Passive immunotherapy for N-truncated tau ameliorates the cognitive deficits in two mouse Alzheimer's disease models

Abstract

Clinical and neuropathological studies have shown that tau pathology better correlates with the severity of dementia than amyloid plaque burden, making tau an attractive target for the cure of Alzheimer's disease. We have explored whether passive immunization with the 12A12 monoclonal antibody (26-36aa of tau protein) could improve the Alzheimer's disease phenotype of two well-established mouse models, Tg2576 and 3xTg mice. 12A12 is a cleavage-specific monoclonal antibody which selectively binds the pathologically relevant neurotoxic NH₂26-230 fragment (i.e. NH₂htau) of tau protein without cross-reacting with its full-length physiological form(s). We found out that intravenous administration of 12A12 monoclonal antibody into symptomatic (6 months old) animals: (i) reaches the hippocampus in its biologically active (antigen-binding competent) form and successfully neutralizes its target; (ii) reduces both pathological tau and amyloid precursor protein/amyloidβ metabolisms involved in early disease-associated synaptic deterioration; (iii) improves episodic-like type of learning/memory skills in hippocampal-based novel object recognition and object place recognition behavioural tasks; (iv) restores the specific up-regulation of the activity-regulated cytoskeleton-associated protein involved in consolidation of experience-dependent synaptic plasticity; (v) relieves the loss of dendritic spine connectivity in pyramidal hippocampal CA1 neurons; (vi) rescues the Alzheimer's disease-related electrophysiological deficits in hippocampal long-term potentiation at the CA3-CA1 synapses; and (vii) mitigates the neuroinflammatory response (reactive gliosis). These findings indicate that the 20-22 kDa NH₂-terminal tau fragment is crucial target for Alzheimer's disease therapy and prospect immunotherapy with 12A12 monoclonal antibody as safe (normal tau-preserving), beneficial approach in contrasting the early Amyloidβ-dependent and independent neuropathological and cognitive alterations in affected subjects.

Keywords: Alzheimer’s disease; immunotherapy; tau cleavage; tau protein; tauopathies.

Graphical Abstract

Figure 1

The i.v.-injected 12A12mAb anti-tau antibody is biologically active into the animals’ hippocampus. (A, B) Western blot analysis carried out on hippocampi from Tg2576 and 3xTg Alzheimer’s disease mice at different ages (1, 3 and 6 months old) and from 6-month-old WT by probing with 12A12mAb (left). β-III tubulin was used as loading control. Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Full uncropped blots are available in Supplementary Fig. 5. Pooled data and relative densitometric quantifications are reported on the right. In this and all other figures, in box-and-whisker plots the centre lines denote median values, edges are upper and lower quartiles, whiskers show minimum and maximum values and points are individual experiments. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). (C) Western blotting analysis was carried out by probing with anti-mouse IgG as primary antibody (Thermo-Fisher 10400C) on hippocampal protein extracts (40μg) from animals of the three experimental groups (WT, ‘naïve’ 3xTg, 3xTg + mAb) which underwent i.v. injection with saline or 12A12mAb (see details in Materials and methods section). β-III tubulin was used as loading control. Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Full uncropped blots are available in Supplementary Fig. 5. Notice that 3xTg animals, which are systemically i.v. injected for 14 days with 12A12mAb (see details in Materials and methods section), exhibit high levels of cerebral mouse IgG when compared to not-vaccinated controls confirming that a fraction of mAb injected into the tail vein is present in the hippocampal parenchyma. Asterisks point to the light and heavy antibody chains (25 and 50 kDa, respectively). (D) Brain levels of anti-tau antibody 12A12mAb were evaluated by ELISA in the TBS-soluble fraction of hippocampal homogenates from WT and 3xTg mice that i.v. received saline or 12A12mAb for 14 days (see details in the Materials and methods section). The ELISA used to measure the anti-tau antibody relies on the plate-immobilized recombinant NH₂26-44aa tau which, being the minimal Alzheimer’s disease-relevant (Borreca et al., 2018) active moiety of the parental longer NH₂26-230 (Amadoro et al., 2004, 2006), was used as catching peptide. Notice that a significant portion of the 12A12mAb in 3xTg brains is bound to endogenous NH₂htau and does non-specifically interact with the large amount of intracellular tau released during homogenization. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant.

