Therapeutic Treatment With OLX-07010 Inhibited Tau Aggregation and Ameliorated Motor Deficits in an Aged Mouse Model of Tauopathy

Abstract

Targeting tau protein is a strategy for the development of disease-modifying therapeutics for Alzheimer's disease (AD) and numerous rare tauopathies. A small molecule approach targeting tau aggregation was used to select and optimize compounds inhibiting tau self-association in vitro that have translated in vivo in preventive studies in htau and P301L tau JNPL3 mouse models of tauopathy. In this therapeutic treatment study, aged JNPL3 mice with pre-existing tau aggregates were used to evaluate the therapeutic effect of OLX-07010. The study had a Baseline group of mice aged 7 months, a vehicle, and two dose groups treated until 12 months by administration in feed. The primary endpoint of the study was the reduction of insoluble tau aggregates with statistical significance. The secondary endpoints were dose-dependent reduction of insoluble tau aggregates, reduction of soluble tau, and improvement of motor behavior. ELISAs and immunoblots were used to determine the levels of tau and its aggregated forms including self-associated tau and Sarkosyl insoluble tau. Effect on motor behavior, as measured by Rotarod assay, was also assessed between the treatment groups. At the end of treatment, reduced levels of self-associated tau, Sarkosyl insoluble tau aggregates, and overall levels of tau in the heat-stable fraction with statistical significance in the cortex were observed. Treatment prevented the accumulation of tau aggregates above baseline, and in parallel, treatment groups had improved motor behavior in a Rotarod assay compared to baseline and vehicle control groups, suggesting that treatment was rescuing motor impairment in aged mice. The functional and biochemical readouts suggest that this small molecule has potential for treating neurodegenerative diseases characterized by tau aggregation such as AD and progressive supranuclear palsy.

FIGURE 1

Schematic of study design. The study was independently performed at FIMR. Female homozygous P301L tau JNPL3 mice were bred at Taconic and aged at FIMR at 3–7 months. Vehicle and Treatment groups were evenly distributed in the three staggered cohorts. The Baseline group was harvested following the motor behavior assay at 7 months of age, and the motor behavior of the Vehicle and 40 and 80 mg/kg groups was assayed at the end of 5 months of treatment. Biochemical studies for tau aggregates were performed for all mice at the same time to minimize inter‐assay variability. Due to attrition during aging, samples for biochemical analysis were obtained from 19 mice in the vehicle group, 22 mice in the 40 mg/kg group, and 20 mice in the 80 mg/kg Treatment groups.

FIGURE 2

Analysis of total and aggregated tau protein in the cortex. Quantification of tau was performed by ELISAs that detect both the endogenous mouse tau and the exogenously expressed human tau construct. The four groups in the study were Baseline (BL; n = 20), sacrificed at 7 months, and Vehicle (Veh; n = 19), 40 mg/kg (n = 22), and 80 mg/kg (n = 20) groups sacrificed at 12 months. Sarkosyl Insoluble total tau (A) and total tau (B) in the heat stable fraction were quantified by ELISA formatted with mAb DA31 for capture and mAb DA9‐HRP for reporter. Self‐associated tau (C) was determined by mono‐Ab ELISA formatted with mAb DA9 for capture and mAb DA9‐HRP for reporter. Results for BL and treatment groups are shown as a percentage of the mean value of the Vehicle group (% Veh). Outliers, identified using the ROUT method (Q = 1%), are shown as open squares. For group comparisons, the significance level of p < 0.05 is considered statistically significant and indicated as * for p < 0.05 or ** for p < 0.01.

FIGURE 3

Immunoblot analysis of tau species using mAb HT7. Immunoblot analysis of tau was performed with mAb HT7 specific for the human tau construct using total protein tissue lysates from cortex. Overall human tau signal from each lane was normalized to Ponceau staining in that lane (A). The signal for tau monomer (B), tau oligomers (C) or very high molecular weight (VHMW) tau (D) was normalized to the total tau signal in each lane for each sample. Results for BL and treatment groups are shown as a percentage of the mean value of the Vehicle group (% Veh). Outliers, identified using the ROUT method (Q = 1%), are shown as open squares. For group comparisons, significance levels are indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001. For pairwise comparisons, p < 0.05 is considered significant, with exact values reported. Exponential growth curves representing the correlation of the levels of VHMW tau (E) to the levels of total human tau in each mouse were plotted for each study group and overlayed to show the effect of 40 mg/kg (green) and 80 mg/kg (purple) treatment relative to the control Baseline (black), and Vehicle (red) groups. (R squared values for correlation exponential curves, N): Red (0.6483, 19); Green (0.7134, 22); Purple (0.7454, 20); Black (0.6359, 20). A representative mAb HT7 immunoblot image (F) indicates the regions of the image that were quantified for monomer, oligomer, and VHMW tau. The signal for monomer, oligomer and VHMW tau at the top of the gel was normalized to the total tau signal in the lane to enable comparison of the tau species between the samples on multiple blots. A representative image of a Ponceau stained membrane has a bracket (G) indicates the region quantified by imageJ (Rasband 1997‐2018) for the normalization of the total tau signal in each lane (A). All immunoblots in this dataset, including the ones shown here, are also presented in Figure S2.

FIGURE 4

Evaluation of motor coordination. Cross‐sectional evaluation of motor coordination was performed with a Rotarod assay. The mice were evaluated prior to sacrifice, the Baseline group at 7 months, and the Vehicle, 40 and 80 mg/kg Treatment groups at 12 months. Data presented as mean ± SEM. Outliers, identified using the ROUT method (Q = 1%), are shown as open squares. For comparison between selected two groups, p < 0.05 is considered significant, with exact values reported.

FIGURE 5

Serum levels of OLX‐07010 and tau protein. (A) Quantification of compound levels in mice sera was performed by LC/MS/MS with an Agilent 6400 LC/Triple Quad. Mice sera was processed from whole blood collected terminally in the middle of the dark cycle. (B) Heat treatment of sera at acidic pH was used to remove interfering antibodies from the samples to enable quantification of tau by ELISA (d'Abramo et al. 2016). Data presented as mean ± SEM. Outliers, identified using the ROUT method (Q = 1%), are shown as open squares. For comparison between selected two groups, p < 0.05 is considered significant, with exact values reported.