High-Content Screening for Protein-Protein Interaction Modulators Using Proximity Ligation Assay in Primary Neurons.

Abstract

The proximity ligation assay (PLA) allows the detection and subcellular localization of protein-protein interactions with high specificity. We recently developed a high-content screening model based on primary hippocampal neurons cultured in 384-well plates and screened a library of ∼1100 compounds using a PLA between tau and bridging integrator 1, a genetic risk factor for Alzheimer's disease. We developed image-segmentation and spot-detection algorithms to delineate PLA signals in the axonal network, but not in cell bodies, from confocal images acquired via a high-throughput microscope. To compare data generated from different plates and through different experiments, we developed a computational routine to optimize the image analysis parameters for each plate and devised a range of quality-control measures to ultimately identify compounds that consistently increase or decrease our read-out. We provide the following protocols. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Routine culture of rat postnatal hippocampal neurons in 384-well plates Basic Protocol 2: Compound incubation using the high-content screening platform Support Protocol 1: Preparation of intermediate plates for compound screening Support Protocol 2: Preparation of intermediate plates for hit validation (dose-response curves) Basic Protocol 3: Proximity ligation assay in 384-well plates Basic Protocol 4: Image acquisition and analysis Support Protocol 3: Optimization of analysis parameters Basic Protocol 5: Identification of hits Basic Protocol 6: Validation of hits based on dose-response curves.