Figure 2

Reduction of the hippocampal NH₂htau in Tg-Alzheimer’s disease (Tg2576) mice immunized with 12A12mAb ameliorates the disease-associated synaptic neuropathology. Representative blots (n = 5) of sodium dodecyl sulphate-polyacrylamide gel electrophoresis western blotting analysis (left) on isolated synaptosomal preparations from hippocampal region of animals from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of Tg2576 strain to assess the content of the NH₂htau fragment (A), total tau full-length (B), AT8-phosphorylated tau (C), APP holoprotein (D) and Aβ monomeric and oligomeric species (E). Data were quantified for molecular weight size ranges for each antibody and normalized to β-III tubulin which was used as loading control (F) and relative densitometric quantifications are reported (right). Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Full uncropped blots are available in Supplementary Fig. 6. Notice that changes in levels of total tau are not statistically significant. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; **** P < 0.0001).

Figure 3

Reduction of the hippocampal NH₂htau in Tg-Alzheimer’s disease mice (3xTg) immunized with 12A12mAb ameliorates the disease-associated synaptic neuropathology. (A–G)Representative blots (n = 5) of sodium dodecyl sulphate-polyacrylamide gel electrophoresis western blotting analysis (left) on isolated synaptosomal preparations from hippocampal region of animals from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of 3xTg strain to assess the content of the NH₂htau fragment (A), total tau full-length (B), AT8-phosphorylated tau (C), APP holoprotein (D) and Aβ monomeric and oligomeric species (E/F). Data were quantified for molecular weight size ranges for each antibody and normalized to β-III tubulin which was used as loading control (G) and relative densitometric quantifications are reported (right). Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Full uncropped blots are available in Supplementary Fig. 7. Notice that changes in levels of total tau are not statistically significant. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001).

Figure 4

Improved cognition in Tg-Alzheimer’s disease (Tg2576) mice immunized with 12A12mAb. (A–C) Fourteen days after i.v. 12A12mAb immunization, the in vivo effect of NH₂htau removal on cognitive performance was investigated in animals from the three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of Tg2576 genetic background in the NOR (A), OPR (B) and Y-maze (C) tasks (top to bottom). For NOR (A) and OPR (B): right and left histograms, respectively, represent the PI (%) of corresponding values measured during the test trial among animals from the different experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of Tg2576 genetic background. The columns refer to objects presented during training and test trial. Analysis of PI (%) measured as time spending in the exploration of the novel/DO/(time spending in the exploration of novel/DO + time spending in the exploration of familiar/SO) × 100. Data were expressed as means ± SEM (n = 6–10). Values are means of at least three independent experiments and statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). For Y-maze (C): right and left histograms, respectively, represent the total entries (the total arm entries correspond to the total number of arms entered) and the spontaneous alternation (the number of alternations corresponds to the successive entries into three different arms in overlapping triplet sets) of animals from the different experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of Tg2576 genetic background. The percentage alternation was calculated as the ratio between number of correct triplets (e.g. ABC) and total entrances minus 2, multiplied by 100. Values are means of at least three independent experiments and statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). FO = familiar object; LO = left object; OPR = object place recognition; RO = right object.

