Targeting tau protein is a strategy for developing 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 that inhibit tau self-association in vitro, which have been translated into preventive studies in htau and P301L tau JNPL3 mouse models of tauopathy. In this 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 seven months, a vehicle, and two dose groups treated until twelve 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 phosphorylated tau, 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 the 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.

Animals

Female homozygous P301L tau JNPL3 mice (Lewis et al. 2000); Research Resource Identifier (RRID): IMSR_TAC:2508; Stock Tg(Prnp-MAPT*P301L)JNPL3Hlmc were purchased from Taconic Biosciences (Rensselaer, NY). The rationale for using females includes their more aggressive and less variable phenotype compared to male mice (Buccarello et al. 2017; Koppel et al. 2019), which can lead to more pronounced and measurable effects of the drug, making it particularly useful in early-stage studies to detect potential therapeutic benefits more clearly. Additionally, using female JNPL3 mice maintains consistency with previous research with OLX-07010 in the preventive study in JNPL3 mice, allowing for more direct comparisons and validation of results. A preventive treatment study with OLX-07010 in htau mice demonstrated its effectiveness in males, as well as its independence from sex (Davidowitz et al., 2023; Davidowitz et al., 2020). Ideally, highly powered studies including males and females should be performed to evaluate translational potential (Dennison et al. 2021) when feasible. A study performed in male JNPL3 mice had a treatment duration of 6 months from 9 to 15 months of age that required a sample size of 49 male mice per group to show an effect (Okuda et al. 2015). The mice were received at Feinstein Institutes for Medical Research (FIMR; Manhasset, NY) at 2 months and aged to 7 months prior to the start of treatment. Mice were examined, handled, and weighed prior to initiation of the study and examined at least once a week during treatment to ensure adequate health and suitability. As recommended by The Guide for the Care and Use of Laboratory Animals, rooms were maintained with 12 hr light/dark cycles, at a temperature of 20 to 23 °C with a relative humidity of approximately 50%. Four mice were housed per cage in standard cages with access to food and water ad libitum for the duration of the study. This study did not use environmentally enriched housing. For all mouse cages, adequate nesting material was provided in addition to the usual cage bedding to alleviate thermal stress and improve overall welfare during the treatment period. Mice were euthanized by cervical dislocation under deep anesthesia induced by an overdose of isoflurane (3-5%) during the middle of the dark cycle. All experiments were conducted in compliance with the FIMR Institutional Animal Care and Use Committee (IACUC; Protocol # 2017-022).

Mouse Diets

Open Standard Diet (Research Diets; New Brunswick, NJ) was used for aging the mice to 7 months and for the Vehicle group. The diet was formulated with test article OLX-07010 manufactured by Albany Molecular Research Inc. (AMRI, Albany, NY); its purity was >99% by HPLC (AUC).  Diets were formulated with 200 or 400 mg of OLX-07010 per kg of diet for the 40 and 80 mg/kg mouse dose treatment groups based on average weight and daily consumption. No dyes were used in diet formulations. Diets were assayed in duplicate to qualify the levels and stability of the compound in the feed by LC-MS/MS (Quintara Discovery, Hayward, CA).

Bioanalytical Analysis 

Quantification of compound levels in mouse sera was performed in-house using LC/MS/MS (Agilent 6400 LC/TQ).  Calibration standards of OLX-07010 were prepared by spiking the compound into blank serum at a series of concentrations. Twenty microliters of the samples or standards were treated with 100 microliters of internal standard (verapamil) in methanol to precipitate the protein. The mixtures were vortexed for 15 minutes and centrifuged at 4,000 rpm at 4 degrees Celsius for 15 minutes. Ten microliters of the supernatant were transferred to an injection plate and mixed with 140 microliters of methanol for analysis via positive electrospray UHPLC/MS/MS in multiple-reaction-monitoring mode. This method has been qualified for the detection and quantification of our lead compound through evaluations of its specificity, selectivity, linearity, accuracy, precision, recovery, carryover, and stability. 

Motor Behavior

Motor function was assayed with the MazeEngineers 6-Lane rotarod for mice (Deacon 2013; Koppel et al. 2019). All mice were acclimated two weeks prior to the experimental trials on the device at 4 rotations/minute (r/min) on the 3 cm rotating rod for 10-minute sessions. For experimental trials, 6 mice were run at a time, on lanes of 6 cm in width, with rotations that began at a start speed of 4 r/min and then accelerated at 8 r/min to a maximum speed of 20 r/min for either 30 minutes or until mice dropped from the rotational drum at a drop height of 16 cm. Utilizing infrared floor sensors that detect true falls, integrated Conductor Software was used to log drop speed, latency to fall, and total distance over the course of the maximum 30-minute trial.

Brain Tissue Preparations

Procedures for preparation of samples and performance of ELISAs are detailed in the publications (Acker et al. 2013; Davidowitz et al. 2020; Forest et al. 2013).  Three types of brain preparations were performed for assays to determine the levels of total self-associated tau, Sarkosyl-insoluble tau aggregates, and soluble tau in the heat-stable (HS) fractions. For self-associated tau, brain tissue from cortex was homogenized in 10 volumes of cold Tris-buffered saline, pH 7.4, containing protease and phosphatase inhibitors, cleared by low-speed centrifugation at 14,000 x g for 10 min at 4 ^oC, and stored at -80 ^oC. To prepare Sarkosyl-insoluble tau, the cleared homogenate was incubated for 10 min with 1% Sarkosyl, the insoluble tau was pelleted by ultracentrifugation at 200,000 x g for 30 mi,n and washed with homogenization buffer. The pellet was suspended in 1x Laemmli sample buffer and heated for 10 min at 100 ^oC to dissociate the tau for analysis by ELISA. HS fractions of brain tissue were prepared by homogenization in 10 volumes of ice-cold buffer (50 mM Tris, pH 7.5, 0.8 M NaCl, 5% β-mercaptoethanol), and the cleared homogenate was heated at 95°C for 10 min and then centrifuged at 20,000 x g at 4°C for 10 min. Tau has the special characteristic of remaining soluble under these conditions, facilitating its purification in the supernatant/HS fraction. Supernatants were cooled and dialyzed against 50 mM Tri,s pH 7.,5 with 1 mM EDTA, 0.1 mM PMSF.

