High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy

Abstract

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

Evaluation of a Selective Chemical Probe Validates That CK2 Mediates Neuroinflammation in a Human Induced Pluripotent Stem Cell-Derived Microglial Model

“…Human induced pluripotent stem cell models have been increasingly utilized to study AD because brain-relevant cell types can be studied in vitro and used to profile molecules that may modify these disease phenotypes. Indeed, hiPSC-derived cells have been employed in several screens of small molecules hypothesized to impact AD processes, however, the majority of these experiments have been performed in neuronal cells and largely focused on the effects of compounds on Aβ and tau ( Brownjohn et al, 2017 ; Kondo et al, 2017 ; van der Kant et al, 2019 ; Cheng et al, 2021 ).…”

Human Induced Pluripotent Stem Cell Models of Frontotemporal Dementia With Tau Pathology

“…Many of these disease phenotypes were reversable providing elegant platforms for high-throughput drug screening for compounds in pre-clinical settings to be able to potentially interfere at an early stage of the disease development process. In fact, human iPSC-derived drug screening platforms have been established ( Wang et al, 2017 ; Cheng et al, 2021 ) and large numbers of patient-derived neural cells can readily be derived, cultured and tested in such dynamic cellular assays in vitro , in contrast to alternative approaches involving genetic mouse models or postmortem brain material from patients. Such drug screening assays may also involve the co-culture of patient iPSC-derived neurons with patient iPSC-derived glial cells such as astrocytes to test glia- or even neuron-mediated non-cell autonomous mechanism of disease development in neurodegenerative diseases, such as FTLD-tau.…”

“…The same group later applied a high-content imaging assay on A152T and control cortical neurons to screen for kinases and small molecules to reduce tau and p-tau expression as well as their somatodendritic redistribution ( Cheng et al, 2021 ). This study revealed that the GSK-3 inhibitor CHIR-99021 and the kinase inhibitors enzastaurin and ruboxistaurin reduced p-tau in cell processes and cell body in A152T and control neurons and that the small molecule kinase inhibitors AT7519 and CGP-60474, as part of a collection of 44 kinase inhibitors, had similar effects in a screen on A152T neurons.…”

“…This study revealed that the GSK-3 inhibitor CHIR-99021 and the kinase inhibitors enzastaurin and ruboxistaurin reduced p-tau in cell processes and cell body in A152T and control neurons and that the small molecule kinase inhibitors AT7519 and CGP-60474, as part of a collection of 44 kinase inhibitors, had similar effects in a screen on A152T neurons. Furthermore, the application of CHIR-99021 and enzastaurin resulted not only in reduced phosphorylation of tau but also in a decrease of total tau in these cells suggesting clearance of tau by these compounds and a potentially beneficial function of kinase inhibitors in patient neurons ( Cheng et al, 2021 ).…”

Proteasomal stimulation by MK886 and its derivatives can rescue tau-induced neurite pathology

“…Inhibiting their formation or enhancing their clearance have been the target of multiple therapeutic discovery campaigns for AD [7][8][9]. Previously, our group identi ed MK886 (MK) as a lead compound capable of modulating tau misfolding and oligomerization in a cellular uorescence-lifetime Förster Resonance Energy Transfer (FLT-FRET) high-throughput screen [10].…”