Figure 5

Improved cognition in Tg-Alzheimer’s disease (3xTg) mice immunized with 12A12mAb. Fourteen days after i.v. 12A12mAb immunization, the in vivo effect of NH₂htau removal on cognitive performance was investigated in animals from the three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of 3xTg genetic background in the NOR (A), OPR (B) and Y-maze (C) tasks(top to bottom). For NOR (A) and OPR (B): right and left histograms, respectively, represent the PI (%) of corresponding values measured during the test trial among animals from the different experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of 3xTg genetic background. The columns refer to objects presented during training and test trial. Analysis of PI (%) measured as time spending in the exploration of the novel/DO/(time spending in the exploration of novel/DO + time spending in the exploration of familiar/SO) × 100. Data were expressed as mean ± SEM (n = 6–10). Values are means of at least three independent experiments and statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). For Y-maze (C): right and left histograms, respectively, represent the total entries (the total arm entries correspond to the total number of arms entered) and the spontaneous alternation (the number of alternations corresponds to the successive entries into three different arms in overlapping triplet sets) of animals from the different experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of 3xTg genetic background. The percentage alternation was calculated as the ratio between number of correct triplets (e.g. ABC) and total entrances minus 2, multiplied by 100. Values are means of at least three independent experiments and statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). FO = familiar object; LO = left object; OPR = object place recognition; RO = right object.

Figure 6

Immunization with 12A12mAb in Tg-Alzheimer’s disease mice is protective against the dendritic spines density loss which affects the memory and learning processes. (A, B) Comparative photomicrographs of Golgi-stained hippocampal CA1 neurons showing dendritic segments from animals from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of both strains (Tg2576, 3xTg) (left, refers to CA1 pyramidal neurons dendrites scale bar: 5 µm). Box-and-whisker plots (right) depict the morphometric analysis of the dendritic spine density from the three experimental groups. Values are expressed as number of spines per 1 µm segment. Statistically significant differences (comparisons were made on single mouse values obtained by averaging the number of spines counted on neurons of the same mouse) were calculated by ANOVA followed by post hoc test for multiple comparison among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001).

Figure 7

Reduction of cognitive deficits in 12A12mAb-immunized Tg-Alzheimer’s disease mice correlates with an increased LTP. (A, D) For Tg2576; (E, H) for 3xTg) time plot of average fEPSP responses (A, E) and changes in magnitude of LTP at CA3-Ca1 synapses (D, H) were calculated among animals (n = 6–10) from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of both strains. At least seven slices from six different mice were recorded for each experimental condition. Data are presented as the mean (±SEM). The traces above the plot show fEPSPs at baseline (1) and at 60 min after LTP induction (2). The box-whisker plots show pooled data. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; **** P < 0.0001). (B, C) For Tg2576; (F, G) for 3xTg) input/output curves show the fEPSP slopes plotted against the corresponding stimulus intensities recorded from hippocampal slices of animals (n = 6–10) from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of both strains (B, F). Comparison of PPF in animals (n = 6–10) from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of both strains (Tg2576, 3xTg) was also determined (C, G). PPF was induced by pairs of stimuli delivered at increasing interpulse intervals (20, 50, 100, 200, 300, 500 ms). Data are presented as the mean (±SEM) facilitation ratio of the second response relative to the first response. Statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). fEPSP = field excitatory post-synaptic potential; PPF = paired-pulse facilitation.

Figure 8

Inflammatory response (activation of astrocytes and microglia) is strongly down-regulated in 12A12mAb-immunized Tg-Alzheimer’s disease mice. (A, B) Neuroinflammation processes (activation of astrocytes and microglia) were assessed on hippocampal extracts from animals from three experimental groups (WT, Tg-Alzheimer’s disease and Tg-Alzheimer’s disease + mAb) of both strains (Tg2576, 3xTg) by western blotting analysis (left) for inflammatory proteins (glial fibrillary acidic protein, Iba1). Relative densitometric quantification of intensity signals (right) indicates lower levels of glial fibrillary acidic protein and Iba1 in Tg-Alzheimer’s disease mice + mAb compared to not-immunized Tg-Alzheimer’s disease. GAPDH housekeeping expression serves as loading control. Arrows on the right side indicate the molecular weight (kDa) of bands calculated from migration of standard proteins. Full uncropped blots are available in Supplementary Fig. 8. Values are from at least three independent experiments and statistically significant differences (see details in the main text) were calculated by ANOVA followed by post hoc test for multiple comparisons among more than two groups. P < 0.05 was accepted as statistically significant (*P < 0.05; **P < 0.01; ***P < 0.0005; ****P < 0.0001). GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GFAP = glial fibrillary acidic protein.