Study The Method Of Produce Parkinson’s Disease Models

Objectives:To better understand the etiology, pathogenesis, molecuar mechanisms underlying the disease process, and the potential treatment strategies of Parkinson’s disease(PD), in this study, we produced Drosophila models of PD involving mutant LRRK2, interaction between VPS35 and α-synuclein mutant, interaction between VPS35 and LRRK2 mutant, and these models mimicked some important characteristics of PD. We also selected relative virus vectors for producing mice model of PD which can mimick some important characteristics of PD.Methods:1. To produced Drosophila models for LRRK2-linked parkinsonism, we expressed UAS-LRRK2-WT, UAS-LRRK2-G2019 S, UAS-LRRK2-R1441 C in all neurons, DA neurons, cholinergic neurons and muscluar by Elav-GAL4, Ddc-GAL4, Chat-GAL4 and 24B-GAL4 lines.2. To produced UAS-VPS35-WT, UAS-VPS35-D260 N transgenic flies, the genes cloned between the Xho I and Bgl II sites of p UAST vector, the resulting constructs were microinjected in w1118 fly embryos, then selected UAS-VPS35-WT, UAS-VPS35-D260 N lines by mini-white.3. We used Elav-GAL4 and Ddc-GAL4 lines to express UAS-VPS35-WT, UAS-VPS35-D260 N, UAS-VPS35-RNAi in all neurons, DA neurons. Then we expressed Elav-GAL4; UAS-VPS35-WT(D260N/RNAi), Ddc-GAL4; UAS-VPS35-WT(D260N/ RNAi) in α-synuclein-A53 T and LRRK2-G2019 S lines. We produced PD Drosophila models involving interaction between VPS35 and α-synuclein mutant and interaction between VPS35 and LRRK2 mutant.4. We observed survival and locomotor activity of these flies, and examined the expression of TH by Immunohistochemistry and Western blot.5. Six virus vectors was injected unilaterally into the striatum of C57 B mice. Then we selected a virus vector for producing mice model of PD.Results:1. Elav-GAL4; UAS-LRRK2-G2019 S, Elav-GAL4; UAS-LRRK2-R1441 C, Ddc-GAL4; UAS-LRRK2-G2019 S, Ddc-GAL4; UAS-LRRK2-R1441 C lines, especially Ddc-GAL4; UAS-LRRK2-G2019 S, all shown shortened lifespan, locomotor dysfunction and loss of dopaminergic neurons.2. VPS35(D260N/RNAi)-α-synuclein(A53T), VPS35(D260N/RNAi)-LRRK2(G2019S) lines all shown shortened lifespan, locomotor dysfunction and loss of dopaminergic neurons.3. There weeks post-intrastriatal injection, rAAV1-EGFP virus vector less expressed in the striatum and no expressed in SN, HSV1/NX01-GFP virus vector expressed in the striatum and SN, HSV1/NX03-GFP virus vector was no expressed in the striatum and SN. Four and five weeks post-intrastriatal injection, rAAV1-EGFP and HSV1/NX01-GFP virus vectors expressed in the striatum and SN, but HSV1/NX03-GFP virus vector less and sporadically expressed in the striatum and SN.Conclusion:1. Ddc-GAL4; UAS-LRRK2-G2019 S line can be a transgenic Drosophila model of PD for further study.2. VPS35(D260N/RNAi)-α-synuclein(A53T), VPS35(D260N/RNAi)-LRRK2(G2019S) lines can be transgenic Drosophila models of PD for further study.3. HSV1/NX01-GFP virus vector can be used for producing mice model of PD for further study.