Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1483-3) contains supplementary material, which is available to authorized users.

German Center for Neurodegenerative Diseases (DZNE), Ludwig-Erhard-Allee, 53175 Bonn, Germany Anle138b effectively crosses the blood-brain-barrier. a Experimental scheme for pharmacokinetic analysis of compound brain level after anle138b treatment. b Brain compound level in ad libitum fed mice on day 4 at 12h, 14h, 16h and 20h (n = 3 mice for each time point). The number of AT8-positive neurons was significantly reduced in the hippocampus and the auditory/somatosensory cortex (n = 10-11 mice/group; 10-12 months).
Anle138b treatment reduces tau pathology in the brain stem of PS19 mice. a Gallyas silver staining showed tangle tau pathology in the brain stem of PS19 mice.
Kinase and Phosphatase activity is unaffected after anle138b treatment.
a Phosphorylation of GSK3 is slightly but not significantly elevated in anle138btreated primary neurons (n = 5; p>0.05, t-test). b PP2A expression is unchanged after anle138b-treatment in primary neurons. c Analysis of PP2A activity in primary neurons revealed that the activity is unaltered in anle138b-treated neurons compared with the vehicle-treated group (n = 3; p > 0.05, t-test).
Autophagy and tau degradation is unaffected after anle138b treatment. Effect of anle138b treatment on mouse weight of PS19 mice. Treatment with anle138b significantly ameliorated weight loss of PS19 mice. The mouse weight was normalized to the peak of body weight. The peak of body weight was defined as the onset of disease. (**; n=43-45 mice/group; p < 0.01; F test).

Pharmacokinetic analysis of brain concentrations of anle138b
Mice (B6SJLF1, Jackson Laboratory) were kept in groups of 4 animals in each cage in an inversed day/night-cycle for 2 weeks before starting and during the experiment (light 18:00 to 6:00, dark 6:00 to 18:00) with free access to food and water. Age for UV absorption at 260 nm. Samples were quantified using peak area ratio of compounds to external standard.

Primary cultures
Primary cortical neurons were isolated from NMRI mouse embryos at day E14.5.
Cortices were collected in DMEM (Invitrogen) and cells were dissociated by incubation with Trypsin/EDTA for 6.5 min at 37 °C. Cells were then diluted in neurobasal medium containing B27 supplement (Gibco / Life Technologies). Cells were plated at a density of 8.0 x 10 5 onto 0.2 mg/ml poly-D-lysin and 2 µg/ml Laminin (Sigma) coated 6 well plates and incubated at 37 °C with 5 % CO 2 . 30 min after seeding a change of medium was performed.

Western blot
Cell

PP2A activity assay
PP2A-holoenzymes, which were purified by immunoprecipitation using a PP2A-Asubunit antibody (Millipore), were incubated with or without 100 µM anle138b or 40 nM okadaic acid for 1 hour. Afterwards PP2A phosphatase activity was determined using a malachite green assay kit (PP2A Immunoprecipitation Phosphatase Assay Kit, Millipore) following the manufacturer's instructions.

Immunohistochemistry
Mice were transcardially perfused with cold PBS after being deeply anesthetized. The brains were removed and weighed. From all animals one brain hemisphere was fixed in 4 % paraformaldehyde for 24 h for histological examination. After fixation the brain tissue was sectioned into 50 µm tissue slices using a Leica VT1000S vibratome (Leica, Wetzlar, Germany). Immunohistochemistry was performed as described by

CHX timecourse
To investigate the effect of anle138b on protein stability, time course experiments with the translation inhibitor cycloheximide (CHX) were performed. Primary cortical neurons were treated with or without 10 µM anle138b in the presence or absence of CHX, f.c. 40 µM. Cells were lysed after 24h, 48h, or 72h of CHX-treatment and the degradation of Tau protein over time was analyzed on a western blot.

Immunoprecipitation
To investigate the effect of anle138b on tau ubiquitination, we immunopurified ubiquitinated proteins and detected ubiquitinated tau protein on a western blot.
Therefore, primary cortical neurons were treated with or without 10 µM anle138b for 24h, harvested and lysed in a buffer containing 50 mM Tris, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 1 % NP40, and complete protease inhibitors (Roche) using the precellys cell homoginator. Immunoprecipitation was carried out using anti-ubiquitin antibodies (Santa Cruz) in combination with protein G agarose beads (Roche) according to the manufacturer's instructions. Ubiquitinated Tau protein was detected on western blots using Tau-antibodies.

Animals and treatment
PS19 human tau transgenic mice [2] were obtained from The Jackson Laboratories conditions. Age and sex matched non-transgenic littermates were used for all experiments and equal numbers of male and female mice were randomly assigned to the experimental groups. Mice had unlimited access to food and water, the light and dark cycle was 12h/12h and the temperature was kept constant at 22°C. The compound anle138b was administered with drug-supplemented food pellets (2 g compound/kg food; Ssniff, Soest, Germany). The compound was added during the manufacturing process of the food pellets as dry powder without a vehicle. Therefore, control mice were treated with non-supplemented food pellets. The treatment was initiated after weaning and lasted until the time of sacrifice. Trained animal caretakers blinded to the study design assessed body weight monthly. The mouse weight was normalized to the peak of body weight development. The peak of body weight was defined as the onset of disease. The experimental procedures were in accordance with guidelines established by the DZNE and were approved by the government of North-Rhine-Westphalia.