In vivo silencing of alpha-synuclein using naked siRNA

Abstract

Background: Overexpression of alpha-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinson's disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD.

Results: We have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion.

Conclusion: We have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for alpha-synucleinopathies resulting from SNCA overexpression.

Figure 1

qRT-PCR analysis of SNCA expression following in vivo RNAi.SNCA siRNA (syn-siRNA), luciferase siRNA (luc-siRNA), or PBS was infused into the right CA1. qRT-PCR was used to determine expression of SNCA following RNAi in treated right side compared to the untreated contralateral side (R:L ratio). SNCA siRNA had a statistically significant decrease of SNCA expression in the right compared to the left side of the brain, and R:L ratios were decreased when compared to controls (vs PBS, p = 0.036; vs. luciferase, p = 0.004). Horizontal lines show medians. Open circles indicate mice in which the cannula was disconnected during treatment or did not function.

Figure 2

RNA and protein analysis of α-synuclein expression following in vivo SNCA siRNA treatment. The right CA1 was infused with either PBS, siRNA to luciferase, or siRNA against our SNCA target. A typical SNCA in situ from an animal treated with (A) PBS or (B) SNCA siRNA on the right side compared to the uninjected left sides. While the cannula tract was evident in the right hippocampi of the infused mice (* in C and F), regardless of treatment group, (C) immunostaining for α-synuclein demonstrates considerable knockdown of protein expression (arrowhead) in the hippocampus when the uninjected control side is compared to the SNCA siRNA-treated side, also shown in higher magnification (D, E), respectively. (F) Inflammatory changes, as shown by Iba-1 immunostaining for microgliosis, were minimal around the infusion site. Sample brain in (F) showed the highest degree of damage from infusion, in this case from SNCA siRNA.

Figure 3

Silencing of SNCA is resilient and target-specific.SNCA and SNCB expression was assessed by in situ hybridization following extended timecourse of in vivo SNCA siRNA treatment. Qualitative densitometric analysis was performed on mice treated with SNCA siRNA on the right side of the brain and a ratio showing either SNCA or SNCB expression in the treated (R) and untreated (L) brain was calculated and plotted for each animal within a group. The knockdown of SNCA expression (black circle) persists in the (A) CA1 and the (B) cortex two weeks after cannula removal with SNCA approaching normal levels by three weeks post-infusion. The closed triangle indicates SNCA levels in a mouse in which the cannula was loose at the end of the study. Non-specific silencing of SNCB (open circle) was not observed at any timepoint.