Passive immunotherapy of tauopathy targeting pSer413-tau: a pilot study in mice

Abstract

Objective: Cellular inclusions of hyperphosphorylated tau are a hallmark of tauopathies, which are neurodegenerative disorders that include Alzheimer's disease (AD). Active and passive immunization against hyperphosphorylated tau has been shown to attenuate phenotypes in model mice. We developed new monoclonal antibodies to hyperphosphorylated tau and sought high therapeutic efficacy for future clinical use.

Methods: Using more than 20 antibodies, we investigated which sites on tau are phosphorylated early and highly in the tauopathy mouse models tau609 and tau784. These mice display tau hyperphosphorylation, synapse loss, memory impairment at 6 months, and tangle formation and neuronal loss at 15 months. We generated mouse monoclonal antibodies to selected epitopes and examined their effects on memory and tau pathology in aged tau609 and tau784 mice by the Morris water maze and by histological and biochemical analyses.

Results: Immunohistochemical screening revealed that pSer413 is expressed early and highly. Monoclonal antibodies to pSer413 and to pSer396 (control) were generated. These antibodies specifically recognized pathological tau in AD brains but not normal tau in control brains according to Western blots. Representative anti-pSer413 and anti-pSer396 antibodies were injected intraperitoneally into 10-11- or 14-month-old mice once a week at 0.1 or 1 mg/shot 5 times. The anti-pSer413 antibody significantly improved memory, whereas the anti-pSer396 antibodies showed less effect. The cognitive improvement paralleled a reduction in the levels of tau hyperphosphorylation, tau oligomer accumulation, synapse loss, tangle formation, and neuronal loss.

Interpretation: These results indicate that pSer413 is a promising target in the treatment of tauopathy.

Figure 1

Specificity of monoclonal antibodies to pathological tau in Alzheimer's disease (AD) brains. Four samples from AD patients (no. 4, 24, 32, 89) and two samples from nondemented control subjects (no. 31, 33) were stained with G2 antibody, a rabbit polyclonal antibody specific to human tau. M, six isoforms of recombinant human tau. Two AD (no. 32, 89) samples and one control (no. 31) sample were selected for the analysis of the specificity of monoclonal antibodies. Tau12, anti-human tau antibody; Ta1505, anti-pSer413 antibody; Ta4 and Ta9, anti-pSer396 antibodies; 11F11, anti-Stx2 antibody.

Figure 2

Effects of monoclonal antibodies on the memory of tauopathy mice. (A) Anti-pSer413 Ta1505 antibody (IgG2a) in 0.1 mol/L citrate buffer (pH5.0) was injected intraperitoneally into 14-month-old tau784 mice (n = 9) once a week at 1 mg/shot 5 times. As the control, antilipopolysaccharide (LPS) mouse monoclonal antibody 4C10F4 (IgG2b) in the same buffer was injected into age-matched tau784 mice (n = 9). The buffer used for the antibody dilution was injected into age-matched non-Tg littermates (n = 8) as the normal control. Spatial reference memory was assessed at 15 months of age by the Morris water maze. *P = 0.0110 versus non-Tg, P = 0.0152 versus Ta1505. No significant difference between non-Tg and Ta1505 was observed. (B) Probe trials with the platform removed were carried out on day 6. Each bar represents the mean time occupancy in the target quadrant for 30 sec. Again, no significant difference between non-Tg and Ta1505 was observed. (C) Anti-pSer396 Ta4 antibody (IgG2b) in PBS was injected intraperitoneally into 14-month-old tau784 mice (n = 9) once a week at 1 mg/shot 5 times. As the control, IgG subtype-matched 4C10F4 antibody in PBS was injected into age-matched tau784 mice (n = 8). PBS was injected into age-matched non-Tg littermates (n = 9) as the normal control. *P = 0.0254 versus non-Tg. (D) Probe trials were carried out on day 5. No significant differences among three groups were observed.

Figure 3

Effects of lower doses of monoclonal antibody on the memory of tauopathy mice. (A) Anti-pSer413 Ta1505 antibody (IgG2a) in PBS was injected intraperitoneally into 10–11-month-old tau784 mice (n = 10) once a week at 0.1 mg/shot 5 times. As the control, IgG subtype-matched anti-Stx2 mouse monoclonal antibody 11F11 (IgG2a) in PBS was injected into age-matched tau784 mice (n = 10). PBS was injected into age-matched non-Tg littermates (n = 9) as the normal control. Spatial reference memory was assessed at 11–12 months of age. *P = 0.0025 versus non-Tg, P = 0.0493 versus Ta1505. No significant difference between non-Tg and Ta1505 was observed. (B) Probe trials were carried out on day 6. Again, no significant difference between non-Tg and Ta1505 was observed. (C) Anti-pSer396 Ta9 antibody (IgG3) in PBS was injected intraperitoneally into 10–11-month-old tau784 mice (n = 10) once a week at 0.1 mg/shot 5 times. As the control, IgG subtype-matched antilipopolysaccharide (LPS) mouse monoclonal antibody 6F11B6 (IgG3) in PBS was injected into age-matched tau784 mice (n = 8). PBS was injected into age-matched non-Tg littermates (n = 8) as a normal control. *P = 0.0065 versus non-Tg; **P = 0.0044 versus non-Tg. (D) Probe trials were carried out on day 6.

