Protective Effects Of TRPML1 On α-synuclein-induced Cell Injury

Parkinson’s disease(PD)is the second most common neurodegenerative disease,characterized by progressive bradykinesia,rigidity,resting tremor,and gait impairment,as well as a spectrum of non-motor symptoms including autonomic and cognitive dysfunction.The main pathological features of PD are the selective loss of dopaminergic neurons in the substantia nigra pars compacta,lewy bodies formed by abnormal aggregation of α-synuclein(α-syn)and iron deposition.The degeneration of dopaminergic neurons in the substantia nigra caused by abnormal aggregation of α-syn is the core pathogenic factor of PD.Aggregated α-syn bodies can cause mitochondrial oxidative stress by affecting cellular calcium homeostasis,and experiments have shown that α-syn pre-formed fibrils(PFF)can cause an increase in intracellular calcium ion concentration,and PFF after entering cells,it is transported along the lysosomal pathway,but the relationship between PFF and calcium channels on lysosomes is rarely reported.Transient receptor potential mucin 1(TRPML1)is the main calcium outflow channel in lysosome.When dopaminergic neurons derived from familial PD upregulate TRPML1,lysosomal exocytosis increases,and blocked α-syn secretion recovers,thus reducing its intracellular accumulation.TRPML1 is also an eactive oxygen species(ROS)sensor on the lysosomal membrane,and when TRPML1 is upregulated,it can inhibit the production of ROS and alleviate mitochondrial damage.However,the protective effects and mechanism of TRPML1 in α-syn-induced cytotoxicity have not been fully elucidated.In this study,we selected two different cell models:(1)doxycycline(DOX)-induced overexpression of SNCA A53 T PC12 cell model(i PC12 cell model),and(2)PC12 cell model with α-syn PFF(α-syn PFF cell model).The effects of overexpression of α-syn or α-syn PFF on cytotoxicity,intracellular calcium levels,TRPML1 and mitochondrial oxidative stress were detected respectively.Furthermore,TRPML1 in the above cell model was activated by MLSA1,and the above indicators were detected.Through the above experiments,the protective effects and possible mechanism of TRPML1 in α-syn induced cell injury were discussed.The specific experimental results are as follows:1、In this study,after i PC12 cells were treated with DOX for different time(3 h,6 h,9 h,12h,24 h,48 h),the results of western blot showed that the levels of α-syn protein increased with the increase of time of DOX treatment of i PC12 cells,and the changes were statistically significant at 12 h,24 h and 48 h(P<0.05).The results of cytotoxicity test kit showed that dehydrogenase(LDH)release increased with the increase of DOX treatment time in i PC12 cells,and the changes were statistically significant at 12 h,24 h and 48 h(P<0.0l).The results of calcium kit showed that the intracellular calcium concentration increased with the increase of DOX treatment time in i PC12 cells,and the changes were statistically significant at 12 h,24 h and 48 h(P<0.05).Western blotting showed that the levels of TRPML1 protein decreased with the increase of time of DOX treatment of i PC12 cells,and the change was statistically significant at 48 h(P<0.05).Therefore,we used DOX to treat i PC12 cells for 48 h to construct an i PC12 cell model.2、Alpha-syn PFF was prepared and α-syn PFF cell model was constructed.The results of transmission electron microscopy showed that the obvious fiber structure could be observed in the unultrasonic α-syn PFF of the two concentration groups(1 mg/m L,0.1 mg/m L),the shorter fiber could be observed in 1 mg/m L α-syn PFF after 1 min ultrasound,and the fiber could not be detected in 0.1 mg/m L α-syn PFF ultrasound,only amorphous polymer could be observed.After PC12 cells were treated with different concentrations of α-syn PFF(0.2 μg/ml,1 μg/ml,5 μg/ml)for 24 h,the results of cytotoxicity test kit showed that the release of LDH from PC12 cells treated with 5 μg/ml α-syn PFF for 24 h was significantly higher than the control group(P<0.05),and the levels of TRPML1 protein were significantly decreased after treated with 5 μg/ml α-syn PFF for 24 h compared with the control group(P<0.05).Therefore,we used 5 μg/ml of α-syn PFF to treat PC12 cells for 24 h to construct an α-syn PFF cell model.3、In this study,we treated PC12 cells with different concentrations of TRPML1 agonistML-SA1(20 μM,40 μM and 60 μM)for 24 h.Western blotting showed that compared with the control group,the protein levels of TRPML1 in PC12 cells treated with 40 μM and 60 μM for 24 h were significantly increased(P < 0.01).The results of flow cytometry showed that compared with the control group,there was no significant change in ROS levels in the ML-SA1 treatment groups with different concentrations.In the follow-up experiments,we commonly used ML-SA1(40 μM)in the literature to treat for 24 h as the experimental condition.4、In order to explore the role of TRPML1 in the cytotoxicity