Figure 6.

Aptamers were Delivered into Cultured Primary Neurons and Rescued the Cell Toxicity Caused by α-syn Overexpression

(A) Alexa594-labeled aptamer F5R1 (10 nM) was complexed with CADY peptide (the molar ratio of CADY/F5R1 at 20), and then incubated with the 3 DIV (day in vitro) primary neurons for 2 hr. The fluorescence was detected by confocal laser microscopy (scale bar, 25 μm). The neurons are stained with MAP2 in green. (B) The 3 DIV primary neurons were treated with CADY/aptamer complex for 2 hr as in (A), then were infected with LV-α-syn cDNA. At 8 DIV, total neuronal proteins were extracted, and the human α-syn was detected by immunoblotting. β-actin was examined as a loading control. (C) Quantitative analysis of α-syn level from (B). Data are presented as the mean ± SD (one-way ANOVA) ***p < 0.001 compared with vector group; ###p < 0.001 compared with α-syn overexpression group (n = 6). (D) The primary neurons were treated as described in (B). At 8 DIV, the mitochondrial transmembrane potential (Δφ_m) was detected by Δφ_m-sensitive probe TMRM under confocal laser microscopy. ZsGreen was used to confirm the virus vector working well in neurons (scale bar, 100 μm). (E). Reactive fluorescence intensity of TMRM in mitochondria from 3 coverslips was quantitative analyzed. Data are presented as the mean ± SD (one-way ANOVA) ***p < 0.001 compared with vector; ###p < 0.001 compared with α-syn overexpression group (n = 3). (F and G) Primary neurons were treated as described in (B). At 8 DIV, cell viability and cytotoxicity were detected in primary neurons with the MTT (F) and LDH (G) assays. Data are presented as the mean ± SD (one-way ANOVA) ***p < 0.001 compared with vector; ###p < 0.001 compared with α-syn overexpression group (n = 6).