Figure 4

Effects of monoclonal antibodies on hyperphosphorylated tau in tauopathy mice based on immunohistochemistry. (A and B) Brain sections from 15-month-old antibody-treated tau784 mice were stained with Ta1505, PHF-1, and AT8 antibodies. Ta1505-treated mice showed an apparent reduction of hyperphosphorylated tau in hippocampal mossy fibers (A). In contrast, Ta4-treated mice exhibited reduced PHF-1-staining but not Ta1505- or AT8-staining (B). CA3 and CA2-3, hippocampal CA3 and CA2-3 regions. Scale bar, 30 μm. (C) Ta1505-, PHF-1-, and AT8-positive areas in each photograph of the hippocampal CA2-3 region were quantified using NIH ImageJ software. n = 5 for each group, except for control IgG in the Ta4 experiment where n = 3. *P = 0.0026, **P = 0.0133, ***P = 0.0364, and †P = 0.0002 versus control IgG.

Figure 5

Effects of monoclonal antibodies on hyperphosphorylated tau in tauopathy mice based on Western blots. Ta4-treated brain samples were obtained from 15-month-old tau784 mice (n = 3–4) after behavioral tests, whereas Ta1505-treated brain samples were newly prepared by injecting Ta1505 and control 11F11 antibodies in PBS into 12-month-old tau784 mice (n = 4 each) once a week at 1 mg/shot 5 times. Brain tissues were homogenized and fractionated into TBS-, sarkosyl-, and GuHCl-soluble fractions. (A and C) TBS-soluble and GuHCl-soluble fractions were subjected to Western blot with G2 (human tau-specific), pool-2 (anti-human/mouse tau), Ta1505, PHF-1, and AT8 antibodies. M, six isoforms of recombinant human tau. Actin was the loading control. (B and D) Signals were quantified using a LAS-3000 luminescent image analyzer (Fujifilm) and are shown in arbitrary units (AU). Ta1505-treated mice showed significantly lower levels of total and Ta1505-, PHF-1-, and AT8-positive tau than did control antibody-treated mice in TBS-soluble and GuHCl-soluble fractions (B). In contrast, Ta4-treated mice exhibited a significant reduction only of PHF-1-positive tau in GuHCl-soluble fraction (D). n = 4 for each group, except for control IgG in the Ta4 experiment where n = 3.

Figure 6

Effects of monoclonal antibodies on synapse loss in tauopathy mice. (A and C) Brain sections from 15-month-old antibody-treated tau784 mice were stained with antisynaptophysin antibody. Ta1505-treated mice showed apparent recovery of synaptophysin levels (A), whereas Ta4-treated mice did not (C). All images were taken from the hippocampal CA3 region. Scale bar, 30 μm. (B and D) Synaptophysin fluorescence intensity in the apical dendritic-somata field (30 × 60 μm, rectangle) of the hippocampal CA3 region was quantified and is shown in arbitrary units (AU). (B) n = 5 for each group. (D) n = 6 for non-Tg, n = 4 for cont IgG, and n = 5 for Ta4.

Figure 7

Effects of monoclonal antibodies on NFT formation in tauopathy mice. (A and C) Brain sections from 15-month-old antibody-treated tau784 mice were examined for NFTs by Gallyas silver staining. Ta1505-treated mice showed apparently reduced levels of NFTs (A), whereas Ta4-treated mice exhibited no changes (C). All images were taken from the EC-II/III region. Scale bar, 30 μm. (B and D) Gallyas silver positive cells in an area (220 × 160 μm) of the EC-II/III region were counted. (B) n = 5 for each group. (D) n = 4 for each group. NFT, neurofibrillary tangle; EC, entorhinal cortex.

Figure 8

Effects of monoclonal antibodies on neuronal loss in tauopathy mice. (A and C) Brain sections from 15-month-old antibody-treated tau784 mice were stained with anti-NeuN antibody. Ta1505 injection partially prevented neuronal loss (A), whereas Ta4 injection did not (C). All images were taken from the entorhinal cortex (EC)-II/III region. Scale bar, 30 μm. (B and D) NeuN-positive cells in an area within 1000 μm along the EC-II (the region between the two broken lines) were counted. (B) n = 5 for each group. (D) n = 6 for non-Tg, n = 4 for cont IgG, and n = 5 for Ta4.