Anti-tau antibody reduces insoluble tau and decreases brain atrophy

Kiran Yanamandra, Hong Jiang, Thomas E Mahan, Susan E Maloney, David F Wozniak, Marc I Diamond, David M Holtzman, Kiran Yanamandra, Hong Jiang, Thomas E Mahan, Susan E Maloney, David F Wozniak, Marc I Diamond, David M Holtzman

Abstract

Objective: We previously found a strong reduction in tau pathology and insoluble tau in P301S tau transgenic mice following intracerebroventricular infusion of the anti-tau antibody HJ8.5. We sought to determine the effects of HJ8.5 in the same model following peripheral administration.

Methods: The primary objective was to determine if HJ8.5 administered at a dose of 50 mg kg(-1) week(-1) by intraperitoneal (IP) injection to 6-month-old P301S mice for 3 months would influence phospho-tau (p-tau) accumulation, tau insolubility, and neurodegeneration.

Results: Treatment with HJ8.5 at 50 mg/kg showed a very strong decrease in detergent-insoluble tau. Importantly, HJ8.5 significantly reduced the loss of cortical and hippocampal tissue volumes compared to control treated mice. HJ8.5 treatment reduced hippocampal CA1 cellular layer staining with the p-tau antibody AT8 and thio-S-positive tau aggregates in piriform cortex and amygdala. Moreover, mice treated with HJ8.5 at 50 mg/kg showed a decrease in motor/sensorimotor deficits compared to vehicle-treated mice. Some effects of HJ8.5, including reduction in brain atrophy, and p-tau immunostaining were also seen with a dose of 10 mg kg(-1) week(-1). In BV2-microglial cells, we observed significantly higher uptake of P301S tau aggregates in the presence of HJ8.5. HJ8.5 treatment also resulted in a large dose-dependent increase of tau in the plasma.

Interpretation: Our results indicate that systemically administered anti-tau antibody HJ8.5 significantly decreases insoluble tau, decreases brain atrophy, and improves motor/sensorimotor function in a mouse model of tauopathy. These data further support the idea that anti-tau antibodies should be further assessed as a potential treatment for tauopathies.

