High Content Screening in Neurodegenerative Diseases

Abstract

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency [1][2][3][4] . Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation 5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known 6,7 . As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long.There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity 8 . This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.High-throughput screening (HTS) is an ideal methodology to address these issues [9][10][11][12] . but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties 13,14 . This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS) 4,[15][16][17] . HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. HCS has many advantages over HTS 18,19 , but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility.Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing 20 and H...

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

“…Cell-based “phenotypic” high-throughput screening (HTS) is a powerful unbiased tool to identify gene targets or small-molecule compounds exerting desired effects. However, HTS requires large numbers of cells and has been largely restricted to immortalized human neuronal lines, such as neuroblastoma SH-SY5Y( Jain et al., 2012 ) and glioma H4 ( Albrecht et al., 2004 ) cells, or non-neuronal lines, such as HeLa cells ( Fatokun et al., 2013 ). Since these cells differ physiologically from post-mitotic neurons, hits identified in these cells might not work in neurons.…”

Impact of RNA-Guided Technologies for Target Identification and Deconvolution

“…In a study using SH-SY5Y neuronal cells, lentiviral shRNA was used to infect dividing cells that were subsequently differentiated, with multiple assays applied to the resulting neurons, including neurite-outgrowth and mitochondrial function. 50 Pooled shRNA screening has been increasingly popular in recent years, driven in part by not requiring the expensive and bulky automation needed for genome-scale arrayed screens. 51 Pooled screens also have a distinct advantage over arrayed screens when studying genes affecting cell proliferation over multiple doublings (and lasting perhaps weeks in length) due to space constraints imposed for cell growth by well geometry and the difficulty in refeeding cells in the plates.…”

“…In a study using SH-SY5Y neuronal cells, lentiviral shRNA was used to infect dividing cells that were subsequently differentiated, with multiple assays applied to the resulting neurons, including neurite-outgrowth and mitochondrial function. 50…”

Neurological Diseases from a Systems Medicine Point of View

“…Currently, the size-scope tradeoff is inevitable. However, as the efforts towards high-quality network curation gain community-scale attention [ 47 , 179 ], and new high-content screening approaches are proposed for network construction [ 180 ], we may expect that disease-specifi c networks will grow in size without sacrifi cing their quality.…”