Abstract
The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.
Highlights
Huntington Disease (HD) is a late-onset, autosomal dominant neurodegenerative disorder characterized at the genetic level by expansion of a CAG repeat in the huntingtin gene
Our candidates fell into several functional groups, the most notable being transport molecules, including those involved in nuclear transport, and nucleotide processing including RNA metabolism
Nuclear transport has been reported to be important in mutant Htt pathogenesis, with Htt itself reportedly shuttling between the nucleus and the cytoplasm [10,13]
Summary
Huntington Disease (HD) is a late-onset, autosomal dominant neurodegenerative disorder characterized at the genetic level by expansion of a CAG repeat in the huntingtin (htt) gene. Expansion of the CAG repeat in exon 1 of the htt gene to greater than 35 repeats results in a disease-causing expanded polyglutamine tract in the Htt protein. CAG repeat diseases, caused by a toxic GOF, can be modeled in the fly by expression of the mutant protein. Drosophila models for SCA 1, SCA 3, MJD, SMBA and DRPLA have been successfully established and mimic various aspects of this group of diseases such as the presence of mutant protein aggregates, progressive neurodegeneration and behavioural abnormalities [2,3,4,5]
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