Figures

Figure 1
Figure 1
Anti-tau antibody markedly reduces insoluble tau. The cortex from vehicle- (n = 15), HJ8.5- 10 mg/kg (n = 13), and HJ8.5- 50 mg/kg (n = 12) treated P301S mice were sequentially extracted by RAB (A), RIPA (B), and 70% formic acid (detergent insoluble) (C). Levels of tau were determined by ELISA. HJ8.5 treatment at 50 mg/kg significantly decreased insoluble human tau (P < 0.0001) compared to vehicle-treated mice. Values represent mean ± SEM. ****P < 0.0001. RAB, reassembly buffer; RIPA, radio immunoprecipitation assay buffer; ELISA, enzyme-linked immunosorbent assay.
Figure 2
Figure 2
Anti-tau antibody decreased phospho-tau staining in the hippocampal CA1 cell layer. (A) Representative coronal sections of biotinylated AT8 antibody staining of phosphorylated tau in the hippocampal CA1 cellular region of 9-month-old P301S mice treated for 3 months with vehicle and HJ8.5 at 50 mg/kg. The lower images are higher power views of the CA1 region in the uppers panels. Red arrows indicate the area magnified in the lower image. Black arrows indicate the hippocampal CA1 cell layer. (B) Quantification of biotinylated AT8 antibody staining of abnormally phosphorylated tau revealed a significant decrease in AT8 staining in mice treated with HJ8.5 at 50 mg/kg in the hippocampal CA1 cellular layer compared to vehicle-treated mice (P = 0.035). HJ8.5 at 10 mg/kg treated mice also showed decreased AT8 staining compared to the vehicle-treated group, but this was not statistically significant (P > 0.05). Values represent mean ± SEM. *P < 0.05. Scale bar represents 300 μm.
Figure 3
Figure 3
Thio-S staining is decreased in anti-tau antibody-treated P301S mice. (A) Representative coronal sections of thio-S staining of tau aggregates in piriform cortex and amygdala in 9-month-old P301S mice treated for 3 months with 10 and 50 mg kg−1 week−1 of HJ8.5 as well as from vehicle-treated mice. (B) Semiquantitative analysis of thio-S staining was rated on a scale of 1 (no staining) to 5 (maximum staining) in all treatment groups. Both doses of HJ8.5 antibody treatment showed significantly lower thio-S staining compared to vehicle-treated mice. Values represent mean ± SEM. *P < 0.05. Scale bar represents 25 μm.
Figure 4
Figure 4
HJ8.5 treatment decreased brain atrophy. (A) Repre-sentative coronal sections of 9-month-old P301S mice treated with vehicle or HJ8.5 (50 mg/kg). Quantification of cortical (B) and hippocampal (C) volumetric analysis were performed among 6-month-old P301S mice (n = 10) and 9-month-old P301S mice treated for 3 months with vehicle (n = 15), HJ8.5 – 10 mg/kg (n = 13), and HJ8.5 – 50 mg/kg (n = 12). HJ8.5-treated mice at 50 mg/kg had increased cortical and hippocampal volumes (*P < 0.05) compared to vehicle-treated mice. Values represent mean ± SEM.
Figure 5
Figure 5
Anti-tau antibody treatment improves sensorimotor function in P301S mice. (A) Mice treated with HJ8.5 at 50 mg/kg exhibited improved performance on the inverted screen test compared to the phosphate-buffered saline (PBS)-treated control group by being able to remain on inverted screen for a significantly longer time compared to the controls. (B) Likewise, the mice treated with HJ8.5 at 50 mg/kg were able to remain on a narrow Plexiglas “ledge” for a significantly longer time relative to the vehicle-treated group. (C) A trend toward improved performance was observed in the mice treated with HJ8.5 compared to controls in terms of freezing levels averaged across the 8-min session during the contextual fear test conducted on day 2, although these differences were not statistically significant. *P < 0.05. Values represent mean ± SEM.
Figure 6
Figure 6
HJ8.5 treatment increased the uptake of aggregated tau in BV2 cells and tau levels in plasma. Alexa-Fluor-647-labeled P301S aggregates (P301S-agg A647) were preincubated with HJ8.5 (P301S-agg A647 + HJ8.5) or control antibody HJ3.4 (P301S-agg A647 + HJ3.4) overnight. Then Alexa-Fluor-647 dye alone (A647) or preincubated labeled P301S aggregates were added to primary cortical neurons (A) or microglial BV2 cells (B). Uptake of labeled P301S tau aggregates were measured by flow cytometry. A significant increase in P301S tau aggregate uptake in microglial BV2 cells was observed in the presence of HJ8.5 compared to either no antibody or control antibody (P < 0.0001, two-way analysis of variance [ANOVA] followed by Tukey's post hoc test). However, there was no difference in uptake of tau aggregates in primary cortical neurons in the presence or absence of HJ8.5. (C) After 3 month administration of two different doses of HJ8.5 (10 and 50 mg/kg) from 6 months till 9 months in P301S mice, tau levels in plasma were analyzed by ELISA. A dose-dependent increase of tau in plasma compared to controls was observed (P < 0.0001). Values represent mean ± SEM.
Figure 7
Figure 7
Increase in plasma tau following anti-tau antibody treatment is bound to antibody. Forty-eight hours after intraperitoneal administration of vehicle or HJ8.5 (10 or 50 mg/kg) in 9-month-old P301S mice (n = 3 per group), tau levels in plasma were analyzed by enzyme-linked immunosorbent assay (ELISA). The levels of total tau were measured in plasma (A), after removal of any tau–antibody complex by immunoprecipitation (B), and in eluates following capture of anti-tau antibody-tau complexes (C). Values represent mean ± SEM.

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