DOI: 10.5061/dryad.1g1jwsv75
title: Data from: Therapeutic treatment with OLX-07010 inhibited tau aggregation and ameliorated motor deficits in an aged mouse model of tauopathy

description: 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.
description: <strong>Animals</strong> Female
 homozygous P301L tau JNPL3 mice<em> </em>(Lewis<em> et
 al.</em> 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<em> et al.</em> 2017; Koppel<em> et
 al.</em> 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<em> et al.</em>, 2023; Davidowitz<em> et
 al.</em>, 2020). Ideally, highly powered studies
 including males and females should be performed to evaluate translational
 potential (Dennison<em> et al.</em> 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<em> et al.</em> 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 <em>ad libitum</em> 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). <strong>Mouse
 Diets</strong> 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 &gt;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). <strong>Bioanalytical
 Analysis </strong> 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.  <strong>Motor
 Behavior</strong> Motor function was assayed with
 the MazeEngineers 6-Lane rotarod for mice (Deacon 2013; Koppel<em>
 et al.</em> 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. <strong>Brain Tissue
 Preparations</strong> Procedures for preparation
 of samples and performance of ELISAs are detailed in the publications
 (Acker<em> et al.</em> 2013; Davidowitz<em> et
 al.</em> 2020; Forest<em> et al.</em> 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 <sup>o</sup>C, and stored at -80
 <sup>o</sup>C. 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 <sup>o</sup>C 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.
 <strong>ELISAs and Antibodies</strong>
 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<em> et al.</em> 2023;
 Forest<em> et al.</em> 2013).
 <strong>Immunoblots</strong>
 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.
 <strong>Statistical Analyses</strong>
 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
 &lt; 0.05, ** for P &lt; 0.01, *** for P &lt; 0.001, and ****
 for P &lt; 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
 &lt; 0.05 (Figure 3E). <strong>Study
 Design</strong> Mice were randomized using a
 block randomization design (Jimenez<em> et al.</em> 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.
description: # Data from: Therapeutic treatment with OLX-07010 inhibited tau
 aggregation and ameliorated motor deficits in an aged mouse model of
 tauopathy
 [https://doi.org/10.5061/dryad.1g1jwsv75](https://doi.org/10.5061/dryad.1g1jwsv75) ## Description of the data and file structure The data was collected to evaluate the effect of therapeutic treatment using a small molecule OLX-07010 on an aged mouse model of tauopathy. ### Files and variables #### File: Davidowitz_et_al._2025_Data_for_DRYAD_for_JNC_revised_07-22-2025.xlsx **Description:** There were 4 Groups of mice in this study. A Baseline (BL) group, sacrificed at 7 months of age; A Vehicle (Veh) group and treatment groups dosed with 40 or 80 OLX-07010 mg/kg/day. Values for the paramaters are presented in columns in an Excel spreadsheet. The in-life treatment and behavioral analyses were completed by end of 2020 and ELISA analyses were completed in 2021.  **Figure 2**: Quantitative ELISAs for A. Sarkosyl insoluble tau, B. heat stable total tau, and C. self-associated tau were performed in the laboratory of Peter Davies, Ph.D., Director, The Litwin-Zucker Research Center for the Study of Alzheimer's Disease, The Feinstein Institutes for Medical Research, Northwell Health, Manhasset, New York, according to the published methods established in his laboratory.  The data is presented as percent of the Vehicle group for ease of interpretation. **Figure 3**: Immunoblot analysis of tau species using monoclonal Aantibody HT7 were subsequently performed at the laboratories of Oligomerix to understand the effect of treatment on the range of the human P301L tau aggregate species relative to the total expression of the construct in each mouse. **Figure 4**: Evaluation of motor coordination by Rotarod was performed by Jeremy Koppel, MD, PhD using a protocol he developed in Dr. Davies's laboratory. **Figure 5**: Serum levels of OLX-07010 were determined by a CRO using LC-MS/MS methods they developed and qualified. The serum levels of tau protein were determined by ELISA in the laboratory of Dr. Davies. ## Code/software Excel software (Microsoft) can be used to view the file