Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening

Abstract

SummaryLowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High-throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) is a powerful tool to identify tau-targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost and low yield, and multi-step differentiation procedures. We engineered an isogenic iPSC line that harbors an inducible neurogenin 2 transgene, a transcription factor that rapidly converts iPSCs to neurons, integrated at the AAVS1 locus. Using a simplified two-step protocol, we differentiated these iPSCs into cortical glutamatergic neurons with minimal well-to-well variability. We developed a robust high-content screening assay to identify tau-lowering compounds in LOPAC and identified adrenergic receptors agonists as a class of compounds that reduce endogenous human tau. These techniques enable the use of human neurons for high-throughput screening of drugs to treat neurodegenerative disease.

CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

“…Differentiation protocols based on induced expression of transcription factors are particularly useful for screens, as they are rapid and yield large numbers of homogeneous neurons. In addition to the Ngn2-driven generation of glutamatergic neurons (Fernandopulle et al, 2018;Wang et al, 2017;Zhang et al, 2013) used here, induced expression of different transcription factors yield other types of neurons, such as motor neurons (Hester et al, 2011;Shi et al, 2018) and inhibitory neurons (Yang et al, 2017). Systematic screens are beginning to uncover additional combinations of transcription factors driving specific neuronal fates (Liu et al, 2018;Tsunemoto et al, 2018).…”

“…We integrated CRISPRi technology with our previously described i 3 Neuron platform (Fernandopulle et al, 2018;Wang et al, 2017), which yields large quantities of highly homogeneous neurons, a prerequisite for robust population-based screens. We decided to use CRISPRi rather than CRISPRn, since CRISPRn-associated DNA damage is highly toxic to iPSCs and untransformed cells (Haapaniemi et al, 2018;Ihry et al, 2018;Schiroli et al, 2019).…”

“…We built on our previously described i 3 Neuron (i 3 N) platform, which enables large-scale production of iPSC-derived glutamatergic neurons. Central to this platform is an iPSC line with an inducible Neurogenin 2 (Ngn2) expression cassette in the AAVS1 safe-harbor locus (Fernandopulle et al, 2018;Wang et al, 2017). To enable stable CRISPRi in iPSC-derived neurons, we generated a plasmid (pC13N-dCas9-BFP-KRAB) to insert an expression cassette for CAG promoter-driven dCas9-BFP-KRAB into the CLYBL safe harbor locus, which enables robust transgene expression throughout neuronal differentiation at higher levels than the AAVS1 locus (Cerbini et al, 2015) ( Fig.…”

The Role of Tau Protein in Diseases

“…The brain can synthesize non-essential fatty acids, but essential PUFA (e.g., arachidonic acid [AA] and docosahexaenoic acid [DHA]) are largely acquired from the peripheral circulation [88]. Arachidonic acid metabolism produces pro-in lammatory lipid metabolites, such as prostaglandins and leukotrienes, whereas metabolism of DHA generates anti-in lammatory mediators, such as resolvins.…”

Concepts toward directing human astroplasticity to promote neuroregeneration

“…The generation of stable transgenic cell lines permits robust production of pure cell types for high‐throughput screening (HTS) of therapeutic candidates as an improvement over immortalized cell lines or sources that are difficult to obtain. For instance, tau‐lowering compounds were screened with induced neurons (iNeurons) from a hPSC line that was generated by inserting an inducible ngn2 cassette into AAVS1 (Wang et al, ). Creating iAstrocytes in a similar fashion, could allow for HTS to discover small molecules that alter reactivity states during various genetic disease paradigms or in response to exogenous insults, such as Zika virus or misfolded prion infection (Retallack et al, ; Krejciova et al, ).…”