocked energy metabolism pathways may be the main cell lethal pathways for DA toxicity.And thus the cells were more vulnerable to the DA and L-DOPA treatments at this time.All data together suggested that dopamine metabolic disorder is a promising factor for the DN death.This study provides new insight in the underlying molecular mechanism of DA metabolic disorders in PD pathogenesis.