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, 73 (1), 147-161

In Vivo Validation of a Small Molecule Inhibitor of Tau Self-Association in Htau Mice

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In Vivo Validation of a Small Molecule Inhibitor of Tau Self-Association in Htau Mice

Eliot J Davidowitz et al. J Alzheimers Dis.

Abstract

Tau oligomers have been shown to transmit tau pathology from diseased neurons to healthy neurons through seeding, tau misfolding, and aggregation that is thought to play an influential role in the progression of Alzheimer's disease (AD) and related tauopathies. To develop a small molecule therapeutic for AD and related tauopathies, we have developed in vitro and cellular assays to select molecules inhibiting the first step in tau aggregation, the self-association of tau into oligomers. In vivo validation studies of an optimized lead compound were independently performed in the htau mouse model of tauopathy that expresses the human isoforms of tau without inherited tauopathy mutations that are irrelevant to AD. Treated mice did not show any adverse events related to the compound. The lead compound significantly reduced the level of self-associated tau and total and phosphorylated insoluble tau aggregates. The dose response was linear with respect to levels of compound in the brain. A confirmatory study was performed with male htau mice that gave consistent results. The results validated our screening approach by showing that targeting tau self-association can inhibit the entire tau aggregation pathway by using the selected and optimized lead compound whose activity translated from in vitro and cellular assays to an in vivo model of tau aggregation.

Keywords: Alzheimer’s disease; drug therapy; pathological; protein aggregation; tau protein.

Figures

Fig. 1.
Fig. 1.
Evaluation of the reproducibility and rigor of the ELISAs in these studies. Heat stable fractions from seven-month-old female tau KO, C57/Bl6 wild type, and JNPL3 mice were used for ELISAs measuring total, phosphorylated, and self-associated tau, and total homogenate was used for the assay for self-associated tau as described in the Methods for ELISAs. Serial dilutions of each sample were tested in duplicate at each concentration (4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 ug/ml); the values from the highest sample concentration within the range of the standard curve for each ELISA format are presented.
Fig. 2.
Fig. 2.
Self-associated tau in the hippocampus of male and female mice (A), male mice (B), female mice (C) was measured by mono-antibody ELISA formatted with capture antibody DA9 (epitope, tau 102–140) and reporter antibody DA9-HRP. Outliers were removed using the ROUT (Q = 1%) method. p-values for analyses between the control group and the group with 25 ng lead cmpd/gm brain are shown above plots. Analysis between the groups determined statistically significant differences from each other for the male and female mice (Kruskal-Wallis test p = 0.0104) and for the male mice (one-way ANOVA p = 0.0086). **p-value < 0.01.
Fig. 3.
Fig. 3.
Differential levels of insoluble tau aggregates in untreated male and female htau mice at 6.5 months of age. These significant results show that the control group of male htau mice developed about twice the level of insoluble tau aggregates as the female control group at the end of the study. This justified the separate analyses of the male mice which developed enough insoluble tau to determine the efficacy of the lead compound. The levels of Sarkosyl-insoluble tau aggregates were determined by ELISA for total tau formatted with capture antibody DA31 (pan-tau) and reporter Ab DA9 (pan-tau) conjugated to HRP. The values were normalized to total tau in the heat stable fractions of forebrain.
Fig. 4.
Fig. 4.
Reduction of insoluble tau levels are dependent on compound concentration in the brain. Sarkosyl-insoluble tau preparations were evaluated by ELISA using pan-tau and p-tau antibodies DA31 (A), PHF1 (B), CP13 (C), and RZ3 (D). Levels of insoluble tau were normalized to total soluble tau in the heat stable fractions of each mouse forebrain and plotted against the average value of compound found in the brains of male C57BL/6 mice. The number of mice in each group: 0 ng/g (n = 12), 6 ng/ng (n = 13), 25 ng/g (n = 14). p-values for pairwise analyses of the control group and the group with 25 ng lead cmpd/gm brain are shown above plots. Immunoblot comparison of vehicle control group with treated group (E). Sarkosyl-insoluble tau preparations from eight mice from each group were run on 4–20% polyacrylamide gradient gel and transferred to a PVDF membrane. Images of short and long capture of chemiluminescent signal are presented in the lower and upper images of the blot, respectively, to show both abundant monomer and less abundant higher molecular weight P-tau species.
Fig. 5.
Fig. 5.
Analysis of brain and plasma levels of lead compound with respect to formulated doses in feed. Groups of male C57BL/6 mice (n = 5) were treated with feed formulated to provide the doses shown on the plot for seven days. Mice were individually caged on the last two days in order to determine the amount of feed each mouse consumed (Murigenics, Vallejo, CA) prior to analyzing compound levels in the brain (A) and plasma (B) by LC-MS/MS (Quintara Discovery, Hayward, CA). Values below quantifiable levels were not included in the groups treated with 20, 30, or 100 mg/kg doses.
Fig. 6.
Fig. 6.
Immunohistochemistry of hippocampal slices of male mice. The mAb MC1, recognizing misfolded pathological tau, was used to stain the hippocampal slices from each mouse. Treated mice with 25 ng compound/g brain had significantly reduced levels of MC1 dependent pathological tau compared to treated mice with 6 ng compound/g brain (A). Representative images of the hippocampal region stained with mAb MC1 are shown for the vehicle control (B) and treatment groups 6 and 25 ng/gm brain (C, D).
Fig. 7.
Fig. 7.
Levels of lead compound in the sera of mice in the confirmatory study. Blood and brain specimens were collected at the end of treatment after sacrifice in the middle of the wake cycle and analyzed by LC-MS/MS for levels of the lead compound. Perfusions of the brains were not performed because that would have affected the post-translational modifications of tau. Thus, the brain lysates contained compound from the blood in the brain tissue that precluded their use for determining levels of the compound in the brain.
Fig. 8.
Fig. 8.
Reduction of insoluble tau in the confirmatory study and combined study analyses. The results for the confirmatory study are shown independently and in combination with the results of the primary study. The most significant result was found in the combined data analysis of the Sarkosyl-insoluble tau aggregates using the ELISA for total tau (E); it indicates the overall efficacy of lead compound in these studies. The levels of phosphorylated insoluble aggregates in the treated group were also reduced. The number of mice for the confirmatory study groups was 15 (A-E) and for the combined analyses were n = 27 for vehicle control mice and n = 29 for treated mice (E-H). The results for the insoluble phosphorylated tau (PHF1, CP13, or RZ3) were normalized to the respective levels of phosphorylated tau in the heat stable fractions (A-E). Analysis of the combined results for phosphorylated tau were normalized to the results of total tau from the pan-tau ELISA using mAb DA31.
Fig. 9.
Fig. 9.
Effect of lead compound on levels of total and phosphorylated soluble tau in the heat-stable fractions from the forebrain. The results from the confirmatory study are shown in panels A-D (n = 15) and from the combination of data from the primary and confirmatory studies in panes E-H (vehicle control group n = 27; treatment group n = 29). The soluble tau from the forebrains of the treated mice showed an increase in phosphorylation at the epitopes indicated. The increase was highly significant in the confirmatory study for pSer-202 (C) and pThr-231 (D), but less significant in combined analyses of the studies (G, H). The results from the P-tau ELISAs using PHF1, CP13, or RZ3 were normalized to the results of total tau from the pan-tau ELISA using mAb DA31.

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