Reduced VMAT2 Expression Contributes To The Non-motor Symptoms In Parkinson's Disease Animal Model

Section 1 Motor disorders and depression-like behavior revealed in VMAT2 heterozygous mice(VMAT2 HT).Objective The aim of the study is to assess the motor ability and depression-like behavior of VMAT2 HT mice with the growing of age.Methods VMAT2 HT mice used in this study were provided by professor Uhl GR at Emory and bred in Huazhong University of Science and Technology Laboratory Animal Center.Male mice of VMAT2 WT and VMAT2 HT with different ages(2 months,6 months,15 months and 30 months)were used for all studies.Behavior test and TH expression as well as DA and 5-HT content of striatum were assessed.Specifically,mice were assessed for motor function with open field test(OFT)and rotarod test,depression-like behavior with sucrose preference test(SPT)and tail suspension test(TST).Then mice were sacrificed for subsequent analysis to assess on TH expression in striatum using immunohistochemical analysis.DA and 5-HT content in striatum were measured via HPLC-ECD.Results As for the motor behavior test,VMAT2 HT mice were similar to WT mice in travel distance in OFT and the latency to fall in rotarod test when at 2 months,6 months and 15 months old(P>0.05).At 30 months old,VMAT2 HT mice displayed a shorter travel distance in OFT and shorter time on rotarod during the 5-minutes test compared to VMAT2 WT mice(P<0.05).As for the depression-like test,VMAT2 HT mice had no difference with WT mice in the percentage of sucrose consumed in SPT and the immobility time in TST when at 2 months old(P>0.05).VMAT2 HT mice displayed a lower percentage of sucrose consumed in SPT and longer immobility time in TST during the 6 minutes test compared to VMAT2 WT mice(P<0.05)at 6 months,15 months and 30 months old,indicating depression-like behavior at these ages.TH immunohistochemistry staining confirmed that there was no difference between VMAT2 HT and VMAT2 WTmice in TH expression in striatum at 2 months,6 months and 15 months.At 30 months old,the optical density of TH positive terminals in VMAT2 HT mice was much lower than VMAT2 WT mice.In line with these results,HPLC showed VMAT2 HT mice were similar to WT mice in DA and 5-HT content in striatum at 2 months,6 months and 15 months.At 30 months old,the DA and 5-HT content in VMAT2 HT mice was lower than VMAT2 WT mice.Conclusions VMAT2 HT mice displayed depression-like behavior at 6 months old and the depression-like behavior continue until 30 months old.However,motor retardation didn't manifest until 30 months old.In addition,at 30 months old,TH expression as well as DA and 5-HT content in striatum was lower than VMAT2 WT mice.Section 2 Reduced VMAT2 expression aggravates the hyposmia and depression-like behavior in the MPTP model of Parkinson's diseaseObjective To examine the effect of subacute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)on mice with a reduced expression of VMAT2 on hyposmia and depression,focusing on the histopathological and biochemical alterations in OB,amygdala and LC as well as the neurogenesis in SVZ/OB system.Methods(1)The observation of non-motor symptoms and change of extranigral change: Male mice of the VMAT2 HT and WT mice(n=10/group)were intraperitoneally injected either with MPTP(30mg/kg)or saline once a day for seven days.On day 18 to 21,the behavioral evaluation was performed.In specific,mice were assessed for motor function with open field test(OFT)and rotarod test,depression-like behavior with sucrose preference test and tail suspension test(TST),hyposmia with buried food pellet test.Then mice were sacrificed for subsequent analysis to assess on nigrostriatal and extranigraldopaminergic systems.TH western blot and immunohistochemical analysis were used to assess the lesion of extranigral structures such as LC,amygdala and OB.DA and its metabolite were analyzed by HPLC-ECD in striatum and OB.(2)The assessment of change of neurogenesis in SVZ/OB system: After the administration of MPTP,on day 9,animal received once injection of Brdu(100mg/kg)per day,a marker of proliferating cells,for 5 consecutive days and sacrificed 24 h after the last injection to investigate cell proliferation in SVZ/OB by measure of count of Brdu positive neurons.Results(1)Behavior test revealed that MPTP can induce locomotor retardation and depression-like behavior in both genotypes but more obvious in VMAT2 HT mice.MPTP exposure only increased the retrieving time in VMAT2 HT mice in buried food pellet test.