Alzheimer's disease (AD) is characterized by the presence of tau protein inclusions and amyloid beta (Aβ) plaques in the brain, with Aβ peptides generated by cleavage of the amyloid precursor protein (APP) by BACE1 and γ-secretase. We previously described a primary rat neuron assay in which tau inclusions form from endogenous rat tau after seeding cells with insoluble tau isolated from the human AD brain. Here, we used this assay to screen an annotated library of ∼8700 biologically active small molecules for their ability to reduce immuno-stained neuronal tau inclusions. Compounds causing ≥30% inhibition of tau aggregates with <25% loss of DAPI-positive cell nuclei underwent further confirmation testing and assessment of neurotoxicity, and non-neurotoxic hits were subsequently analyzed for inhibitory activity in an orthogonal ELISA that quantified multimeric rat tau species. Of the 173 compounds meeting all criteria, a subset of 55 inhibitors underwent concentration-response testing and 46 elicited a concentration-dependent reduction of neuronal tau inclusions that were distinct from measures of toxicity. Among the confirmed inhibitors of tau pathology were BACE1 inhibitors, several of which, along with γ-secretase inhibitors/modulators, caused a concentration-dependent lowering of neuronal tau inclusions and a reduction of insoluble tau by immunoblotting, although they did not decrease soluble phosphorylated tau species. In conclusion, we have identified a diverse set of small molecules and related targets that reduce neuronal tau inclusions. Notably, these include BACE1 and γ-secretase inhibitors, suggesting that a cleavage product from a shared substrate, such as APP, might affect tau pathology.

The hallmark pathological features of Alzheimer's disease (AD) brains are senile plaques, comprising β-amyloid (Aβ) peptides, and neuronal inclusions formed from tau protein. These plaques form 10-20 years before AD symptom onset, whereas robust tau pathology is more closely associated with symptoms and correlates with cognitive status. This temporal sequence of AD pathology development, coupled with repeated clinical failures of Aβ-directed drugs, suggests that molecules that reduce tau inclusions have therapeutic potential. Few tau-directed drugs are presently in clinical testing, in part because of the difficulty in identifying molecules that reduce tau inclusions. We describe here two cell-based assays of tau inclusion formation that we employed to screen for compounds that inhibit tau pathology: a HEK293 cell-based tau overexpression assay, and a primary rat cortical neuron assay with physiological tau expression. Screening a collection of ∼3500 pharmaceutical compounds with the HEK293 cell tau aggregation assay, we obtained only a low number of hit compounds. Moreover, these compounds generally failed to inhibit tau inclusion formation in the cortical neuron assay. We then screened the Prestwick library of mostly approved drugs in the cortical neuron assay, leading to the identification of a greater number of tau inclusion inhibitors. These included four dopamine D2 receptor antagonists, with D2 receptors having previously been suggested to regulate tau inclusions in a Caenorhabditis elegans model. These results suggest that neurons, the cells most affected by tau pathology in AD, are very suitable for screening for tau inclusion inhibitors.