The Protective Effect Of Caffeine On Dopaminergic Neurons: Mitophagy Promoted By The Dephosphorylation Of DRP1-Ser637

The mitochondrion is the main site for energy synthesis. Maintaining normal mitochondrial morphology and functions are important foundations for the well functioning of other cells. In a large number of neurodegenerative diseases, such as Parkinson’s disease(PD), a common etiological factor is the impaired mitochrondiral function in the brain. Autophagy is a very important ―self digestion‖ mechanism, which can realize energy recycling and reuse of organelles. It is the only way to remove any damaged organelles within a cell, and is though to be involved in a most of the neurodegenerative diseases.One of the most commonly used PD cellular models is the rotenone cell model. Rotenone can disrupt mitochondrial homeostasis, influence quality of protein synthesis and affect autophagy lysosome pathway. Studies have found that different concentrations of rotenone had varied effects on autophagy in PD cell model. However, the relationship between the molecular mechanisms of autophagy and rotenone concentration is still not clear. Therefore, the current study is aimed to elucidate the relationship between rotenone and autophagy through the mitochondrial dynamics point of view. The phosphorylation / dephosphorylation process of DRP1, one of the key proteins related to the maintenance of mitochondrial dynamic balance, was discussed.The effect of rotenone on autophagy was determined by Western blot. Results showed that 3 μM rotenone could significantly increase the level autophagy in SH-SY5 Y cells. It was also found that 3 μM rotenone could reduce the expression of Ser637p-DRP1 protein, i.e. promote Ser637p-DRP1 dephosphorylation. Numerous studies have shown that, Ser637p-DRP1 dephosphorylation could lead to increased levels of mitochondrial fission. The results of confocal microscopy showed that 3 μM rotenone increased the colocalization of autophagosome and mitochondria. Transmission electron microscopy also showed that contents in the autolysosomes appeared to be the degrading mitochondria, suggesting that mitophagy was induced by rotenone. To further prove this idea, DRP1 specific inhibitor Mdivi-1 and FK506 were used to investigate the relationship between rotenone increased mitotophagy and Ser637p-DRP1 phosphorylation. The results of Western blot, laser confocal and transmission electron microscopy all demonstrate that inhibiting DRP1 Serine 637 phosphorylation would prevent mitochondrial autophagy in SH-SY5 Y cells. These results indicated that the dephosphorylation of Ser637p-DRP1 might have contributed to the rotenone induced mitophagy.Caffeine is a common purine alkaloid with central nervous activity. Investigation on biological epidemiology has demonstrated that a certain amount of caffeine intake can reduce the risk of PD. However, the mechanism of caffeine on PD has yet to be fully elucidated. The current study aimed to investigate the effect of caffeine on neurotoxin-induced rat model. The spontaneous activity and morbidity showed that different degree of behavioral disorders was found in rotenone-treated model rats and morbidity was significantly increased. Pre-adminisration of caffeine could significantly improve these behavioral disorders and morbidity. Meanwhile, the content of dopamine(DA) in striatum of rats were determined by HPLC-ECD. In model rat, the content of DA was significently decreased. However, caffeine could significantly raise the content of DA.Finally, rotenone cell model was developed to verify the alleviating mechanism of caffeine in PD from the view of mitophagy. Results showed that 10 μM rotenone could reduce the expression of TH and increase the expression of α-syn, while pre-administration of caffeine could significantly reverse the effect of rotenone on TH and α-syn. Subsequent experiment also found that caffeine produced similar effects on cell autophagy and Ser637p-DRP1 dephosphorylation as 3 μM rotenone. In order to further verify our conjecture, autophagy inhibitor 3-MA and DRP1 specific inhibitor FK506 were used. Western blot, laser confocal and transmission electron microscopy results showed that inhibition of Ser637p-DRP1 dephosphorylation could also inhibit the mitophagy that induced by caffeine.In summary, caffeine could alleviate rotenone-induced PD in both animal and SH-SY5 Y cellular model. The attenuation of PD by caffeine was probably related to autophagy, especially mitophagy. Ser637p-DRP1 dephosphorylation might be the molecular mechanism involved in caffeine-induced mitophagy.