MPTP-induced vulnerability of dopamine neurons in A53T α-synuclein overexpressed mice with the potential involvement of DJ-1 downregulation

Abstract

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the α-synuclein (α-syn) gene. Mutant α-syn expression increases the vulnerability of neurons to exogenous insults. In this study, we developed a new PD model in the transgenic mice expressing mutant hemizygous (hemi) or homozygous (homo) A53T α-synuclein (α-syn Tg) and their wildtype (WT) littermates by treatment with sub-toxic (10 mg/kg, i.p., daily for 5 days) or toxic (30 mg/kg, i.p., daily for 5 days) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase and Bcl-2 levels were reduced in the α-syn Tg but not WT mice by sub-toxic MPTP injection. In the adhesive removal test, time to remove paper was significantly increased only in the homo α-syn Tg mice. In the challenging beam test, the hemi and homo α-syn Tg mice spent significantly longer time to traverse as compared to that of WT group. In order to find out responsible proteins related with vulnerability of mutant α-syn expressed neurons, DJ-1 and ubiquitin enzyme expressions were examined. In the SN, DJ-1 and ubiquitin conjugating enzyme, UBE2N, levels were significantly decreased in the α-syn Tg mice. Moreover, A53T α-syn overexpression decreased DJ-1 expression in SH-SY5Y cells. These findings suggest that the vulnerability to oxidative injury such as MPTP of A53T α-syn mice can be explained by downregulation of DJ-1.

Keywords: Apoptosis; DJ-1; MPTP; Parkinson's disease; Synuclein.

Fig. 1. Evaluation of synaptophysin and synuclein expression in the SN of WT and α-syn Tg mice.

(A) SN tissue lysates were immunoblotted with each antibody. (B, C) The intensity of each band was normalized to that of β-actin and presented in bar graphs. The values represent the means±SD (n=7). ^*p<0.05, ^**p<0.01.

Fig. 2. Evaluation of TH expression in the SN of MPTP-treated Tg mice.

Mice were treated with MPTP for 5 days. (A) SN tissue lysates were immunoblotted with each antibody. The intensity of each band was normalized to that of β-actin and presented in bar graphs. (B) Immunostaining of TH was evaluated in the SN of MPTP-treated Hemi Tg mice. The number of TH-positive cells in the SN is shown in bar graphs (right panel). (Bar, 200 µm) The values represent the means±SD (n=7). ^*p<0.05, ^**p<0.01.

Fig. 3. Evaluation of Bcl-2 expression in the SN of control and MPTP-treated mice.

Mice were treated with MPTP for 5 days. (A) SN tissue lysates were immunoblotted with each antibody. (B) The intensity of each band was normalized to that of β-actin and presented in bar graphs. The values represent the means±SD (n=7). ^*p<0.05, ^**p<0.01.

Fig. 4. Behavioral tests of MPTP-treated Tg mice.

Mice were treated with MPTP for 5 days. Adhesive removal test (A) and challenging beam traversal test (B) were performed at day 12. The values represent the means±SD (n=7). ^*p<0.05 vs WT, ^**p<0.01 vs WT, ^†p<0.05 vs Hemi, ^※p<0.05 vs Home.

Fig. 5. Evaluation of DJ-1 and UBE2N expression in the SN of WT and α-syn Tg mice.

(A) SN tissue lysates were immunoblotted with each antibody. (B) The intensity of each band was normalized to that of β-actin and presented in bar graphs. The values represent the means±SD (n=7). ^*p<0.05 vs. WT.

Fig. 6. Reduced cell viability and DJ-1 down-regulation in the A53T synuclein overexpressed SH-SY5Y cells.

(A) SH-SY5Y cells were infected with A53T synuclein lentivirus for 72 h and the cells were treated with various concentration of 6-OHDA for 24 h. The expression level of synuclein was examined by immunoblotting (upper panel). Then, cell viability was examined by MTT assay. (B) SH-SY5Y cells were infected with 1, 5, or 10 m.o.i. of A53T synuclein lentivirus for 72 h and the cell lysates were electrophoresed and immunoblotted with indicated antibody. The intensity of each band was normalized to that of β-actin and presented in bar graphs. (C) SH-SY5Y cells were transfected with control or DJ-1 siRNA for 48 h. Then, cell viability was examined by MTT assay cells and DJ-1 expression was examined by immunoblotting. The values represent the means±SD (n=4). ^**p<0.01 vs. Control. ^***p<0.001 vs. Control.