Preliminary Study Of Effect And Its Mechanism Of Glucocerebrosidase Knockdown On α-synuclein

Parkinson’s disease (PD) is a progressive degeneration diseases of the central nervoussystem occurs in the elderly, the major clinical manifestations for static tremor,bradykinesia, myotonia, abnormal gait and posture. Since the first reported in1817, themedical profession of PD has been up to nearly200years. The gold standard for diagnosisof PD pathology is the formation of Lewy bodies (LB), while the study found α-synucleinis an important component of LB. On physiological conditions, the function of α-syn isparticipate in the formation of the SNARE (Soluble N-ethyl-maleimide sensitive fusionprotein attachment protein receptor) complex and regulate neurotransmitter release; in thespecific case, α-syn were polymerizing to insoluble oligomeric form (starch like fibrosis),rise to dopaminergic neurons neurotoxicity, leading to neuronal death. Has been made itclear that, α-syn gene (SNCA) mutation is a pathogenic factor of PD with autosomaldominant inheritance. Animal studies reported that in the transgenic mice and drosophilawith overpressed α-syn can reproduce the pathology and symptoms of PD. Thus, thescientific community agreed that α-syn is a causally related to PD, although the mechanismof α-syn cause PD is not entirely clear,Gaucher disease (GD) is an autosomal recessive genetic disease is caused by mutationsin the glucocerebrosidase gene (GBA), cause to lysosomal enzyme-beta-glucocerebrosidase(GCase) shortage, deficiency in the enzyme glucocerebrosidase lead to the accumulation ofits Glucosylceramide (GluCer)substrate in vivo mononuclear phagocyte system, ultimatelyresult in a metabolic disorder. Over the past decade, identification of rare GD patients withparkinsonian symptoms appearing in case reports, larger patient series, and prospectivestudies in some cases. Next, clinical study reported that PD patients have a highermutation rate of GBA; other study also observed that the frequency of GBA mutations inPD patients was20%by autopsy. Mechanism study for the relationship of GBA mutationsand PD by immunofluorescence show that dual-positive Lewy bodies of GCase and α-synin the GD patients with Parkinsonism. recent studies show that，the effection of GBA mutation to α-synuclein may have closerelationship with GD become PD，but the mechanism of the relationship between Gaucherdisease and Parkinson’s disease was not clearly. Currently there are two main hypotheses:Gain-of-function hypothesis believe that GBA mutations leading to protein misfolding,increasing the ubiquitin-proteasome system (ubiquitin proteasome system, UPS) burdenreduction that abnormal protein degradation caused mutant a-syn levels, thereby increasingthe susceptibility of PD; according to the loss-of-function hypothesis, GBAhaploinsuffiency might cause its substrate glucocerebroside and other polyunsaturatedlipids to accumulate, the membrane lipid structure changes, interferes with the α-synbinding to receptors at the cell membrane, making the α-syn’s abnormal accumulation, butthe lack of direct evidence.The above studies have shown that GBA mutations affect the level of α-syn may beclosely related to the incidence of PD, but its mechanism is not clear. This subject build theGCase RNAi lentiviral vector interference GCase expression analog GBA-functionmutation at first; then explore the possible mechanisms on GBA loss-of-function mutationsof α-syn by GCase interference α-syn for clinical treatment PD looking for a new target toprovide a theoretical basis. The experiments were divided into three parts, as follows:Part1: The construction of the RNAi vector and itssilencing effect on GCase expressionObjective: To construct GCase RNA interference targeting vector and transfectedSH-SY5Y cells, identification silencing effect for subsequent experiments provide researchtools.Methods: According to previously reported in the literature and the design andsynthesis of published data Genbank3siRNA, transient infection of SH-SY5Y cells usingWestern blotting preliminary evaluation of three siRNA interference effect. Better filter outinterference by siRNA synthesis shRNA, build carry GCase shRNA plasmid pLKO.1,lentivirus packaging. The packaged viral particles infect slow SH-SY5Y cells werescreened using the stable puromycin-resistant cells infected (GCase shRNA group).Negative control group (Control) carrying shRNA no homology with the target gene, the normal control group (Normal) without lentiviral transfection. Reverse transcriptionpolymerase chain reaction (RT-PCR) to detect the group GCase mRNA levels. Detect theGCase protein levels in each group by Western blotting, and to detect GCase activity.Detect the GluCer level by fluorescent immunohistochemistry. MTT assay was used todetect the cell survival and growth capacity.Results: The constructed three siRNA interference effects are, after Western blottingpreliminary testing, siRNA1(1452) has good interference effect. To siRNA1(1452)constructed pLKO.1-GCase-shRNA, and lentivirus packaging for subsequent experiments.Lentivirus infected