Evaluating the efficacy of purchased antisense oligonucleotides to reduce mouse and human tau in vivo

Abstract

Many preclinical and clinical studies support the use of antisense oligonucleotides (ASOs) as effective therapeutic strategies. However, acquiring ASOs for research purposes may be limited by partnerships with the pharmaceutical companies. Our lab previously developed an effective ASO strategy to lower human tau and reverse pathology in aged tauopathy model mice. Testing the efficacy of purchased tau lowering ASOs would provide support for these reagents as broad research tools. Purchased mouse and human tau lowering ASOs were infused or injected intracerebroventricularly into wildtype and tau transgenic mice. Following treatment, brain tissue evaluated for ASO distribution and levels of tau mRNA, protein, and phosphorylated tau. We show that purchased ASOs enter cell types of the brain and effectively decrease mouse or human tau mRNA and protein levels. Human tau lowering ASO treatment in PS19 mice decreased phosphorylated tau and gliosis relative to saline-treated PS19 mice, consistent with our previous study using a non-commercial tau lowering ASO. The results of this study demonstrate the efficacy of purchased tau targeting ASOs in vivo to support their broad use by researchers.

Keywords: Alzheimer’s disease; antisense oligonucleotides; human tau mouse model; tau protein; tauopathies.

FIGURE 1

Purchased ASOs distribute to neurons and glia within the brain. Immunofluorescent images within the CA3 region of the hippocampus from mice treated with purchased ASOs showing ASO distribution within neurons (NeuN), astrocytes (GFAP), and microglia (Iba1). (A) Representative image of ASO, GFAP, and NeuN and (B) ASO, Iba1, and NeuN immunoreactivity, with orthogonal projection image insets to show cell-specific localization. Scale bars = 50 μm, insets 5 μm.

FIGURE 2

Purchased mouse tau-targeting ASO reagents reduce mouse tau mRNA and protein in C57BL/6 mice. (A) Mouse tau mRNA expression was significantly decreased following administration of purchased mouse tau knockdown (mTau KD) ASO in C57BL/6 (C57) mice relative to saline and control ASO treated mice (n = 4–5/group). Mouse tau mRNA levels were normalized to Gapdh and shown relative to saline treated mice. One-way ANOVA, F = 74.68, P < 0.0001; Tukey’s multiple comparisons test. (B) Mouse tau protein expression was significantly decreased in mTau KD ASO-treated mice relative to saline or control ASO-treated mice (n = 4–5/group). One-way ANOVA, F = 12.69, P < 0.01; Tukey’s multiple comparisons test. Data are presented as mean ± SEM. **P < 0.01, *⁣*⁣**P < 0.0001.

FIGURE 3

Purchased human tau-targeting ASO reagents reduce human tau mRNA and protein in hTau and PS19 mice. (A) Human tau mRNA and (B) human tau protein expression were significantly decreased following purchased human tau knockdown (hTau KD 1) ASO administration in hTau mice relative to control ASO treated hTau mice (n = 3/group). Human tau mRNA levels were normalized to Gapdh and shown relative to control ASO. For Panel (A): Two-tailed t-test, P < 0.0001; Panel (B): Two-tailed t-test, P < 0.05. (C) Human tau mRNA expression was significantly decreased following treatment with purchased human tau knockdown (hTau KD 2) ASO in PS19 mice relative to saline treatment (n = 4–5/group). Human tau mRNA expression was undetected in non-transgenic (NT) littermates treated with saline (n = 5). One-way ANOVA, F = 164.9, P < 0.0001; Tukey’s multiple comparisons test. (D) No difference in mouse tau mRNA levels was observed among treated mice. mRNA levels were normalized to Gapdhand expressed relative to saline-treated mice. One-way ANOVA, F = 1.258, P = 0.3221. (E) Human tau protein expression was significantly reduced in hTau KD 2 ASO-treated PS19 mice relative to saline-treated PS19 mice. Two-tailed t-test, P < 0.001. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001.

FIGURE 4

hTau KD ASO treatment in PS19 mice shows a reduced trend in phosphorylated tau accumulation and reduces gliosis. Representative images of phosphorylated tau (AT8) immunohistochemistry within the cortex and hippocampus from (A–C) saline-treated PS19 mice and (B–D) human tau knockdown (hTau KD 2) ASO-treated PS19 mice. Scale bar = 500 μm, insets 250 μm. Quantification of the percentage of AT8 immunoreactivity within the (E) cortex and (F) hippocampus of saline or hTau KD 2 treated PS19 mice (n = 4–5 mice/group). Panel (E), Cortex: One-way ANOVA, F = 10.50, P = 0.0028, Tukey’s multiple comparison test, P = 0.1032; Panel (F), Hippocampus: One-way ANOVA, F = 8.162, P = 0.0067, Tukey’s multiple comparison test, P = 0.3126. Representative images from the CA3 region of the hippocampus showing (G,H) Iba1 and (I,J) GFAP immunofluorescence for microglia and astrocytes, respectively, from PS19 mice treated with saline or human tau knockdown (hTau KD 2) ASO. Scale bars = 100 μm. Quantification of the percentage of (K) Iba1 and (L) GFAP immunoreactivity within the hippocampus of non-transgenic (NT) and saline or hTau KD treated PS19 mice (n = 4–5 mice/group). Panel (K), Iba1: One-way ANOVA, F = 9.216, P < 0.01, Tukey’s multiple comparison test, P < 0.01; Panel (L), GFAP, F = 15.20, P < 0.001; Tukey’s multiple comparison test, P < 0.01. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, non-significant.