Figure 3.
Intranasal Indatraline-Conjugated 499-siRNA Treatment Downregulates α-Synuclein Expression
(A) Schematic representation of the treatment. Mice were intranasally administered with PBS, indatraline-conjugated nonsense siRNA (IND-NS-siRNA), or indatraline-conjugated 499-siRNA (IND-499-siRNA) at 30 μg/day for 4 days and were sacrificed at 1, 3, or 7 days after last administration (1, 3, or 7 days, respectively; n = 5–10 mice/group). (B) Coronal brain sections showing reduced α-synuclein mRNA levels in the SNc, VTA, DR, and LC of mice treated with IND-499-siRNA (4 days) and sacrificed at 1 day post-administration were assessed by in situ hybridization (ISH). Signal represents the relative optical density (ROD) of autoradiograms as indicated at the right-hand side of the image. White arrowheads show α-synuclein mRNA expression in SNc, VTA, DR and LC. Scale bar: 500 μm. (C) Time course of α-synuclein mRNA suppression in the monoaminergic nuclei after intranasal IND-499-siRNA administration. Bar graphs showing a significant reduction of α-synuclein mRNA level compared with their respective controls at day 1 post-administration. Conversely, no difference was detected at days 3 and 7 post-administration. **p < 0.01, ***p < 0.001, versus PBS-treated mice; +p < 0.05, ++p < 0.01, +++p < 0.001 versus IND-NS-siRNA (two-way ANOVA followed by Tukey’s post hoc test). (D) Image of immunoblot of α-synuclein, β-actin, and tyrosine hydroxylase (TH) in SNc/VTA of mice treated with PBS or IND-conjugated siRNAs. (E) Bar graphs showing α-synuclein protein levels in SNc/VTA normalized against β-actin or TH, and TH protein levels normalized against β-actin. IND-499-siRNA reduced α-synuclein protein level in SNc/VTA 24 hr after last administration; then α-synuclein level was recovered 3 days later. *p < 0.05 versus control groups (two-way ANOVA followed by Tukey’s post hoc test). Data are mean ± SEM. DR, dorsal raphe nucleus; LC, locus coeruleus; SNc/VTA, substantia nigra compacta/ventral tegmental area.