cells, after puromycin obtained stably infected GCase shRNA lentiviralSH-SY5Y cells. After infection GCase mRNA changes: by RT-PCR identification, Normalgroup and Control group, GCase mRNA levels were not significantly different; whileGCase shRNA group GCase mRNA levels were significantly lower than the Control groupand Normal group and Control group, GCase shRNA Groups mRNA inhibition rate of68%.GCase protein levels: by Western blotting identified Normal group and Control groupGCase protein levels were not significantly different, and GCase shRNA group GCaseprotein levels were significantly lower than the Control group and Normal group andControl group, GCase shRNA group GCase protein levels decreased70%. GCase activitychanges: Normal group and Control group, GCase activity was no significant difference;compared with the Control group, GCase shRNA group GCase activity was significantlydecreased activity decreased65%. GluCer levels: Normal group and Control group, GluCerno significant change; compared with the Control group, GCase shRNA groupGluCer-positive cells was significantly increased (171%). Cell survival and growth capacity:The MTT assay, Normal group and Control group, cell viability was no significantdifference; and Control comparison, GCase-shRNA group cell activity decreased by6.15%(24h),18.43%(48h),25.54%(72h),25.43%(7d).Conclusion: The constructed three GCase siRNA, transient transfection can reach theinterference effect; After initial screening will interfere with the better-siRNA constructshRNA, lentivirus packaging. GCase interference lentivirus infections SH-SY5Y cells canbe stable and effective interference GCase mRNA expression, decreased expression ofGCase. GCase levels down after GCase activity decreased, causing GluCer decompositiondecreased accumulation of intracellular GluCer,7d inner cell growth and survivalcapabilities are subject to different degrees. Part2: Study on the effect of GCase RNAinterference on α-synuclein levelsObjective: The first part of the carrier RNA interference experiments, after observingGCase RNAi cells α-syn level changes, and explore the impact of α-syn GCase RNAipossible mechanisms, in order to order to clarify the link between GD and PD.Methods: The experiment was divided into four groups, Normal group, Control group,GCase shRNA groups, and leupeptin treated group (Leu); Normal group, Control group andGCase shRNA group of cells derived from the first part of the experiment, Leu groupControl group Leu treatment given cell. To detect the levels of α-syn mRNA and proteinlevels by Realtime PCR and Western blotting respectively. To detect the expression ofLAMP1(lysosomal markers) and α-syn expression, and it’s coexistence situation byimmunofluorescence. To detect soluble α-syn (T-soluble α-syn) and insoluble α-syn(T-insoluble α-syn) protein levels in each group furtherly by Western blotting.Results: Western blotting results showed that: α-syn in normal SH-SY5Y cells hasbeen expressed in visible18KD protein bands; compared with the Normal group, Controlgroup α-syn protein levels were no significant differences; while the Control group andNormal group, GCase shRNA lentiviral transfection α-syn protein expression wassignificantly increased (for the Control group133.9%±5.573%). Realtime PCR analysisshowed that: Normal group, Control group, GCase shRNA group, three groups α-synmRNA levels no significant difference. Immunofluorescence staining showed: with thenegative control group (30.96%±3.184%) compared, GCase shRNA lentiviral infectionα-syn-positive cells was significantly increased (46.49%±2.849%). With GCase shRNAgroup, giving bright lysosomal inhibitors aprotinin (Leu) treated α-syn positive cellscontent (65.78%±3.208%) increase further. Further analysis revealed that Leu groupLAMP-1and α-syn double positive cells content was25.78%±2.475%, compared withcontrol group (17.24%±2.332%) increased significantly; while GCase shRNA groupLAMP-1and α-syn double positive cells content38.99%±2.602%, compared with Leugroup increased significantly. Further adoption by Western blotting T-soluble α-syn protein levels results show that: Compared with Normal, Control group T-soluble α-syn proteinlevels did not change significantly, gray value analysis to Normal group94.33%±4.86%;and Control group, GCase shRNA group T-soluble α-syn protein levels were significantlyincreased, the gray level of a Control group132.0%±6.26%. Leu given after T-solubleα-syn protein levels, compared with GCase shRNA group was significantly higher grayvalue analysis Control group152.7%±6.323%. Western blotting analysis T-insolubleα-syn results show that: compared with the Normal group, Control group T-insoluble α-synprotein levels did not change significantly, gray value analysis to Normal group99.75%±5.12%. Compared with the Control group, GCase shRNA group T-insoluble α-syn proteinincreased significantly, gray value analysis Control group298.3%±15.82%. Leu givenafter T-insoluble α-syn protein levels, compared with GCase interference group hadsignificantly decreased gray value analysis Control group211.5%±15.01%.Conclusion:1, RNA