DRAM1 Protects Against Rotenone-induced Dopaminergic Damage

Aim:To clarify if overexpression of DNA damage-regulated autophagy modulator1(DRAM1) can protect the dopaminergic MES23.5 cells from rotenone-induced damage and clarify the influence of DRAM1 overexpression on autophagy in dopaminergic neurons. Reveal the roles of DRAM1 on the degradation of α-synuclein and the protection to neurons, and their molecular mechanism.Methods: Choose the appropriate concentration and action time of rotenone by testing the survival rate of cells, and futher construction a rotenone-induced Parkinson cell model. The protein levels of α-synuclein and DRAM1 were tested, also, RT-PCR amplification was used to test the m RNA levels of α-synuclein and DRAM1. The impact of rotenone on α-synuclein accumulation in MES23.5 cells was detected with immunohistochemical analysis. Rotenone-induced PD-like rat model was constructed to examine the protein levels of α-synuclein and DRAM1 in midbrain of rat model. To evaluate whether DRAM1 could affect the rotenone-induced reduction of cell survival rate, cells were transfected with DRAM1 si RNA with or with not rotenone. The protein levels of p62 and LC3 Ⅱwere tested when MES23.5 cells were treated with rotenone. The protein levels of p62, LC3 Ⅱ and α-synuclein were also examined in DRAM1 overexpression model. DRAM1 overexpression model cells were further treated with rotenone to detect the changes in α-synuclein protein level. Lentivirus-overexpression of DRAM1 was injected into the substantia nigra of α-synuclein A53 T mutation transgenic mice, and the protein levels of α-synuclein were tested. Stable overpression α-synuclein cells were transfected with Flag-DRAM1, the protein expressions of exogenous α-synuclein were detected with Western blot. Autophagy inhibitors were used to examine the impact of DRAM1 on α-synuclein protein level, and evaluate whether DRAM1 could still induce the degradation of α-synuclein. Control cells and stable overpression DRAM1 cells were treated with rotenone, and lysosome-related index, such as Cathepsin, NAG and acid phosphatase were examined. Using stable overpression DRAM1 cells, the impact of DRAM1 on CMA-related protein Lamp2 A and GAPDH was detected.Results: Here we found that rotenone would concentration- and time-dependent damage MES23.5 cells. Based on this result, we successfully construction a rotenone-induced Parkinson cell model. Also, we found out rotenone prevented α-synuclein degradation and induce its abnormal accumulation, accordingly upregulated the protein level of α-synuclein. Datas from animal experiment further confirmed rotenone could upregulate α-synuclein protein level. Interestingly, we also found DRAM1, an essential protein in TP53-dependent autophagy and apoptosis, was involved in this phenomenon. The rotenone-induced damage in dopamine neurons, observed in the MES23.5 cells as well as in rat model, was reversed by overexpression DRAM1, confirmed by datas from cell viability test and cell morphological observation. On the contrary, inhibition of DRAM1 expression by sh RNA aggravated the damage induced by rotenone. Results showed DRAM1 upregulated the protein level of LC3 II, downregulated the protein level of p62 and α-synuclein at the same time. Further more, was obviously inhibited when DRAM1 was overexpression. Inhibition of autophagy activation by Bafilomycin or 3-MA blocked DRAM1-induced degradation of α-synuclein. Results from stable overexpression DRAM1 cell demonstrated DRAM1 promoted the activation of Cathepsin B. Also, the release of symbolic enzyme NAG from lysosomes was also significantly increased inrotenone-treated MES23.5 cells, and overexpression of DRAM1 reduced the releaseof NAG from lysosomes. Overexpression of DRAM1 would also increase the activity of lysosomal acid phosphatase. Results showed DRAM1 upregulated the protein level of Lamp2 A, downregulated the rotenone-induced accumulation of GAPDH at the same time. These results indicated that DRAM1 enhanced CMA in MES23.5 cells.Conclusion: Rotenone induces the accumulation of α-synuclein and damages the MES23.5 cells at low concentration. DRAM1 downregulats the protein level of α-synuclein and rescues the MES23.5 cells from rotenone. DRAM1 enhances the degradation fuction of lysosomes and CMA in α-synuclein. These results identify DRAM1 as a promising target for therapies aimed at neuroprotection and disease modification in PD.