A high throughput RNAi screen to identify modifiers of ALS8 aggregation using automated computational analysis

Abstract

Protein aggregation is a hallmark feature of human neurodegenerative diseases (NDs). In familial NDs the affected genetic locus is perturbed by point/missense mutation that codes for a protein which misfolds and forms intracellular inclusions. Large-scale whole genome RNAi screens using cells in culture are useful to identify proteins/genes that may modulate (enhance or suppress) the formation of cellular inclusions in disease conditions. These modulators could help us understand relationship between aggregation and neuronal cell death. For my thesis work, I present the standardization and validation of a Drosophila S2 cell based high-throughput screen for identifying genes that can modulate the aggregation of the ALS8 locus VAP. We have established stable S2 cells expressing inducible VAPBP58S:GFP and VAP:GFP fusions that can be monitored for aggregate formation over time, by epifluorescence microscopy. Using high-throughput gene knockdown and automated imaging, we have collected data for the effect of knockdown of 1200 independent genes on VAPBP58S:GFP aggregation. The imaged data has been analyzed using a custom MATLAB program and a list of modifiers has been generated. One key result is that modifiers include other ALS loci such as ALS1/SOD1, ALS2/Alsin and ALS10/TDP43. As a final step of my study, I have characterized and validated the interaction of ALS1/SOD1 with VAPBP58S. My investigations suggest that different ALS loci can interact with VAPBP58S and their levels can affect the propensity / degree of protein aggregation. This finding has major implications as it may lead to a better mechanistic understanding of onset and progression of motor neuron disease.