The Mechanism Research Of Inhibiting Glucocerebrosidase Leads To Parkinson's Disease In Mice Via Decreasing Cathepsin D

BackgroundParkinson's disease(PD)is the most common neurodegenerative disease.The pathological hallmarks of PD are loss of dopaminergic(DA)neurons in the substantia nigra of the midbrain,the appearance of Lewy bodies(LB)and pathological accumulation of alpha-synuclein(?-syn)in LB which has toxic effect on neurons.There are many risk factors for PD,studies have showed that glucocerebrosidase(GCase;GBA)deficiency is one of the risk factors for developing PD in recent years.GCase is a soluble sugar ester which is widely present in cell lysosomes.It is involved in sphingolipid metabolism and encoded by GBA gene.Mutations in GBA make the expression and activity of GCase decrease.The studies have confirmed that the decline of GCase expression and activity are found in the substantia nigra of sporadic PD patients.Animal models have exhibited that inhibiting the expression of GCase leads to the accumulation of ?-syn in substantia nigra and the occurrence of PD.Two major pathways are used to maintain protein homeostasis in central nervous system: autophagy-lysosome pathway(ALP)and ubiquitin-proteasome system.ALP is the main pathway for the degradation of ?-syn.Cathepsin D(CTSD)is a lysosomal hydrolase which plays an important role in the degration of protein aggregates.The expression of CTSD reflects the function of lysosomes.Modified by phosphorylation and nitration,?-syn changes into toxic and insoluble protein aggregates in the presynaptic membrane of cells.Studies have showed that serine129(S129)phosphorylated ?-syn is involved in the transformation and pathological accumulation of ?-syn.The specificity of S129 phosphorylated ?-syn is higher than ?-syn in pathological process of PD.ObjectiveIn this study,male C57BL/6 mice were injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and subacute PD model was established.Intraperitoneal injection of CBE(conduritol ?-epoxide)was used to inhibit GCase level in mice brain.The changes of DA neurons in the substantia nigra of mice and the changes of S129 phosphorylated ?-syn,CTSD and GBA were observed by immunohistochemistry and Western blotting respectively.Material and methodsIn this study,male C57BL/6 mice weighing 20-25 g and 8 weeks were used.These mice were divided into 3 groups(n=30 for each group): Control group,MPTP treatment group and CBE treatment group.The mice in MPTP treatment group were injected with 30mg/kg MPTP in normal saline at 9 A.M.for 5 days consecutively to induce subacute PD model mice.The mice in the Control group were subjected to intraperitoneal(i.p.)injection of normal saline with the same volume.The mice in CBE group received a single i.p.injection of 200mg/kg of CBE in deionized water at 9 A.M.for 2 days consecutively.A pole test was employed to evaluate the motor ability.The amount of time to orient downwards(t-turn)and the total time to descend the pole(t-descend)were measured.T-turn time reflects extent of motor coordination and t-descend time reflects extent of bradykinesia.Western blotting was used to evaluate the expressions of S129 phosphorylated ?-syn,CTSD and GBA in substantia nigra of mice.To explore the change of DA neurons in the substantia nigra of mice,immunohistochemistry was used to observe the number of Tyrosine Hydroxylase(TH)-positive cells.All statistical analyses were carried out using GraphPad Prism 6.0 software.We used one-way analysis of variance(ANOVA)analysis to compare changes before and after treatment.P values of less than 0.05 were considered statistically significant.Results1.Compared with the Control group,MPTP and CBE groups showed vertical hair,vertical tail,tremble,bradykinesia and postural instability symptoms.Pole test results showed significantly increased time to turn and descend in these two groups(P<0.001).2.Immunohistochemistry results showed that the numbers of TH-positive cells in the bilateral substantia nigra of MPTP and CBE groups were significantly decreased compared with the Control group(P<0.001).3.The result of Western blotting showed that in comparison to the Control group,the level of S129 phosphorylated ?-syn were increased(P<0.01,P<0.05),but the expressions of CTSD and GBA were decreased in MPTP and CBE groups(P <0.001).Conclusion1.Lysosomal dysfunction results in the accumulation of S129 phosphorylated ?-syn in substantia nigra.2.GCase deficiency is a risk factor for the occurrence of PD.3.Inhibition of GCase leads to the accumulation of S129 phosphorylated ?-syn and loss of DA neurons in substantia nigra via decreasing CTSD,which result in the occurrence of PD in mice.