caused by α-syn,the following experimental groups were designed in i PC12 cell model: control group,DOX group andDOX+MLSA1 group.The results of cytotoxicity test kit showed that the release of LDH in DOX+ML-SA1 group was significantly lower than in DOX group(P<0.01).Western blotting showed that the levels of TRPML1 protein in DOX+ML-SA1 group was significantly higher than that in DOX group(P<0.05).The levels of α-syn protein in DOX+ML-SA1 group was significantly lower than that in DOX group(P<0.05).The results of calcium detection kit showed that the intracellular calcium concentration of DOX+ML-SA1 group was significantly lower than that of DOX group(P<0.05).The results of flow cytometry showed that the levels of ROS in DOX group were significantly higher than that in control group,while the levels of ROS in DOX+ML-SA1 group were significantly lower than that in DOX group(P<0.05).The levels of mitochondrial membrane potential in DOX group were significantly lower than that in control group,while the levels of mitochondrial membrane potential in DOX+ML-SA1 group were significantly higher than that in DOX group(P<0.01).These results suggest that MLSA1 can effectively reduce the accumulation of α-syn and the increase of intracellular calcium ion caused by the increased expression of α-syn,the increase of ROS levels and the decrease of mitochondrial membrane potential in i PC12 cells.5、The following experimental groups were designed in α-syn PFF cell model: control group,α-syn PFF group and α-syn PFF+ML-SA1 group.The results of cytotoxicity test kit showed that the release of LDH in α-syn PFF+ML-SA1 group was significantly lower than in α-syn PFF group(P<0.05).Western blotting showed that the levels of TRPML1 protein in α-syn PFF+ML-SA1 group were significantly higher than in α-syn PFF group(P<0.05).The levels of α-syn protein in α-syn PFF+ML-SA1 group were significantly lower than that in α-syn PFF group(P<0.05).The results of laser confocal microscope showed that the intracellular calcium concentration of the control group increased significantly with time after perfusing α-syn PFF(P<0.01),while the intracellular calcium concentration of the ML-SA1 pretreatment group did not change significantly after the perfusion of α-syn PFF.The results of flow cytometry showed that the levels of ROS in α-syn PFF group were significantly higher than that in control group,while the levels of ROS in α-syn PFF+ML-SA1 group were significantly lower than that in α-syn PFF group(P<0.05).The levels of mitochondrial membrane potential in α-syn PFF group were significantly lower than that in control group,while that in α-syn PFF+ML-SA1 group were significantly higher than that in α-syn PFF group(P<0.05).The results of JC-1 kit showed that the mitochondrial membrane potential was higher in the control group, and JC-1 aggregation in the mitochondrial matrix showed red fluorescence,while in the α-syn PFF group,the mitochondrial membrane potential decreased,JC-1 showed green fluorescence,and in the α-syn PFF+ML-SA1 group,the mitochondrial membrane potential was improved.These results suggest that ML-SA1 can effectively reduce the accumulation of α-syn and the increase of intracellular calcium ion caused by the increased expression of α-syn,the increase of ROS levels and the decrease of mitochondrial membrane potential in PC12 cells.In conclusion,with the extention of DOX treatment time,the expression of α-syn gradually increased and the expression of TRPML1 gradually decreased in i PC12 cells.When i PC12 cells were treated with DOX for 48 h,TRPML1 decreased most significantly.In PC12 cells,TRPML1 decreased most when treated with 5 μg/m L α-syn PFF for 24 h.These results suggest that TRPML1 may be significantly down-regulated as a protective factor when α-syn overexpression or abnormal aggregation plays a toxic role.Overexpression or abnormal aggregation of α-syn could lead to the increase of intracellular calcium ion,the production of ROS and the decrease of mitochondrial membrane potential,while the treatment with MLSA1,an agonist of TRPML1,could increase the expression of TRPML1 and ameliorate the above changes induced by α-syn.It is suggested that up-regulation of TRPML1 in PD may increase the secretion of α-syn by increasing lysosomal exocytosis,reduce the accumulation of intracellular α-syn,and then reduce the increase of intracellular calcium induced by α-syn,and reduce the production of ROS and the change of mitochondrial membrane potential.On the other hand,it may promote the clearance of damaged mitochondria and ROS by increasing mitochondrial signal transmission,thus reducing the change of mitochondrial membrane potential.The exact mechanism is worthy of further study.This study not only revealed the protective effects of TRPML1 in α-syn induced cytotoxicity,but also provided experimental basis for the development of ML-SA1,a TRPML1 agonist,as a potential drug for the prevention or treatment of PD.