Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy

Abstract

Accumulation of hyperphosphorylated tau directly correlates with cognitive decline in Alzheimer's disease and other primary tauopathies. One therapeutic strategy may be to reduce total tau expression. We identified antisense oligonucleotides (ASOs) that selectively decreased human tau mRNA and protein in mice expressing mutant P301S human tau. After reduction of human tau in this mouse model of tauopathy, fewer tau inclusions developed, and preexisting phosphorylated tau and Thioflavin S pathology were reversed. The resolution of tau pathology was accompanied by the prevention of hippocampal volume loss, neuronal death, and nesting deficits. In addition, mouse survival was extended, and pathological tau seeding was reversed. In nonhuman primates, tau ASOs distributed throughout the brain and spinal cord and reduced tau mRNA and protein in the brain, spinal cord, and cerebrospinal fluid. These data support investigation of a tau-lowering therapy in human patients who have tau-positive inclusions even after pathological tau deposition has begun.

Fig. 1. Antisense Oligonucleotides Reduce Human Tau and Prevent Tau Pathology in vivo

(A, B) Saline, Scrambled ASO, or Tau^ASO-12 was delivered via intracerebroventricular (ICV) infusion at 30μg/day for 1 month into 3–6month old adult PS19 mice. Human tau (huTau) (A) and mouse tau (muTau) (B) mRNA was analyzed at 4 (control n=10; Tau^ASO-12 n=3), 8 (control n=5; Tau^ASO-12 n=5), and 12 (control n=8; Tau^ASO-12 n=11) weeks after pump implantation and ASO delivery. Two-way ANOVA, Bonferroni post hoc analysis. (C) Mouse brains collected 12 weeks after pump implantation were stained with an ASO-antibody (red) and counterstained with nuclear stain DAPI (blue) on the contralateral hemisphere of the catheter. ASO was found throughout the entire hippocampus and brain (see also Fig. S2). Scale bar: 500μm. (D) PS19 mice at 6 months of age were treated with 30μg/day Scrambled ASO (n=5) or Tau^ASO-12 (n=10) for 1 month (Scrambled treated non-transgenic, NT, was the control, n=7). Pumps were removed after 1 month and mouse brain tissue was collected 2 months later. Total huTau protein was reduced in the Tau^ASO-12 group. (E, F) ImageJ threshold quantification of AT8 positivity in the mouse hippocampus (E) and whole brain (F) showed a reduction in AT8 in the Tau^ASO-12 treated mice. (G–J) Representative images of AT8 tau pathology from treated 6–9 month old PS19 mice. Scalebar for hippocampal panel: 500μm, for CA1, Dentate Gyrus (DG) panels: 50μm. Scale bar for whole brain: 2.5mm. One-way ANOVA, Sidak post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.

Fig. 2. Reducing Human Tau in Aged PS19 Mice Reverses Tau Pathology

(A) PS19 mice at 9 months of age were treated with Scrambled (Scram) ASO (n=6) or Tau^ASO-12 (n=6) for 1 month at 30μg/day. Scrambled ASO-treated non-transgenic (NT) mice served as control (n=5). Alzet osmotic pumps were removed after 1 month and mouse brain tissue was collected 2 months later. Total human tau (huTau) protein was measured. One-way ANOVA, Sidak post hoc analysis. (B, C) Sarkosyl extraction was performed on mouse brain lysates and then run on western blots and probed for total human tau (antibody: Tau13) and actin (B). Quantification of western blots indicated less insoluble tau in PS19 mice treated with Tau^ASO-12 compared to PS19 mice treated with scrambled ASO (C). Two-tailed Student T-test. (D, E) Mouse brain lysate was run on semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and probed for total tau (D) and phosphorylated-tau (PHF-1; pS396/pS404). (E). (F–H) SDD-AGE high molecular weight (HMW) and low molecular weight (LMW) bands were quantified in ImageJ. Two-tailed Student T-test. (I, J) Representative images of AT8 pathology in 9 month old PS19 mice (pathology baseline). (K–N) Representative images of AT8 tau pathology in the mouse hippocampus and full brain sections from 9 to 12 month old PS19 mice treated with Scrambled ASO or Tau^ASO-12. Scale bar for hippocampus panel: 500μm. Scalebar for CA1, dentate gyrus (DG): 50μm. Scale bar for whole brain: 2.5mm. (O, P) Quantification of AT8 positive staining in the hippocampus (O) and whole brain (P) demonstrates a reversal in AT8 positivity in the Tau^ASO-12 treated PS19 9–12 month old cohort of mice compared to the 6–9 month old scrambled ASO control group. One-way ANOVA, Sidak post hoc analysis. (Q) Representative images of Thioflavin S staining in the mouse piriform cortex showing reversal of Thioflavin S positivity in Tau^ASO-12 treated PS19 mice. (Quantification is shown in Fig. S5). White arrowheads indicate neurons positive for neurofibrillary tangles. Scale bar: 100μm. *p<0.05, ****p<0.0001. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.