interference GCase after a-syn protein levels, but a-syn mRNAlevels did not change significantly, suggesting that RNA interference GCase does not affectthe transcription of a-syn levels. Leu lysosomal inhibitors given treatment, SH-SY5Y cells,a-syn protein levels, which is a lysosomal enzyme GCase, after which the RNA interferenceactivity decreased, so that the a-syn changes in protein levels possible mechanism isreduced GCase activity after impact lysosomal degradation pathway, causing a-synaccumulation.2, GCase interference main elevated T-insoluble a-syn, and lysosomalinhibitors Leu deal mainly elevated T-soluble a-syn. So that there may be other mechanismsGCase interference, causing a-syn aggregation occurs, resulting insoluble a-syn.Part3: The effect of GluCer on α-synuclein amyloid fibrosisObjective: To study the effection of GluCer on α-syn amyloid fibrosis, to clarify theimpact of RNA interference GCase α-syn possible mechanisms further.Methods: Normal SH-SY5Y cells were cultured by adding GluCer were divided intothree groups, the control group (Control), GluCer low group (PC90/GluCer10), GluCerhigh level group (PC25/GluCer75), the control group was normal culture cells remainingtwo groups were joined by the corresponding proportion of L-α-phosphatidylcholine (PC)and GluCer,24h and72h in the co-culture was observed by Western blot T-insoluble α-syn expression. In vitro cell-free environment GluCer and α-syn has grown to simulate in vivolysosomal environment (pH=5,37℃), the experimental group setting case above, thecontrol group only contains α-syn, I2GluCer groups in addition to equimolar α-syn, thecorresponding proportions were joined by PC and GluCer. At the corresponding point intime, using Thioflavine T fluorescence detection and analysis of Congo red absorbance wasmeasured in each group α-syn protein amyloid fibrosis situation. Using SDS-PAGEelectrophoresis and Coomassie blue staining of protein molecular weight changedcircumstances.Results: SH-SY5Y cells were cultured by adding GluCer, Western blot results:cultured24h and72h,3groups T-insoluble α-syn expression levels were not significantlydifferent. In vitro cell-free environment GluCer and α-syn has grown, Thioflavine Tfluorescence detection RESULTS: Control group, PC90/GluCer10group at each time pointThioflavine T fluorescence values did not change significantly; with the Control group andPC90/GluCer10group, PC25/GluCer75group Thioflavine T fluorescence intensity wassignificantly lower in the16h,24h when the fluorescence intensity was significantly higherfluorescence intensity increases with hysteresis. Congo red absorbance analysis results withsimilar Thioflavine T: compared with the Control group, PC90/GluCer10group at eachtime point of Congo red absorbance values did not change significantly; PC90/GluCer10with the Control group and the group, PC25/GluCer75groups Congo red absorbance valuewas significantly lower at16h,24h when the absorbance increased significantly elevatedabsorbance also has hysteresis. SDS/PAGE gel electrophoresis and Coomassie bluestaining results showed that, a-syn protein bands appear in the35-50kD range frommolecular point of view meet insoluble a-syn characteristics.Conclusion: In vitro cell-free environment, GluCer can accelerate α-syn fibrosis,causing α-syn molecular weight increases, and the occurrence of amyloid fibrosis; α-synchanges with GluCer concentration. Clnclusions1: Successfully construct three GCase siRNA, the interference effect can reached bytransient transfection, the siRNA1has better interference effect (1452).2: GCase shRNA packaging of lentivirus infected SH-SY5Y cells, can interferedGCase mRNA level, decreaseed the GCase protein level stablely and effectively. ThenGCase activity was reduced, GCase causing decomposition to reduce GluCer, accumulationof GluCer in cell can affect cell survival and growth capacity.3: After expression of the RNA interference GCase, a-Syn protein level was increased,but no significant change in a-Syn mRNA level. Mean that GCase knockdown does notaffect the transcription level of a-syn. Treat with lysosomal inhibitors Leu, a-Syn proteinlevels in SH-SY5Y cells can be increased. GCase is a lysosomal enzyme; its activity wasdecreased by RNA interference, so that the possible mechanism of a-syn protein levelsaccumulation may be owing to the impaction of lysosomal degradation pathway by GCaseenzyme activity reduced. So regulate the lysosomal protein degradation pathway may be atherapeutic target for Parkinson’s disease.4: The GCase interference elevated T-insol a-syn apparent; and treat with lysosomalinhibitors Leu was elevated T-sol a-SYN more apparent. That means there may be the othermechanisms causing a-syn aggregation by GCase interference, resulting a-syn-insoluble occurs.Changing cell lipid composition may be therapeutic targets for Parkinson’s disease5: GluCer and a-syn were incubate in vitro cell-free environment can cause a-synfibrosis. GCase interference is considered, GluCer accumulation, may be constituted by achange of cell lipids, causing the formation of insoluble a-syn.