Genetic Analysis Of LRRK2 In Parkinson ’S Disease In Han Chinese Population And Curcumin Protects Against LRRK2 G2385R-induced Neurotoxicity

Part Ⅰ.Genetic analysis of LRRK2 in Parkinson’s disease in Han Chinese populationBackground:The pathogenesis of Parkinson’s disease(PD)is unclear until now.At present,and it is believed that the interactions between genetics,environmental factors and aging are involved in the development of PD.Genetics plays more and more important role in this process.Mutations in leucine-rich repeat kinase 2 gene(LRRK2)are recognized as the most frequent genetic factors contributing to familial and sporadic PD.More than 100 variants of LRRK2 have been associated with PD,indicating an important role for LRRK2 in PD susceptibility.The large coding regions of LRRK2 have made it difficult to conduct genetic analysis of the entire gene.The aim of this study was to systematically explore variants in the coding regions of LRRK2 in PD patients within the Han Chinese population.Methods:The whole coding regions of LRRK2 from 296 Han Chinese PD patients were sequenced by targeted regions sequencing and exome sequencing.The data were compared to the 1000 Genome Project and Exome Aggregation Consortium(ExAC)datasets to distinguish rare(minor allele frequency(MAF)<0.5%)and common variants of LRRK2.The identified variants were confirmed by Sanger sequencing.The association between LRRK2 G2385R and R1628P were analyzed in sporadic PD and 643 healthy controls.Collect all the basic demographics,motor and non-motor manifestations in the idiopathic PD(iPD,n=216),PD with common LRRK2 variants(common LRRK2-PD,n=56)and PD with rare LRRK2 variants(rare LRRK2-PD,n=16),and studied the genotype-phenotype association between the three groups.Results:Eighteen rare variants were identified in 27 PD patients,and 13 of them(M100T,L153W,A459S,S722N,R792K,C925Y,R981K,S1007T,V1447M,R1677S,N2308D,N2313S,S2350I)were firstly reported in PD.We found the frequency of the common G2385R variant to be significantly higher than the frequency reported in the 1000 Genomes database for the Han Chinese population(P<0.001).In contrast,there was no significant difference between PD and controls for the R1628P variant in our sample population(P=0.532).We found one LRRK2 G2019S mutation in a sporadic,late onset PD case in the Han Chinese population,suggesting that the G2019S variant could also contribute to PD in the Asian population.We also tried to explore the genotype-phenotype associations of LRRK2 variants in our data,and found that PD patients with common and rare LRRK2 variants were more likely to have motor fluctuation and non-motor symptoms.Conclusion:This is the first systematic analysis of LRRK2 coding region variants in mainland Han Chinese PD patients using next generation sequencing.The identification of novel variants in LRRK2 suggests that this gene plays an important role in the pathogenesis and phenotype of PD in Han Chinese population,and our data also rang the alarm bell-more attention should be paid to the whole coding regions of LRRK2.Part Ⅱ.Curcumin protects against LRRK2 G2385R-induced neurotoxicityBackground:Mutations in LRRK2 are recognized as the most frequent genetic factors contributing to Parkinson’s disease(PD).The frequency of LRRK2 variants in different races is significantly different.Our study found the frequency of LRRK2 G2385R in Chinese population is about 18.58%in sporadic PD,OR is 2.707 compared to healthy controls.The pathogenesis role of LRRK2 G2385R is not clear.Unlike G2019S mutation,G2385R may have loss of kinase activity.The main pathological mechanism of PD is the loss of dopaminergic neurons and the aggregation of a-synclein.Methods:We studied the G2358R toxicity by cell viability assay in human neuroblastoma cells(SH-SY5Y).GFP(green fluorescent protein)and various LRRK2 plasmids[Vector,Flag-wild-type(WT)、Flag-G2385R pcDNA3.1-LRRK2(LRRK2 G2385R)]were transfected at a 1:10 ratio for 48h.Healthy viable cells were counted by possessing at least one neurite twice the length of the cell body.We used MitoSOX,a specific dye for mitochondrial ROS to explore the effect of hydrogen peroxide(H2O2)on LRRK2 G2385R mutation.We detected the difference of caspase3/7 activity in the three groups in SH-SY5Y cells by Caspase3/7 activity assay.We screened out three possible protective drugs(curcumin,CDK-5 inhibitor Roscovitine、microtube stablizer Epothilone D)to explore whether they could rescue the toxicity of G2385R mutation.By Caspase3/7 activity assay and Western blot,we explored the changes of Caspase3/7 activity and the levels of the activated caspase-3 and PARP,and further study the possible protective mechanism of curcumin for LRRK2 G2385R mutation.Results:1.In SH-SY5Y cells,the percentage of cell viability was significantly decreased in those expressing LRRK2 G2385R mutation compared with WT group;2.After the incubation of H2O2 for 12hours,we found LRRK2 G2385R increased Mitochondrial ROS levels after treatment with H2O2 in HEK293 cells;3.Elevated Caspase 3/7 was found in LRRK2 G2385R transfected SH-SY5Y cells;4.Pretreated with curcumin for 1 h at different concentration.We found that 1 μM curcumin could rescue LRRK2 G2385R toxicity in SH-SY5Y cells.However,we did not find the protective effects of roscovitine and epothilone D;5.Curcumin prevented Mitochondrial ROS after H2O2 incubation in LRRK2 G2385R transfected HEK293 cells.Conclusion:We found that curcumin can be used as a scavenger of reactive oxygen species,which may protect LRRK2 G2385R-induced cell injury by anti-oxidative stress and apoptotic pathways.The researches on the PD pathogenesis and protective drugs are of great value to the precise treatment in PD patients.