Fig. 3. Reducing Human Tau Prevents Hippocampal Neuron Loss in PS19 Mice

(A, C) Brain sections systematically selected at 300μm intervals throughout the hippocampus were taken from 9 month old non-transgenic mice (NT) (n=6), 12 month old NT mice (n=6), 9 month old PS19 mice (n=10), 9–12 month old PS19 mice [both untreated (n=6) and treated with scrambled ASO n=6], and 9–12 month old PS19 mice treated with Tau^ASO-12 (n=6). Representative images (A) and volume quantification (C) show hippocampal loss prevention in the Tau^ASO-12 treatment group. (B, D) Representative images (B) and quantification (D) of the CA1 region of the hippocampus stained with the neuronal marker NeuN (red) and counterstained with the nuclear marker DAPI (blue) in a predefined CA1 region. 9–12 month old Tau^ASO-12 treated PS19 mice had more DAPI positive neurons in CA1 than the age matched PS19 controls treated with scrambled ASO. One-way ANOVA, Sidak post hoc analysis. *p<0.05, **p<0.01. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.

Fig. 4. Tau Seeding Activity is Reversed After Human Tau Reduction

(A) Schematic of the in vitro FRET tau seeding assay. (B, C) Representative images of HEK293 Tau^RD-CFP/Tau^RD-YFP cells sensor not treated with mouse brain lysate (B) or treated with brain lysate from 9–12 month old non-transgenic (NT) mice (C). (D–G) Representative confocal FRET images of sensor cells treated with brain lysates from 6–9 month old (D, E) or 9–12 month old (F, G) PS19 mice treated with scrambled ASO or Tau^ASO-12. Tau aggregation was visible in all conditions as evident by the accumulation of FRET positive inclusions detected with confocal microscopy, but was reduced in sensor cells exposed to brain lysates from Tau^ASO-12 treated mice in both the 6–9 month old and 9–12 month old age groups. (H) Normalized Integrated FRET density (the percent of FRET positive cells multiplied by the Median Fluorescence Intensity of those FRET positive cells and normalized to cells not treated with lysate) quantitative “tau seeding” values for all samples. Tau seeding activity was reduced in both the 6–9 month old and 9–12 month old PS19 Tau^ASO-12 treated brain lysates as compared to age-matched PS19 scrambled ASO treated brain lysates. Tau seeding activity in the 9–12 month old Tau^ASO-12 treated brain lysate was reversed when compared to the younger 6–9 month old scrambled ASO control treated lysate. One-way ANOVA, Sidak post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.

Fig. 5. Human Tau Reduction Extends PS19 Mouse Survival and Prevents Nesting Behavioral Deficits

(A) 9 month old PS19 mice were treated with Scrambled ASO or Tau^ASO-12 (n=17/treatment) for 1 month at 30μg/day (grey bar) and survival was monitored. Tau^ASO-12 treatment prolonged survival (median survival 348 days) compared to Scrambled ASO control treatment (median survival 312 days). Log-rank (Mantel Cox) test. (B) PS19 mice at 8–9 months of age were treated with saline (n=11), Scrambled ASO (n=11) or Tau^ASO-12 (n=25) for 1 month at 30μg/day. Treatment of non-transgenic (NT) mice with 30μg/day Scrambled ASO served as the control (n=30). Alzet osmotic pumps were removed after 1 month and nestlets were provided to the mice 6 weeks later. PS19 mice treated with saline or Scrambled ASO showed no significant difference in untorn nestlet weight or nestlet score, and thus these data were combined (control). A perfect nest will have no untorn nestlet left and will weigh less, whereas a poor nest will have more untorn nestlets that will weigh more. PS19 mice treated with Tau^ASO-12 had significantly less untorn nestlet material than control treated PS19 mice. One-way ANOVA, Sidak post hoc analysis. (C) Nestlet score was blindly measured on a scale from 0 (untouched nestlet) to 7 (perfect nest). PS19 mice treated with Tau^ASO-12 constructed better nests than did control PS19 mice. Kruskal-Wallis test, Dunn’s post hoc analysis. *p<0.05. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.

Fig. 6. Tau ASOs Reduce Tau Protein in the Non-Human Primate Brain and CSF

(A) Experimental design for 2 week dosing study. SAC: Sacrifice. (B) Quantification of total endogenous tau mRNA in the spinal cord and brain of Cynomologus monkeys after ASO treatment. Data expressed as percent vehicle (artificial CSF). Nonhuman primates received a single dose via lumbar puncture of either vehicle (n=4), 30mg (n=3) or 50mg (n=3) ASO against monkey Tau. Tissue was collected 2 weeks after the single dose treatment. One-way ANOVA, Sidak post-hoc analysis. (C) Experimental design for 6 week Duration of Action study. Nonhuman primates were dosed with 10mg Tau ASO followed 1 week later by a repeat dose of 30mg Tau ASO. Control monkeys were treated with Vehicle for both injections. Tissue from half of the monkeys was collected at 2 weeks post-treatment (Control n=4; Tau ASO n=4) and tissue from the remaining monkeys was collected at 6 weeks after the initial dose (Control n=4; Tau ASO n=4). (D, E) Total tau mRNA (D) and protein (E) was measured in spinal cord and brain regions at 2 and 6 weeks post initial dose. (F) Experimental design for 5 week brain and CSF tau protein analysis. (G) Quantification of total endogenous tau protein in the spinal cord and brain of nonhuman primates 5 weeks after the initial dose of ASO or vehicle (Control n=4; Tau ASO n=4). One-way ANOVA, Sidak post-hoc analysis. (H) CSF was drawn just prior to the initial dose and again just prior to necropsy. The percent change in total tau in the CSF between the two time points for each treatment group was calculated. Two-way Repeated Measures ANOVA, Sidak post-hoc analysis. (I) CSF tau protein directly correlated with the total tau protein in the hippocampus. Linear Regression. #p<0.01, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Graphical data are represented as box and whisker plots with individual points overlaid, where error bars represent maximum and minimum values and the boxed line represents the median.