Anti-acetylated-tau immunotherapy is neuroprotective in tauopathy and brain injury

Abstract

Background: Tau is aberrantly acetylated in various neurodegenerative conditions, including Alzheimer's disease, frontotemporal lobar degeneration (FTLD), and traumatic brain injury (TBI). Previously, we reported that reducing acetylated tau by pharmacologically inhibiting p300-mediated tau acetylation at lysine 174 reduces tau pathology and improves cognitive function in animal models.

Methods: We investigated the therapeutic efficacy of two different antibodies that specifically target acetylated lysine 174 on tau (ac-tauK174). We treated PS19 mice, which harbor the P301S tauopathy mutation that causes FTLD, with anti-ac-tauK174 and measured effects on tau pathology, neurodegeneration, and neurobehavioral outcomes. Furthermore, PS19 mice received treatment post-TBI to evaluate the ability of the immunotherapy to prevent TBI-induced exacerbation of tauopathy phenotypes. Ac-tauK174 measurements in human plasma following TBI were also collected to establish a link between trauma and acetylated tau levels, and single nuclei RNA-sequencing of post-TBI brain tissues from treated mice provided insights into the molecular mechanisms underlying the observed treatment effects.

Results: Anti-ac-tauK174 treatment mitigates neurobehavioral impairment and reduces tau pathology in PS19 mice. Ac-tauK174 increases significantly in human plasma 24 h after TBI, and anti-ac-tauK174 treatment of PS19 mice blocked TBI-induced neurodegeneration and preserved memory functions. Anti-ac-tauK174 treatment rescues alterations of microglial and oligodendrocyte transcriptomic states following TBI in PS19 mice.

Conclusions: The ability of anti-ac-tauK174 treatment to rescue neurobehavioral impairment, reduce tau pathology, and rescue glial responses demonstrates that targeting tau acetylation at K174 is a promising neuroprotective therapeutic approach to human tauopathies resulting from TBI or genetic disease.

Keywords: Acetylated tau; Human plasma; Immunotherapy; TBI; Tauopathy.

Fig. 1

Characterization of new anti-acetylated tau (ac-tauK174) antibodies. (A) Sequence of the antigen peptide (human tau amino acids 163–185) and workflow used to generate monoclonal antibody against acetylated tau (Ac-K174). (B, C) Immunoblot of HEK293T cells transfected with p300 and WT human tau (hTau), K174R, or K274R mutant. ActauK174 signal is readily detected where WT hTau is co-transfected with p300, while K174R, but not K274R mutation blocks Clone 1 and Clone 2 signal. (D, E) Immunoblots of hippocampal lysate from age-ranged WT, PS19, and Tau KO mice showing that Clone 1 and Clone 2 detect ac-tauK174 immunoreactivities. Human tau (hTau, arrows) migrates at a higher molecular mass than murine tau (∼50 kDa). Asterisk indicates a non-specific band in Clone 1. (F-H) Specific binding affinity of Clone 1 (F) and Clone 2 (G) for ac-tauK174. Dissociation constant between clones and ligand was measured by Surface Plasmon Resonance (SPR) binding assay (H) using the ac-tauK175 peptide as the ligand. Kon, association rate; Koff, dissociation rate; KD, dissociation constant

Fig. 2

Anti-ac-tauK174 antibody Clone 1 rescues neurobehavioral impairment in P301S (PS19) mice. (A) Experimental timeline. At 6 months of age, male and female P301S mice were peripherally (IP) injected with Clone 1 (25 mg/kg) or vehicle (PBS). Non-transgenic (WT) littermates were injected with PBS and are included as controls. All mice received a single injection weekly for a period of 15 weeks. Behavioral analysis was performed during the last 4 weeks of treatment. Mice were sacrificed at 10 mo following the last injection. (B) Percentage of weight loss at the endpoint (week 15) among three groups of animals, normalized to the start point (week 1). ***p < 0.001, *p < 0.05 by one-way ANOVA, Sidak’s multiple comparison test. (C, D) Motor coordination impairment was measured by hindlimb extension test. Each mouse was suspended by its tail for 10 s and its hind-limb posture was scored as 1, 0.5, or 0 (C). The average score for each group is presented (D). ***p<0.001, **p < 0.01 by one-way ANOVA, Sidak’s multiple comparison test. (E, F) Spatial learning and memory were measured by Morris water maze (MWM). (E) Learning curve during the training phase. ***p < 0.001, *p < 0.05 by two-way ANOVA, Tukey’s multiple comparison test. (F) Probe trial 72 hr post-training performed on Day 8. **p < 0.001, *p < 0.05 by paired t-test. n = 14 per group

