Inhibition of Tau aggregation with BSc3094 reduces Tau and decreases cognitive deficits in rTg4510 mice

Abstract

Background: One of the major hallmarks of Alzheimer's disease (AD)is the aberrant modification and aggregation of the microtubule-associated protein Tau . The extent of Tau pathology correlates with cognitive decline, strongly implicating Tau in the pathogenesis of the disease. Because the inhibition of Tau aggregation may be a promising therapeutic target, we tested the efficacy of BSc3094, an inhibitor of Tau aggregation, in reducing Tau pathology and ameliorating the disease symptoms in transgenic mice.

Methods: Mice expressing human Tau with the P301L mutation (line rTg4510) were infused with BSc3094 into the lateral ventricle using Alzet osmotic pumps connected to a cannula that was placed on the skull of the mice, thus bypassing the blood-brain barrier (BBB) . The drug treatment lasted for 2 months, and the effect of BSc3094 on cognition and on reversing hallmarks of Tau pathology was assessed.

Results: BSc3094 significantly reduced the levels of Tau phosphorylation and sarkosyl-insoluble Tau. In addition, the drug improved cognition in different behavioral tasks and reduced anxiety-like behavior in the transgenic mice used in the study.

Conclusions: Our in vivo investigations demonstrated that BSc3094 is capable of partially reducing the pathological hallmarks typically observed in Tau transgenic mice, highlighting BSc3094 as a promising compound for a future therapeutic approach for AD.

Keywords: Alzet pump; Alzheimer's disease; BSc3094; P301L Tau mutation; Tau pathology; aggregation inhibitor.

FIGURE 1

Direct intracerebral administration of BSc3094 significantly reduced sarkosyl‐insoluble Tau. (A) Intravenous administration of BSc3094 (3 mg/kg in PEG400/ddH₂O (60:40)) demonstrated poor BBB permeability, as the concentration of drug in the brain was very low compared to the initial administered drug concentration, and the half‐life of the drug in the brain equaled 0.8 h, meaning its potential therapeutic effect wears off in an extremely short period of time. (B) Direct intraventricular administration of three increasing doses of BSc3094 over 28 days into the brains of rTg4510 mice led to a significant reduction (≈70%) of sarkosyl‐insoluble Tau achieved with the 1.5 mM drug concentration (one‐way ANOVA with uncorrected Fisher's LSD post hoc test; P < .0001). All numerical data are shown as mean ± SEM; * denotes the significance compared to 100% (no drug). **** P < .0001

FIGURE 2

Total brain and body weight in rTg4510 and control mice used in the study. (A) Representation of the total brain weight (in mg) at the moment of sacrifice. A two‐way ANOVA showed an overall effect of the genotype [F(1, 46) = 112.8; P < .0001], with double transgenic rTg4510 mice presenting a lower brain weight (≈−15%) compared to control mice. Further post hoc analysis with Tukey's multiple comparison test showed that vehicle‐treated rTg4510 mice had lower brain weight than vehicle‐treated controls (P < .0001). Similarly, BSc3094‐treated rTg4510 mice also presented lower brain weight than BSc3094‐treated controls (P < .0001). There was no effect of the drug treatment. (B) Representation of the brain weight over time, from the pump implantation until the sacrifice date, showed that double transgenic mice had a decreased brain weight compared to the control littermates [F(1, 472) = 69.92; P < .0001]. Similar to the brain weight, the drug treatment did not reverse the brain weight loss observed in the transgenic mice. All numerical data are shown as mean ± SEM; * denotes the effect of the genotype; **** P < .0001

FIGURE 3

Treatment with BSc3094 reduces Tau phosphorylation in rTg4510 mice. (A) A two‐way ANOVA analysis demonstrated an interaction between genotype and treatment on the levels of 12E8 [F(1, 22) = 4.32; P = .0495]. Further post hoc analysis with uncorrected Fisher's LSD test showed that rTg4510 mice presented an increase in the levels of 12E8 compared to control animals (P = .429), an effect that was reversed by BSc3094 treatment (P = .0160). (B) An overall effect of the genotype was detected regarding the levels of PHF‐1 [F(1, 22) = 14.58; P = .0009]. Uncorrected Fisher's LSD post hoc test showed that the phosphorylation at the epitope Ser396/ser404, detected with PHF‐1 antibody, was increased in vehicle‐treated rTg4510 ≈15‐fold compared to controls (P = .0007). This effect was reversed down to half (≈7‐fold with respect to (w.r.t.) controls) by treatment with BSc3094 (P = .0452). All numerical data are shown as mean +/‐ SEM; * denotes the effect of the genotype; $ denotes the effect of BSc3094 treatment. *, $ P < .05; *** P < . 001

