| Parkinson's disease(PD)is a common neurodegenerative disease.In China,the prevalence of PD is about 1.7% in people over 65 years old.The clinical symptoms of PD are mainly dyskinesia,memory loss,constipation,and sleep disorders.The main pathological features are a loss of dopaminergic neurons in the substantia nigra(SN)and observed that ?-synuclein aggregates constituting Lewy bodies(LBs).Inflammation,oxidative stress,iron accumulation,abnormal protein aggregation and mitochondrial dysfunction,might contribute to the degeneration of dopaminergic neurons.The etilology of PD is unknown,however,it is accepted that a combination of aging,genetic and environmental factors was involved.Among all of the genetic factors related to PD,Gaucher disease(GD)is caused by mutations of the GBA gene,which encodes the lysosomal gluococerebrosidase(GCase).GD patients and GD carriers are predisposed towards the development of PD;whether or not they carry GBA mutations,GCase activity and protein levels are significantly reduced in the SN in PD patients,suggesting that there might be a correlation between GCase and the onset of PD.It has been suggested that the absence of GCase leads to accumulation of glucosylceramide(GlcCer)in the lysosomes causing to lysosomal dysfunction,inhibiting ?-synuclein degradation resulting in pathological aggregation-the neuropathological hallmarks of PD.Accumulated GlcCer directly promotes the formation of the ?-synuclein oligomer;aggregated ?-synuclein inhibits the endoplasmic reticulum(ER)–Golgi trafficking of newly synthesized GCase resulting in lysosomal localization normal GCase,which forms a vicious cycle and eventually leading to the death of dopaminergic neurons.Abnormal iron accumulation is another neuropathological feature of PD.Iron mediates generation of hydroxyl radicals by Fenton reaction,inducing oxidative stress and mitochondrial dysfunction,resulted in dopaminergic neuronal death.Our laboratory previously proved that iron up-regulated the expression level of ?-synuclein and abnormal aggregation,which was partially blocked by antioxidant,in iron overload cell model and iron dextran overload rat model.It was reported small interfering RNA(siRNA)silencing of PIKN1 in SHSY-5Y cells induced oxidative stress and mitochondrial dysfunction,leading to decreased GCase activity and protein levels.GCase activity is significantly decreased,the protein levels of ferroportin1(FPN1)was down-regualted and iron was accumulated,in the GCase inhibitors-Conduritol B Epoxide(CBE)treated the J774 macrophage cell line.These evidences indicated that GCase expression or activity and iron metabolism might be correlated,but the effect of PD iron metabolism abnormality on GCase expression or activity is not elucidated.In the present study,western blots and luciferase assay were used to detect GCase protein expression and GCase enzyme activity in SH-SY5 Y dopaminergic cells with ferric ammonium citrate(FAC),CBE or FAC+CBE treatment.The autophagy receptor protein p62/SQSTM1 and the autophagy markers LC3 II were detected.The lysosomal marker lysosomal-associated membrane proteins-1(LAMP-1)and GCase transporter protein lysosomal integral membrane protein-2(LIMP-2)were detected for lysosomal function.Alpha-synuclein expression was also evaluated.In iron overload rat model with intraperitoneal injection of iron dextran,western blots were used to detect the expression of GCase protein,LIMP-2 protein,?-synuclein protein in the olfactory blub(OB)and the striatum(Str).Injection of adeno-associated virus-shRNA(Gba)(AAV-shRNA(Gba))was adopted to interfer GCase protein expression in the Str.The changes of the above indicators were observed in iron-dextran treated mice.The results were shown as follows:1.SH-SY5 Y cells were treated with 100 ?mol/L FAC for 4 h,12 h,24 h and 48 h.GCase protein levels began to increase at 4 h;GCase protein levels were up-regulated(1.13 times,1.18 times,and 1.32 times)at 4 h,12 h and 24 h compared with the control group(F=11.47,P<0.05,P<0.01,P<0.01).GCase protein levels returned to normal level at 48 h.SH-SY5 Y cells were treated with 100 ?mol/L FAC,100 ?mol/L CBE or 100 ?mol/L FAC+CBE for 24 h,respectively.Analysis of variance(ANOVA)of factorial design showed that there was no interaction between FAC and CBE effects on GCase protein expression,GCase protein levels were up-regulated(64%,73%)treated with FAC or CBE groups compared with the control group(F=4.475,F=12.270;P<0.05,P<0.01).GCase enzyme activity were down-regulated(38%,61%)treated with FAC or CBE compared with the control group(F=6.026,F=4.392;P<0.05,P<0.05).The results indicated that intracellular iron overload could up-regulate GCase protein expression and decrease GCase enzyme activity.2.SH-SY5 Y cells were treated with 100 ?mol/L FAC,100 ?mol/L CBE or 100 ?mol/L FAC+CBE for 24 h,respectively.ANOVA of factorial design showed that there was no interaction between FAC and CBE effects on p62,LC3 II protein expression.p62 protein levels were up-regulated respectively(48%,36%)treated with FAC or CBE groups compared with the control group(F=10.215,F=5.001;P<0.01,P<0.05).LC3 II protein levels were up-regulated(54%,50%)compared with the control group(F=6.026,F=4.392;P<0.05,P<0.05).The results indicated that intracellular iron overload or GCase activity inhibition could up-regulate expression of autophagy receptor protein p62 and autophagy marker LC3 II protein.3.SH-SY5 Y cells were treated with 100 ?mol/L FAC for 24 h.LAMP-1 protein levels were down-regulated(30%)compared with the control group(t=3.272,P<0.01),however,LIMP-2 protein levels were up-regulated(92%)compared with the control group(t=3.737,P<0.001).The results indicated that intracellular iron overload could down-regulate lysosomal marker LAMP-1 protein expression and up-regulate GCase transporter LIMP-2 expression.4.SH-SY5 Y cells were treated with 100 ?mol/L FAC,100 ?mol/L CBE or 100 ?mol/L FAC+CBE for 24 h,respectively.ANOVA of factorial design showed that there was no interaction between FAC and CBE effects on ?