Skip to main content
. 2020 Feb 14;23(3):100910. doi: 10.1016/j.isci.2020.100910

Figure 8.

Figure 8

Nuclear Localization of α-Syn Is Linked to Parkinson Disease

(A) qPCR detection of ATPase cation-transporting 13A2 (ATP13A2) and PTEN-induced kinase 1 (PINK1) in SH-SY5Y cells. Cells were transduced to express human α-Syn or a mock-GFP virus and treated for 16 h with RA (5 μM) or DMSO. Cells were collected and analyzed following 5 days from viral infection. Mean ± SD of n = 4 experiments. ∗p< 0.05 One way ANOVA.

(B) Inducible SH-SY5Y cells conditioned with doxycyline for 72 h to induce α-Syn expression, control cells were conditioned and treated in parallel but without doxycyline. Cells treated with RA (5 μM) or DMSO (0.1%) for 16 h; incubated for additional 24 h in standard conditioning medium and lysed in RIPA buffer. Protein samples (25 μg) were analyzed by quantitative Western blotting. The immunoblot was reacted with the indicated antibodies. Representative blot out of n = 4.

(C) Quantitation of the immunoreactive signal obtained for ATP13A2 (B) normalized to α-tubulin. Mean ± SD. ∗p< 0.05. One way ANOVA.

(D) Quantitation of the immunoreactive signal obtained for PINK1 (B) normalized to HSP-60. Mean ± SD. ∗p < 0.05, T test.

(E) MCF7 cells transiently expressing the indicated α-Syn mutations. Cells were treated with RA (1 μM) or DMSO for 3 h and processed for ICC 48 h post-transfection. Bar graph showing the nuclear to cytosolic ratio of each α-Syn form following RA treatment, relative to the respective DMSO-treated cells (WT α-Syn set at 100%, vertical bar). Mean ± SD of n = 26–53. ∗p< 0.05, one-way ANOVA.

(F) PSer129 α-Syn levels, determined by Lipid-ELISA, in inducible SH-SY5Y cells, conditioned with doxycycline for 48 h, and treated with RA (1 μM) or DMSO for 24 h before cell harvest. The soluble fraction obtained by cell fractionation was analyzed. Mean ± SD of n = 4 experiments. ∗p< 0.01.

(G) PSer129 α-Syn signal determined in primary cortical neurons from A53T α-Syn at 14 DIV. Neurons incubated with RA (1 μM) or DMSO (0.1%) for 16 h, fixed and analyzed by ICC using anti PSer129 α-Syn antibody. Total PSer129 levels and nuclear to cytosolic ratio are presented. Mean ± SD of n = 15–17 cells. ∗p< 0.05 T test. (H) ICC of primary cortical neurons immunoreacted with anti PSer129 α-Syn ab (as in (G). An image of the entire neuron is presented in Figure S6). Bar = 50 μm.