FIGURE 4

No motor impairments observed in rTg4510 mice, but an increase in anxiety‐like behavior compared to control mice. rTg4510 mice presented similar distance covered values on days 1 (A) and 2 (B) of the OF test compared to control mice, showing that they did not have significant motor impairments that could affect the results of the behavioral assessment. On the other hand, an overall effect of the genotype was observed in the time spent in the center of the arena on day 1 (C) (two‐way ANOVA [F(1, 62) = 7.745; P = .0071]). Uncorrected Fisher's LSD post hoc test revealed that vehicle‐treated rTg4510 mice presented lower percentage of time in the center of the arena compared to vehicle‐treated controls (P = .0306), which is an indicator of anxiety behavior. On day 2, a similar pattern was observed, with vehicle‐treated rTg4510 mice presenting a lower percentage of time spent in the center of the arena (D) compared to vehicle‐treated controls (P = .0330). This reduction in the time spent in the center of the arena was not observed in rTg4510 mice on days 1 and 2 after treatment with BSc3094, showing that the drug produced a positive effect in anxiety‐like behavior. All numerical data are shown as mean ± SEM; * denotes the effect of the genotype; * P < .05

FIGURE 5

Treatment with BSc3094 partially reversed the memory deficits in rTg4510 mice. (A) Vehicle‐treated rTg4510 mice spent a lower percentage (≈−20%) of time exploring the novel object in the NOR test compared to controls (P = .0291). The memory impairment was reversed back to control levels by the treatment with BSc3094, as drug‐treated rTg4510 spent a significantly higher percentage of time exploring the novel object compared to vehicle‐treated transgenic mice (P = .0171). Two‐way ANOVA followed by uncorrected Fisher's LSD post hoc test. All numerical data are shown as mean ± SEM; * denotes the effect of the genotype; $ denotes the effect of BSc3094 treatment. *, $ P < .05. (B) BSc3094 did not improve spatial reference memory in rTg4510 mice. Vehicle‐ and BSc3094‐treated control mice presented a lower preference for the old arm in the y‐maze test. A two‐way ANOVA revealed an interaction between genotype and treatment [F(3,122) = 3.414; P = .0197]. Post hoc analysis with uncorrected Fisher's LSD test showed a decreased preference for the old arm in vehicle‐treated control mice (P = .024) and in BSc3094‐treated control mice (P = .0112). In contrast, vehicle‐treated rTg4510 mice showed no preference for the new or old arm reflecting impaired spatial reference memory. These deficits in the transgenic mice were not reversed by BSc3094 treatment. All numerical data are shown as mean ± SEM. * P < .05 when compared to new arm preference. (C) BSc3094 treatment slightly improved long‐term memory in the MWM. Two‐way ANOVA analysis revealed an overall effect of the genotype on the latency to escape in the MWM F(3, 305) = 42.88. Vehicle‐treated rTg4510 mice presented a significantly increased latency to escape in the MWM test compared to vehicle‐treated controls (P < .0001). This memory deficit was not reversed by BSc3094 treatment, as drug‐treated controls (P < .0001). (D) By comparing the percentage of time each group spent in the target quadrant with the 25% chance of exploration we observed that transgenic mice spent a lower percentage of time in the target quadrant of the MWM in the three probe trials performed compared to control mice, an effect that was not reversed by treatment with BSc3094. (E) A two‐way ANOVA analysis of the percentage of time in the target quadrant in the long‐term probe trial denoted an overall effect of the genotype [F(1, 62) = 5.309; P = .0246]. Further post hoc analysis with uncorrected Fisher's LSD test showed that vehicle‐treated rTg4510 mice spent a significantly lower percentage of time (−28%) in the target quadrant in the long‐term probe trial compared to vehicle‐treated controls (P = .0246), an effect that was partially reversed by BSc3094 treatment. All numerical data are shown as mean ±; * denoted the effect of the genotype in A, C, and E; in D, * denotes the significance compared to 25% chance of exploration of the target quadrant. *, $ P < .05; **P < .01; ***P < .001; ****P < .0001

FIGURE 6

BSc3094 treatment did not reverse the loss of synaptic markers in rTg4510 mice. (A) A two‐way ANOVA showed an overall effect of the genotype on the levels of GluR1 [F(1, 22) = 35.08; P = .0001]. Further uncorrected Fisher's LSD post hoc test showed that vehicle‐treated rTg4510 mice presented significantly lower levels of GluR1 compared to vehicle‐treated control mice (P = .0030), a decrease that was also observed in BSc3094‐treated rTg4510 mice (P = .0039), demonstrating the BSc3094 fails to reverse the loss of GluR1 observed in the transgenic mice. (B) An overall effect of the genotype was observed on the levels of PSD95 [F(1, 22) = 8.892; P = .0066]. Uncorrected Fisher's LSD post hoc test revealed that vehicle‐treated rTg4510 mice presented significantly lower levels of PSD95 compared to vehicle‐treated control mice (P = .0456), a decrease that was also observed in BSc3094‐treated rTg4510 mice (P = .0153), demonstrating that the drug treatment did not reverse the loss in the expression of PSD95. (C) Analysis of the expression of synaptophysin with two‐way ANOVA showed an overall effect of the genotype [F(1, 22) = 34,24; P < .0001]. Further post hoc analysis with uncorrected Fisher's LSD test showed that vehicle‐treated rTg4510 mice presented significantly lower levels of synaptophysin compared to control mice (P < .0001), an effect that was not reversed by BSc3094 treatment (P < .0001). All numerical data are shown as mean ± SEM; * denotes the effect of the genotype; * P < .05; ** P < .01; **** P < .0001