-synuclein protein expression.Alpha-synuclein levels were up-regulated respectively(90%,87%)treated with FAC or CBE groups compared with the control group(F=5.915,F=4.901;P<0.05,P<0.05).The results indicated that intracellular iron overload or GCase activity inhibition could up-regulate expression of ?-synuclein.5.Rats were intraperitoneally injected with iron dextran for 1,2,3,4 weeks.GCase protein levels were down-regulated(35% and 21%)in the OB in 1-week and 2-week compared with the control group(t=2.119,t=2.969;P<0.05,P<0.05).GCase expression did not change in the OB at 3-week and 4-week compared with the control group.GCase expression did not change in the Str at 1-week compared with the control group.GCase protein expression levels were down-regulated(41%,29% and 43%)in the Str at 2-week,3-week and 4-week compared with the control group(t=2.342,t=2.835,t=3.513;P<0.01,P<0.05,P<0.01).The results indicated that intraperitoneal injection of iron dextran could down-regulate expression of GCase protein in the OB and Str.6.LIMP-2 protein levels did not no significantly change in the OB of iron dextran-treated rats for 1,2,3,4 weeks compared with the control group.LIMP-2 protein levels were respectively down-regulated(54%,42% and 34%)in the Str at 1-week,2-week and 3-week compared with the control group(t=3.618,t=3.529,t=2.942;P<0.05,P<0.05,P<0.05).LIMP-2 protein levels did not change in the Str in 4-week.The results indicated that intraperitoneal injection of iron dextran could down-regulate the expression of LIMP-2 protein in the Str.7.Alpha-synuclein levels were up-regulated(69%)in the OB of iron dextran treated rats for 4 weeks compared with the control group(t=3.070,P<0.01).Alpha-synuclein expression levels did not change in the OB at 1-week,2-week and 3-week compared with the control group.Alpha-synuclein expression levels were up-regulated(36% and 45%)in the Str in 3-week and 4-week compared with the control group(t=2.425,t=3.473;P<0.05,P<0.01).Alpha-synuclein expression levels did not change in the Str at 1-week and 2-week compared with the control group.The results indicated that intraperitoneal injection of iron dextran could up-regulate the expression of ?-synuclein in the OB and Str.8.AAV-shRNA(Gba)or AAV-shRNA(NC)virus was injected into the Str of rats for 2 weeks,and then iron dextran was injected intraperitoneally for 4 weeks.ANOVA of factorial design showed that there was no interaction between iron and AAV-shRNA(Gba)effects on GCase protein expression.GCase protein levels were down-regulated(38%,34%)in iron+AAV-shRNA(NC),NS+AAV-shRNA(Gba)groups,respectively,compared with the NS+AAV-shRNA(NC)group(F=14.715,F=12.064;P<0.05,P<0.05).The results indicated that injection of iron dextran or AAV-shRNA(Gba)could down-regulate the expression of GCase protein in the Str.9.ANOVA of factorial design showed that there was no interaction between iron and AAV-shRNA(Gba)effects on LIMP-2 protein expression.LIMP-2 protein expression levels did not change in iron+AAV-shRNA(NC)group compared with the NS+AAV-shRNA(NC)group.LIMP-2 protein expression levels were up-regulated(1.2 times)in NS+AAV-shRNA(Gba)groups compared with the NS+AAV-shRNA(NC)group(F=32.574,P<0.01).The results indicated that injection of AAV-shRNA(Gba)could up-regulate the expression of LIMP-2 protein.10.ANOVA of factorial design showed that there was interaction between iron and AAV-shRNA(Gba)effects on ?-synuclein protein expression.Alpha-synuclein expression levels were up-regulated(88%,1.5 times,1.8 times)in iron+AAV-shRNA(NC),NS+AAV-shRNA(Gba)or iron+ AAV-shRNA(Gba)groups,respectively,compared with the NS+AAV-shRNA(NC)group(F=5.404,F=66.455,F=10.767;P<0.05,P<0.001,P<0.001).Alpha-synuclein expression levels were up-regulated(48%)in iron+AAV-shRNA(Gba)group compared with the iron+AAV-shRNA(NC)group(F=9.675,P<0.05).The results indicated that iron dextran or AAV-shRNA(Gba)could synergistically up-regulate the expression of ?-synuclein protein in the Str.The above results indicated that iron overload in SH-SY5 Y cells could down-regulate GCase enzyme activity and up-regulate the protein levels of GCase,as well as autophagy receptor protein p62 and autophagy marker LC3 II and ?-synuclein,consistent with CBE results.These results suggested that iron overload or absence of GCase activity impaired autophagy flux leading to abnormal autophagy function,which may be related to the up-regulation expression of ?-synuclein.Iron could down-regulate the expression of lysosomal marker LAMP-1 and up-regulate the expression of LIMP-2,suggesting that iron might decrease GCase enzyme activity by affecting the lysosomal function.Low GCase enzyme activity might cause the compensatory expression of GCase and LIMP-2.GCase and LIMP-2 were down-regulated and ?-synuclein was up-regulated in the OB and Str of iron dextran treated rats.Adeno-associated virus-shRNA(Gba)(AAV-shRNA(Gba))interfers GCase expression in the Str,cooperating with iron to up-regulate ?-synuclein expression.This study explored the relationship between iron overload and GCase expression and activity in vivo and vitro,and provided a new experimental basis for the interaction of iron metabolism disturbance in PD and GCase in the pathogenesis of PD. |
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