Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format

Abstract

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation

“…NFTs were also absent following overexpression of full-length 4-repeat (4R) tau (Mertens et al, 2013). Tau aggregation, as demonstrated on Western blot, can be induced with exogenous tau fibrils in hiPSCs overexpressing full-length tau; albeit, the resultant NFTs do not exhibit thioflavin staining, a confirmation of β -pleated sheet formation (Medda et al, 2016; Verheyen et al, 2015). However, overexpression of both the presenilin-1 (PS-1) familial mutations and the amyloid precursor protein (APP) have been shown to initiate hyperphosphorylation of tau as well as formation of SDS-insoluble, silver stain-positive NFTs in 3-dimensional (3-D) cultures (Choi et al, 2014).…”

Application of Human‐Induced Pluripotent Stem Cells (hiPSCs) to Study Synaptopathy of Neurodevelopmental Disorders

“…To fully mimic the natural architecture within the human brain and develop a method to generate human neurons on a large scale for clinical research, a threedimensional (3D) neuronal culture was developed from NPC or rosette aggregates (Mariani et al, 2012;Hogberg et al, 2013;Gouder et al, 2015;Hookway et al, 2016;Chandrasekaran et al, 2017;Hofrichter et al, 2017;Chen et al, 2018b) under the support of 3D scaffolds (Edgar et al, 2017;Gu et al, 2017;Hoveizi et al, 2017;Ishikawa et al, 2017;Lei and Schaffer, 2013;Leong et al, 2016;K. Li et al, 2017;Lu et al, 2012;Medda et al, 2016;Pellett et al, 2015;Uemura et al, 2011;Zhang et al, 2016). Neurons derived from 3D scaffolds, which are mostly made of hydrogel, demonstrated less retention of undifferentiated cells, lower contamination of astrocytes, higher expansion rate, enhanced neuronal differentiation efficiency, and formation of more homogeneous neural tissues containing functional migrating neurons and neuroglia (Edgar et al, 2017;Gu et al, 2017;Ishikawa et al, 2017;Lei and Schaffer, 2013;Lu et al, 2012;Medda et al, 2016;Zhang et al, 2016).…”

“…Li et al, 2017;Lu et al, 2012;Medda et al, 2016;Pellett et al, 2015;Uemura et al, 2011;Zhang et al, 2016). Neurons derived from 3D scaffolds, which are mostly made of hydrogel, demonstrated less retention of undifferentiated cells, lower contamination of astrocytes, higher expansion rate, enhanced neuronal differentiation efficiency, and formation of more homogeneous neural tissues containing functional migrating neurons and neuroglia (Edgar et al, 2017;Gu et al, 2017;Ishikawa et al, 2017;Lei and Schaffer, 2013;Lu et al, 2012;Medda et al, 2016;Zhang et al, 2016). Moreover, co-culture of iPSC-derived astrocytes and neural stem cells in 3D organoid-like spheres contains more astrocytes and synapses, suggesting the benefits of using 3D culture systems in studies of neural circuits, disease modeling, and drug screening (Krencik et al, 2017).…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…This point raises the more general issue of how closely hiPSCderived neuronal models can mimic the clinical phenotype presented in patients suffering from neurodegenerative conditions. Certainly, hiPSC-derived neurons have be used in novel high-throughput screening efforts, making them potentially useful for rapid preclinical studies [Medda et al, 2016;Brownjohn et al, 2017]. In light of these studies, it remains unclear whether inclusion of all central nervous system cell types in physiologically representative ratios and organizations is necessary to produce data that is truly predictive.…”

“…In this regard, examination of epigenetic factors may represent a plausible target as epigenetic regulation is not only a defined "hallmark of aging" [López-Otín et al, 2013] but also plays a critical role in neuronal function and cognition. Importantly, HDAC inhibitors can ameliorate a broad range of neurodegenerative phenotypes [Hommet et al, 2007;Xuan et al, 2012;Falkenberg and Johnstone, 2014] and hiPSC-derived neurons are amenable to preclinical screening paradigms [Medda et al, 2016;Brownjohn et al, 2017].…”