Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra - PubMed (original) (raw)

Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra

Alison L McCormack et al. PLoS One. 2010.

Abstract

The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

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Conflict of interest statement

Competing Interests: One of the authors (D.B.) is an employee of Alnylam Pharmaceuticals, a for-profit company focused on the development of RNA interference-based therapeutics.

Figures

Figure 1

Figure 1. Treatment of squirrel monkeys with siRNA.

Animals were unilaterally implanted with a cannula connected to an Alzet minipump delivering siRNA into the left substantia nigra. (A) Sequence of the α-synuclein siRNA. “A,C,G,U” indicate ribonucleotides, “T” designates deoxythymidine, “c” and “u” specify 2′-O-Me-modified pyrimidines and “s” denotes a phosphorothioate linkage. (B) Midbrain sections were immunostained for tyrosine hydroxylase (brown) and counterstained with cresyl violet (purple). A representative section shows placement of the cannula approximately 1 mm dorsal to the substantia nigra (SN). The location of the cannula is indicated by the square box, and the asterisk denotes the exit of the third nerve. Scale bar = 800 µm.

Figure 2

Figure 2. Reduction of α-synuclein mRNA in the substantia nigra infused with α-synuclein siRNA.

Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein (A) or luciferase (B). Midbrain sections at the level of the exit of the 3rd nerve were used for α-synuclein in situ hybridization using digoxigenin-labeled antisense riboprobes. Representative images compare α-synuclein mRNA in the right (untreated) vs. left (siRNA-infused) substantia nigra. Scale bar = 100 µm.

Figure 3

Figure 3. α-Synuclein siRNA decreases α-synuclein mRNA within pigmented nigral neurons.

siRNA against α-synuclein was infused into the left substantia nigra of squirrel monkeys. Right (A) and left (B) midbrain sections at the level of the exit of the 3rd nerve were used for α-synuclein in situ hybridization. Representative images show nigral dopaminergic neurons containing neuromelanin (brown granules). The hybridization signal (purple) was markedly reduced in the left (siRNA-infused) as compared to the right (untreated) hemisphere. Scale bar = 10 µm.

Figure 4

Figure 4. Measurement of nigral α-synuclein mRNA by qPCR.

Squirrel monkeys received unilateral nigral infusion of siRNA targeting α-synuclein (A) or luciferase (B). Nigral tissue was dissected from midbrain sections rostral and caudal to the exit of the 3rd nerve. Values are the ratio of α-synuclein mRNA levels measured by qPCR in the left (siRNA-infused) and right (untreated) substantia nigra (L∶R ratio). Bars represent mean values.

Figure 5

Figure 5. Effect of α-synuclein siRNA on α-synuclein protein in the monkey substantia nigra.

α-Synuclein or luciferase siRNA was unilaterally infused into the left substantia nigra. Midbrain sections were immunostained with an antibody against α-synuclein. Representative images from an animal receiving α-synuclein siRNA show more robust α-synuclein immunoreactivity within the neuropil of the right (untreated, A) vs. left (siRNA-infused, B) substantia nigra. Scale bar = 5 µm. (C) Optical density measurements of nigral α-synuclein immunoreactivity. Data are expressed as percent of the control value in the right (untreated) substantia nigra and represent mean ± SEM. A significant decrease is caused by α-synuclein but not luciferase siRNA in the left (siRNA-infused) hemisphere. *p<0.03.

Figure 6

Figure 6. Lack of microglial activation following siRNA infusion.

α-Synuclein siRNA was unilaterally infused through a cannula positioned approximately 1 mm dorsal to the substantia nigra. Representative midbrain sections were immunostained for microglial cells using an antibody against ionizing calcium-binding adaptor molecule 1 (Iba-1, brown) and counterstained with cresyl violet (purple). Images are from the right (untreated, A and C) and left (siRNA-infused, B and D) substantia nigra. At higher magnification (C and D), Iba-1-positive cells with morphological features of resting microglia are shown close to dopaminergic neurons containing neuromelanin (black granules). The arrows indicate one of these neurons in each panel. Scale bars = 20 µm (A and B) and 10 µm (C and D). (E) The number of Iba-1-immunoreactive cells was counted in the right (R) and left (L) substantia nigra. Data are shown as mean ± SEM. (F) A representative section from the left midbrain shows Iba-1 immunoreactivity close to the tip of the infusion cannula (arrow) but not within the nearby parenchyma. This robust immunoreactivity was observed within cells with morphological characteristics of activated microglia (inset). Scale bars = 250 µm (panel F) and 10 µm (inset).

Figure 7

Figure 7. The number of nigral dopaminergic neurons is not affected by siRNA-induced α-synuclein suppression.

Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein. Both the number of TH-immunoreactive cells and the total number of dopaminergic neurons were counted stereologically in the substantia nigra. Values (mean ± SEM) were not different between the right (untreated) and left (siRNA-infused) hemisphere.

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