Abstract

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.

Highlights

  • Huntington’s Disease (HD) is a dominantly inherited fatal neurodegenerative disorder

  • The severity of the Htt138Q1 allele suggests that this model may correspond to juvenile-onset HD observed in humans [20]

  • We have used Drosophila primary neural cultures isolated from an HD model to screen for RNAi and small molecule suppressors of expanded polyQ Htt-induced toxicity

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Summary

Introduction

Huntington’s Disease (HD) is a dominantly inherited fatal neurodegenerative disorder. It results from expansion of a polygluatamine (polyQ) tract in the Huntingtin (Htt) protein which alters its conformation and function [1]. Linkage analysis using large affected families is the gold standard for identifying disease modifying loci [10]. These investigations require lengthy study periods and considerable resources. Primary neuronal disease models have many advantages, since cellular growth and differentiation states are more similar to the in vivo situation, yet they are amenable to high-throughput chemical and genetic suppressor screens

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