(2)Reduced VMAT2 expression displayed enhanced MPTP-induced neurotoxicity in nigrostriatal system.Western blotting analysis of SN lysates showed that administration of MPTP induced decrease of TH expression in both genotypes and this trend is more obvious in MPTP-treated VMAT2 HT mice.This observation was further confirmed by the TH positive cell counting.MPTP decreased expression of TH in the striatum of VMAT2 HT and WT mice and reduced expression of VMAT2 further aggravated this effect.Analysis of HPLC results showed both the genotype exhibited an obvious decrease in DA content after exposure to MPTP and the amplitude was more prominent in VMAT2 HT mice compared to WT mice(82% VS 67%).(3)Reduced VMAT2 expression exacerbated MPTP-induced neurotoxicity in the extranigral structures,such as LC,amygdala and OB.MPTP slightly reduced TH expression of LC in WT animals without significance(P>0.05)as measured by Western blot.By contrast,significant decrease was observed in VMAT2 HT mice(P<0.01).TH immunostaining in LC further confirmed the results.Administration of MPTP also decreased TH fibers in the amygdala of VMAT2 HT and WT mice and reduced expression of VMAT2 further aggravated this effect.TH expression slightly decreased in the OB of VMAT2 WT mice after the administration of MPTP(P>0.05)while VMAT2 HT animals experienced an obvious decrease(P<0.001)compared to saline-treated controls asmeasured by western blotting.TH positive counting in OB confirmed the significant decrease in the VMAT2 HT mice after MPTP.HPLC results of OB showed that DA content in OB slightly decreased in VMAT2 WT mice while VMAT2 HT mice displayed a 66% reduction in response to MPTP.(4)MPTP decreased Brdu+ cell counting in SVZ and OB in both genotypes but more obvious in VMAT2 HT mice.Conclusions Reduced expression of VMAT2 further aggravates the MPTP lesion inducing more obvious motor and non-motor symptoms,in this study depression and hypomisa,accompanied by extended histopathological and biochemical alterations of extranigral dopaminegic system,LC,amygdala and OB.Furthermore,the neurogenesis of OB was also further impaired in the MPTP-treated VMAT2 HT mice.Section 3 The toxic effect of DOPAL on dopaminergic cellObjective To investigate the change of DOPAL/DA in VMAT2 HT mice with the growing age and in response to MPTP.We also aim to study the effect of DOPAL on SHSY-5Y cell focusing on cell survival,cell morphology,?-synuclein aggregation and the autophagy lysosome pathway.Methods(1)DOPAL and DA content of striatum in VMAT2 WT and VMAT2 HT mice with different ages(2 months,6 months,15 months and 30 months)were measured by HPLC-ECD.Striatum of MPTP-treated 6-month-old male VMAT2 WT and HT mice were also analyzed by HPLC-ECD.(2)SHSY-5Y cell were treated with DOPAL with different concentration and different time.Inverted microscope was employed to observe the cell morphology.The cell survival was detected via CCK8.Western blot and immunofluorescence staining were used to confirm the aggregation of ?-synuclein.Theprotein level of autophagy-related molecule,such as P62,Beclin 1 and LC3,were measured by Western blot.Results(1)DOPAL/DA in VMAT2 HT mice was slightly higher than VMAT2 WT mice at 2 months,6 months and 15 months old with no statistical difference(P>0.05).At 30 months old,DOPAL/DA in VMAT2 HT mice was significantly higher than VMAT2 WT mice(P<0.05).Administration of MPTP increased the DOPAL/ DA in both VMAT2 HT and VMAT2 WT mice and the trend was more obvious in VMAT2 HT mice.(2)SH-SY5 Y cell shaped as short fusiform,with the great soma,equal plasm and clear nucleolus.After DOPAL intervention,cell became shrinkage and turned round and chromatin brought together.CCK8 showed that DOPAL decreased the cell survival in a concentration and time dependent manner.Western blot and immunofluorescence staining revealed that DOPAL can induce the aggregation of ?-synuclein.The LC3-II/LC3-I and P62 were increased after the administration of DOPAL and in a concentration dependent manner when the dose was below 100 ?M as measured by Western blot.In a similar way,the protein level of Beclin 1 and P62 began to increase after the DOPAL treatment for 24 h and the increase trend might last for 48 h.Conclusions DOPAL/DA increase with age in VMAT2 WT and HT mice but more obvious in VMAT2 HT mice.MPTP increased the DOPAL/ DA in both VMAT2 HT and VMAT2 WT mice and the trend was more obvious in VMAT2 HT mice.DOPAL could decrease the cell survival,induce the aggregation of ?-synuclein and the autophagy lysosome pathway might be involved in the course of ?-synuclein aggregation.