Fig. 3

Anti-ac-tauK174 antibody ameliorates tau pathology and neurodegeneration in PS19 mice. (A) Representative Nissl-staining showing hippocampal morphology of WT mice treated with PBS, PS19 mice treated with PBS, and PS19 mice treated with Clone 1 antibody. Scale bar: 500 µm. (B) Quantification of hippocampal volume. **p<0.01 by one-way ANOVA, Kruskal-Wallis test. (C) Pearson correlation analysis of Morris water maze performance score (rank summary latency during learning) and hippocampal volume. (D) Representative immunohistochemistry staining of p-tau (AT8) in the hippocampi. Scale bar: 250 µm. (E) Quantification of hippocampal AT8-positive area. ***p<0.001 by one-way ANOVA, Kruskal-Wallis test. (F) Schematic diagram showing the injection of tau fibrils to the hippocampus of PS19 mice induces spreading of tau pathology from the ipsilateral (seeding) side to the contralateral (spreading) side of the brain. (G) Representative immunostaining showing MC1-positive tau inclusions in the seeding side and the spreading side after treatment with IgG2a or Clone 2 antibody. Scale bar: 100 µm. (H) Quantification of relative MC1-positive signal in IgG2a and Clone 2 treated groups, normalizing the spreading side to the seeding side. n = 5 mice for both groups. ***p<0.001, STATA mixed model

Fig. 4

Anti-ac-tauK174 treatment ameliorates behavioral impairments and neuropathology in P301S mice with TBI. (A) 8-month PS19 mice were exposed to sham or traumatic brain injury (closed head concussive) surgery. One day prior to trauma surgery animals received PBS or Clone 1 administered intraperitoneally (IP). Treatment continued weekly throughout the duration of experimentation until termination 5 weeks post-injury. (B) Schematic diagram showing the injury site and areas of pathology analysis. (C) Percentage of weight loss at Day 28 among three groups of PS19 animals showing a trend of reduction in mice with TBI and treated with antibody. (D, G) Representative immunohistochemistry staining of AT8-positive p-tau (F) and Iba-1 (I) in the upper (LOI 1) and lower (LOI 2) cortex of PS19 mice with sham surgery or TBI, treated with PBS or Clone 1 antibody. Scale bar: 100 µm. (E, F, H, I) Quantification of AT8 positive % area (E, F) and Iba-1 positive % area (H, I) in upper and lower cortex of the three sections adjacent to the injury site. **p<0.01, *p<0.05 by one-way ANOVA, Tukey’s multiple comparison test. (J) Pearson correlation analysis of AT8-positive p-tau levels (by western blot) and Iba-1 signal in the lower cortex. n = 12 per group

Fig. 5

snRNA-seq reveals microglial activation and oligodendrocyte myelination impairment in TBI are rescued by anti-ac-tauK174 immunotherapy. (A) Subcluster analysis of microglia (resolution 0.15) between conditions: WT, PS19-Sham-PBS, PS19-TBI-PBS, and PS19-TBI-Ab Clone 1. (B) Bar graph of cell ratio per condition within each microglia cluster. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons correction within each subcluster. (C) Ingenuity Pathway Analysis (IPA) upstream regulators of MG1 markers. (D) Percentage and average expression levels of disease-associated microglia (DAM) genes (Tyrobp, B2m), and cytokine gene (C1qa) across 4 conditions; scale, log2 fold-change. (E) Subcluster analysis of oligodendrocytes (resolution 0.2) between conditions. (F) Bar graph of cell ratio per condition within each oligodendrocyte cluster. ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons correction within each subcluster. (G) Simple linear regression analysis with standard error showing a positive correlation between OL5 markers and disease-associated oligodendrocyte markers (DAO) (R = 0.4, p = 2.7e-6). (H) Heat map of average expression levels of myelination-related genes (Mpb, Sox10, Tcf7l2), IFN-gamma hallmark genes (Arid5b, Auts2, Bpgm, Usp18), and IFN-responsive oligodendrocyte (IRO) markers (Ifi27l2a, B2m) across 4 conditions; scale, log2 fold-change

Fig. 6

Anti-ac-tauK174 treatment ameliorates behavioral impairments and reduces tau seeding in vitro. (A, B) Trauma-induced memory deficits were measured by novel object recognition (NOR) test. Three weeks post-injury mice were exposed to two identical objects, five minutes later one of the objects was replaced with a new object (A). Memory deficits were calculated by decrease in time spent with the novel object graphed as a discrimination index (B). *p< 0.05 by one-way ANOVA, Sidak’s multiple comparison test. n=9 (Sham-PBS), 8 (TBI-PBS), 12 (TBI-Ab Clone 1). (C, D) Pearson correlation analysis of NOR discrimination index (DI) and AT8-positive p-tau levels (by western blot) (C), and Iba-1 signal (D) (by western blot). n=12 per group. (E) Tau RD P301S FRET Biosensor cells were treated with brain lysates and liposomes, incubated for 72 hours, and imaged to assess tau seeding activity by measuring % FRET+ cells. (F, G) Representative immunocytochemistry staining (D) and quantification of % CFP/YFP FRET cells (E) after incubation with PS19 mouse lysate. *p<0.05 by one-way ANOVA, Tukey’s multiple comparison test. n=4 (Sham-PBS), 4 (TBI-PBS), 4 (TBI-Ab Clone 1). Scale bar: 50 µm. (H) Representative immunoblot of acetylated tau (K174) (Clone 2) in control (Normal) and TBI human plasma samples. (I) Quantification of plasma ac-tau (K174) levels normalized by total protein levels. The mean level of ac-tau (K174) was significantly higher in the TBI cohort at 24 h in comparison to the controls (1.57 ± 0.48 versus 1.13 ± 0.27,***p< 0.001). n=24 (normal), 44 (TBI),**p< 0.01 by one-way ANOVA, Dunnett’s multiple comparison test