Abstract
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.
Highlights
The hallmark pathological features of Alzheimer’s disease (AD) brains are senile plaques, comprising -amyloid (A) peptides, and neuronal inclusions formed from tau protein
Our laboratories previously demonstrated [32] that intracellular tau inclusions can be promoted by the cytosolic delivery of pathological seeds composed of pre-formed synthetic fibrils assembled from recombinant tau into HEK293 cells that express the longest form of human recombinant tau containing the P301L mutation found in inherited frontotemporal lobar degenerative (FTLD) [40]
Insights into the possible reasons for these clinical failures have been provided by advances in our understanding of brain changes that occur as AD progresses from prodromal to symptomatic stages
Summary
Our laboratories previously demonstrated [32] that intracellular tau inclusions can be promoted by the cytosolic delivery of pathological seeds composed of pre-formed synthetic fibrils assembled from recombinant tau (tau pre-formed fibrils or PFFs) into HEK293 cells that express the longest form of human recombinant tau containing the P301L mutation found in inherited FTLD (referred to as T40PL) [40]. Neuron cultures were treated with AD-tau and 10 M compound in triplicate as above, but underwent the milder Triton X-100 and PFA permeabilization/fixation protocol to allow visualization of NeuN-positive nuclei and MAP2-positive dendritic processes This additional analysis revealed that a number of the hits affected dendritic outgrowth and/or reduced neuron counts by Ͼ2 S.D., thereby resulting in likely false-positive reductions in tau inclusions. Both alizapride and metoclopramide have reported D2 receptor affinities of ϳ300 nM [46], which is roughly the IC50 values obtained with these compounds in the neuronal tau assay We assessed whether these D2 receptor antagonists act to reduce overall tau protein expression by measuring the amount of soluble tau by ELISA in compound-treated rat cortical neuron cultures, and these compounds did not generally affect soluble tau, bromopride and azaperone caused modest decreases with the latter reaching statistical significance (Fig. S4). The screening of larger compound libraries in this assay may lead to the identification of additional small molecules and associated molecular targets that regulate the formation or clearance of tau aggregates
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