ELISAs and Antibodies

Monoclonal antibodies (mAbs) used in the ELISAs were all developed, produced, and formatted for assays in the laboratory of Peter Davies, Ph.D., Director, Litwin-Zucker Center for Alzheimer’s Disease & Memory Disorders, FIMR. Pan-tau antibodies mAb DA31 (epitope, amino acids 150–190 in 4R2N tau) and mAb DA9 (epitope, amino acids 102-140). ELISAs were performed for total tau (capture Ab DA31, reporter Ab DA9HRP) and self-aggregated tau by monoantibody tau ELISA (DA9-DA9HRP) as described (Davidowitz et al. 2023; Forest et al. 2013).

Immunoblots

Thirty µg of total protein from each brain lysate sample was loaded onto 4-20% gradient polyacrylamide precast Criterion Gels (Bio-Rad) with 0.1% final SDS under non-reducing conditions, electrophoresed, and transferred onto PVDF membrane using the Transblot Turbo semi-dry system (Bio-Rad). The blots were ponceau-stained, followed by blocking in 5% non-fat milk in PBST for 1 hour at ambient temperature. The blots were then incubated overnight at 4°C in primary antibody HT7 (product # MN1000, Invitrogen) diluted 1:5,000 in blocking buffer on a rocking platform. Following (3) 10-minute washes in blocking buffer, the blots were incubated for 1 hour in goat-anti-mouse HRP conjugated (Jackson Immunochemicals) diluted 1:10,000 in blocking buffer at ambient temperature on a rocking platform. After several washes with decreasing concentrations of Tween-20, the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher) was added, and chemiluminescent images were captured on the FluorChem R (Protein Simple) using movie mode exposures for quantification of chemiluminescent signal performed using AlphaView software (ProteinSimple). To measure the monomeric and aggregated tau from the blots, the luminescence counts for monomer, oligomer, and very high molecular weight (VHMW) tau were normalized to the total tau signal in the lane to enable comparison of the tau species between the samples on multiple blots.

Statistical Analyses

The study was powered by assuming a β risk of 0.8 and an α risk of 0.05. The primary outcome was the reduction of insoluble tau aggregates in the brains of the treated mice compared to untreated mice, with statistical significance. Results were expressed as mean ± standard error of the mean and presented as the percentage of the Vehicle group. All statistical analyses were performed using Graph-Pad Prism 10.2 (GraphPad Software, San Diego, CA). Outliers, identified using the ROUT method with a false discovery rate (Q) set at 1%, are depicted as open squares in the figure. Normality was assessed beforehand using the D'Agostino & Pearson test and the Anderson-Darling test. For data that met the assumptions of normality, multiple group comparisons were performed using the Brown-Forsythe and Welch ANOVA tests, followed by the Dunnett T3 post-hoc test (Supplemental Tables 3 and 4). For comparisons between two groups, the two-tailed Unpaired t-test with Welch’s correction was applied (Supplemental Table 5). These parametric tests assume that the data are normally distributed and do not have equal variances or standard deviations. In cases where the data did not meet the assumptions for parametric tests, non-parametric methods were employed. The Kruskal-Wallis test followed by Dunn's multiple comparisons test was used for multiple group comparisons (Supplemental Tables 3 and 4), while the Mann-Whitney test was used for two-group comparisons (Supplemental Table 5). These non-parametric tests do not assume a normal distribution. For multiple group comparisons, a P-value less than 0.05 was considered statistically significant and indicated by the following symbols: * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001. While for comparison between two groups, a P-value less than 0.05 was considered statistically significant, and the exact P-values were reported for these comparisons. The correlation of the levels of VHMW tau to the levels of total human tau in each mouse was plotted for each study group using exponential growth curves, and correlation analysis was performed with Spearman’s rank correlation (r). Comparisons were considered statistically significant at an α-level of P < 0.05 (Figure 3E).

Study Design

Mice were randomized using a block randomization design (Jimenez et al. 2023). Random assignment generators were used to assign animals born at the same time to different treatment arms in blocks until all the slots were filled. Mice were divided into 4 groups: a Baseline group (n = 20, harvested at 7 months of age), a Vehicle group (n = 25, harvested at 12 months of age), and two treatment groups (40 and 80 mg/kg doses, n = 25 per group, harvested at 12 months-of age). The Vehicle and Treatment groups were evenly divided into 3 cohorts. Mice treated with vehicle or drug were started in a group-balanced manner over 7 days to permit tissue collection exactly 5 months later, with a practical number of mice each day. The diets for the Vehicle and Treatment diets were coded prior to delivery to FIMR so that investigators were blind to treatment group identity. No exclusion criteria were pre-determined. There was attrition of 14 mice that was not test article related, 6 from the Vehicle group, 3 from the 40 mg/kg group, and 5 from the 80 mg/kg group. There were no drug-related side effects of this compound during the entire length of the study. Samples for biochemical analysis were obtained from the remaining mice. Data were unblinded after the analyses were completed. 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 3 staggered cohorts. The Baseline group was harvested following 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 group.