A Genomewide RNA Interference Screen for Modifiers of Aggregates Formation by Mutant Huntingtin in Drosophila

Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Genetics (Impact Factor: 5.96). 04/2010; 184(4):1165-79. DOI: 10.1534/genetics.109.112516
Source: PubMed Central


Protein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington's disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.

Download full-text


Available from: Richard Binari, Aug 14, 2014
  • Source
    • "Equally, the finding that QPCT inhibition could increase ␣␤-crystallin chaperone levels HEK293 cells and Drosophila (Jimenez-Sanchez et al., 2015), warrants further investigation in mice. Other candidates from screens that have yet to be tested in mammalian systems are TCF, a protein involved in Wnt signaling, rpi, a metabolic enzyme, TDO, a member of the kynurenine pathway, Tra1, a component of the HAT complex, and DJ-1␣, an oxidation-sensitive chaperone protein (Campesan et al., 2011; Dupont et al., 2012; Sajjad et al., 2014; Wang et al., 2012; Zhang et al., 2010). Considering the successful translation of many other candidates discovered or validated in Drosophila models of HD (Table 2), these remain prime candidates for evaluation in mammalian systems. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Huntingtin (Htt) protein is essential for a wealth of intracellular signalling cascades and when mutated, causes multifactorial dysregulation of basic cellular processes. Understanding the contribution to each of these intracellular pathways is essential for the elucidation of mechanisms that drive pathophysiology. Using appropriate models of Huntington's disease (HD) is key to finding the molecular mechanisms that contribute to neurodegeneration. While mouse models and cell lines expressing mutant Htt have been instrumental to HD research, there has been a significant contribution to our understating of the disease from studies utilizing Drosophila melanogaster. Flies have a Htt protein, so the endogenous pathways with which it interacts are likely conserved. Transgenic flies engineered to overexpress the human mutant HTT gene display protein aggregation, neurodegeneration, behavioural deficits and a reduced lifespan. The short life span of flies, low cost of maintaining stocks and genetic tools available for in vivo manipulation make them ideal for the discovery of new genes that are involved in HD pathology. It is possible to do rapid genome wide screens for enhancers or suppressors of the mutant Htt-mediated phenotype, expressed in specific tissues or neuronal subtypes. However, there likely remain many yet unknown genes that modify disease progression, which could be found through additional screening approaches using the fly. Importantly, there have been instances where genes discovered in Drosophila have been translated to HD mouse models. Copyright © 2015. Published by Elsevier B.V.
    Journal of Neuroscience Methods 08/2015; DOI:10.1016/j.jneumeth.2015.07.026 · 2.05 Impact Factor
  • Source
    • "tionally flexible genetics, has been employed consistently and successfully to study the process of neurodegeneration and to identify genes that enhance or suppress degeneration from various toxic proteins (Jaiswal et al., 2012). By using the fruit fly, multiple groups have modeled human diseases and have identified key players in the pathogenesis of Alzheimer's and Parkinson's diseases, as well as polyQ-dependent disorders (Jackson et al., 1998; Warrick et al., 1998, 2005; Fernandez-Funez et al., 2000; Taylor et al., 2003; Pandey et al., 2007; Romero et al., 2008; Zhang et al., 2010; Casas-Tinto et al., 2011; Whitworth, 2011; Rincon-Limas et al., 2012; Choksi et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Age-related neurodegeneration has been studied extensively through the use of model organisms, including the genetically versatile Drosophila melanogaster. Various neurotoxic proteins have been expressed in fly eyes to approximate degeneration occurring in humans, and much has been learned from this heterologous system. Although Drosophila expedites scientific research through rapid generational times and relative inexpensiveness, one factor that can hinder analyses is the examination of milder forms of degeneration caused by some toxic proteins in fly eyes. Whereas several disease proteins cause massive degeneration that is easily observed by examining the external structure of the fly eye, others cause mild degeneration that is difficult to observe externally and requires laborious histological preparation to assess and monitor. Here, we describe a sensitive fluorescence-based method to observe, monitor, and quantify mild Drosophila eye degeneration caused by various proteins, including the polyglutamine disease proteins ataxin-3 (spinocerebellar ataxia type 3) and huntingtin (Huntington's disease), mutant α-synuclein (Parkinson's disease), and Aβ42 (Alzheimer's disease). We show that membrane-targeted green fluorescent protein reports degeneration robustly and quantitatively. This simple yet powerful technique, which is amenable to large-scale screens, can help accelerate studies to understand age-related degeneration and to find factors that suppress it for therapeutic purposes. © 2014 Wiley Periodicals, Inc.
    Journal of Neuroscience Research 09/2014; 92(9). DOI:10.1002/jnr.23395 · 2.59 Impact Factor
  • Source
    • "The majority of functional genomic applications in human cells rely upon RNAi loss-of-function screens (reviewed by Mullenders and Bernards, 2009). RNAi in Drosophila cell lines has been previously utilized to study cellular toxicity—as examples, Eggert et al. (2004) identified small molecules inhibiting the cell cycle, while Zhang et al. (2010) discovered genes that increased the aggregation of mutant Huntingtin proteins. Barcoded short hairpin RNA (shRNA—a method to accomplish RNAi) libraries enable identification of shRNAs that elicit a specific phenotype under toxicant selection. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The increased presence of chemical contaminants in the environment is an undeniable concern to human health and ecosystems. Historically, by relying heavily upon costly and laborious animal-based toxicity assays, the field of toxicology has often neglected examinations of the cellular and molecular mechanisms of toxicity for the majority of compounds-information that, if available, would strengthen risk assessment analyses. Functional toxicology, where cells or organisms with gene deletions or depleted proteins are used to assess genetic requirements for chemical tolerance, can advance the field of toxicity testing by contributing data regarding chemical mechanisms of toxicity. Functional toxicology can be accomplished using available genetic tools in yeasts, other fungi and bacteria, and eukaryotes of increased complexity, including zebrafish, fruit flies, rodents, and human cell lines. Underscored is the value of using less complex systems such as yeasts to direct further studies in more complex systems such as human cell lines. Functional techniques can yield (1) novel insights into chemical toxicity; (2) pathways and mechanisms deserving of further study; and (3) candidate human toxicant susceptibility or resistance genes.
    Frontiers in Genetics 05/2014; 5:110. DOI:10.3389/fgene.2014.00110
Show more