In order to test the ability of naked siRNA to reduce
SNCA expression
in vivo, we identified the hippocampus and the cortex as having the highest expression of
SNCA in the murine brain (data not shown). We subsequently chose to target siRNA against
SNCA expression in the hippocampus to decrease variability in our measurements that could be introduced when dissecting out smaller, less defined structures. We delivered Mayo8S2M siRNA against murine
SNCA, siRNA against luciferase (luc), or PBS into the right CA1 of the hippocampi of wild-type C57BL6 female mice. Infusions were performed on these inbred female mice to reduce variability that can be introduced by combining genders or by having genetically heterogeneous backgrounds. Continuous infusion of the siRNA or PBS solutions was performed over a period of 15 days with Alzet mini pumps connected to cannulae which were surgically implanted into the right CA1. After 15 days, two pumps containing the
SNCA siRNA and three pumps containing the luc siRNA had disconnected. These mice, represented by open circles, are included in the data analysis in Figure
1. The left CA1 was not injected and was therefore utilized for an untreated control. Hippocampal infusion of the Mayo8S2M siRNA resulted in significant knockdown of
SNCA when assessed by Taqman quantitative real-time PCR. Normalization was performed against
HPRT and
GAPDH as endogenous controls. Quantitative RT-PCR analysis demonstrated that
SNCA expression was significantly decreased in the right (treated) hippocampus of animals which have received
SNCA siRNA when compared to the left (untreated) hippocampus (p = 0.037) as demonstrated by the R:L ratio of
SNCA expression. Additionally, the
SNCA-specific siRNA reduced
SNCA expression when compared to luciferase-siRNA (p = 0.004) and PBS (p = 0.036) treated control mice (Figure
1).
Although
SNCA expression was significantly reduced in the
SNCA siRNA treated hippocampi, we hypothesized that the efficacy of the siRNA in the brain might be underestimated due to partial diffusion of the siRNA into the contralateral hippocampus. In order to address this issue, we endeavored to examine the distribution of
SNCA knockdown by
in situ hybridization so that the extent of
SNCA reduction could be fully appreciated. Utilizing a mouse
SNCA specific probe, we determined if the knockdown of
SNCA expression extended beyond the CA1 cannulation site (Figure
2).
SNCA knockdown with
SNCA siRNA was observed an average of 2.67 mm ± 0.57 mm from the cannula tip. While the cannulation tract was evident in the infused animals, in general, little damage nor increase in inflammation (Figure
2C, D, E) was noted at the site of infusion regardless of treatment group, and no animals were removed from the study due to hippocampal damage. Figure
2F shows the hippocampus from the mouse with the most damage at the infusion site. Ratios were calculated for each animal between the treated (right) side (
SNCA siRNA, luc siRNA, and PBS) and the untreated (left) side and then compared across each of the three groups (Table
1). The least reduction in
SNCA levels, shown as a reduction in right (R):left (L) ratio, was observed in the cortex (71% between PBS and
SNCA siRNA treated animals, p = 0.067), likely reflecting the fact that the cortex spans from regions adjacent to the infusion site to regions quite distant from the infusion site and thus less likely to be affected by the siRNA. Significant reductions in
SNCA levels were observed in the CA1 (66%, p < 0.001), CA2 (59%, p < 0.001), CA3 (77%, p < 0.001), and dentate gyrus (81%, p = 0.001) when
SNCA siRNA treated animals were compared to PBS treated animals. Similar results were obtained when
SNCA siRNA treated animals were compared to luc siRNA treated control mice. Reduction in
SNCA levels was confirmed by immunostaining for murine α-synuclein protein (Figure
2C and
2D). Notably, α-synuclein levels in the cell bodies of the hippocampus were decreased, while α-synuclein in projections from distal regions persisted. Toluidine Blue staining of
in situ sections showed that decreased
SNCA levels in mice treated with
SNCA siRNA were not due to neuronal loss (data not shown).
Table 1
Quantitative densitometry of SNCA in situ hybridization
Cortex
| 0.92 (0.82 – 0.98) | 0.90 (0.86 – 1.00) | 0.27 (0.20 – 0.90) | 0.036 | 0.067 |
CA1
| 1.02 (0.98 – 1.14) | 0.97 (0.93 – 1.06) | 0.35 (0.14 – 0.54) | <0.001 | <0.001 |
CA2
| 1.02 (0.93 – 1.05) | 1.01 (0.97 – 1.15) | 0.42 (0.19 – 0.62) | <0.001 | <0.001 |
CA3
| 1.15 (0.99 – 1.24) | 1.09 (1.01 – 1.15) | 0.27 (0.12 – 0.71) | <0.001 | <0.001 |
DG
| 1.02 (0.99 – 1.11) | 0.99 (0.94 – 1.04) | 0.19 (0.10 – 0.68) | 0.008 | 0.001 |