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RNAi screening in Drosophila cells identifies new modifiers of mutant huntingtin aggregation.

Doumanis J, Wada K, Kino Y, Moore AW, Nukina N - PLoS ONE (2009)

Bottom Line: 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.Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.

View Article: PubMed Central - PubMed

Affiliation: Lab for Structural Neuropathology, RIKEN Brain Science Institute, Wako, Saitama, Japan.

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.

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Related in: MedlinePlus

Selected screen candidates modify mutant Htt-induced toxicity in the fly eye.GMR-Gal4-driven expression of UAS-Nhtt(152Q)EGFP, UAS-Nhtt(48Q)NLS in the compound eye results in a progressive loss of pigmentation compared to expression of UAS-Nhtt(18Q)EGFP, UAS-Nhtt(18Q)NLS (A). Carrying an extra UAS transgene does not noticeably alter this phenotype (B). Female flies carrying a UAS-CG1109 RNAi transgene on the X chromosome show a clear suppression of the mutant Htt-induced toxicity phenotype, whereas no clear modification is seen in male flies lacking the UAS-CG1109 RNAi transgene (C). Expression of UAS-CG5537 RNAi on the X chromsome (D), and an autosomal UAS-CG4738 RNAi transgene (E) also suppress the mutant Htt-induced degenerative phenotype, with a rescue of pigmentation in these flies. Note: both males and females carry a copy of the for the UAS-CG5537 RNAi transgene as explained in Materials and Methods. Flies carrying a UAS-hiw RNAi transgene showed a slight rough eye phenotype that was also observed when coexpressed with normal Htt (F). Overexpression of Rheb resulted in a drastic enhancement of the Htt phenotype with an increase in black necrotic patches, further loss of pigmentation and a rough eye with bristle disorganization (G). Overexpression of Rheb in flies expressing normal Htt also resulted in a rough eye phenotype with a mild loss of pigmentation. The chaperone molecule, dhdJ1 clearly suppressed Htt-induced loss of pigmentation when overexpressed (H). All flies were aged between 21 and 22 days.
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pone-0007275-g004: Selected screen candidates modify mutant Htt-induced toxicity in the fly eye.GMR-Gal4-driven expression of UAS-Nhtt(152Q)EGFP, UAS-Nhtt(48Q)NLS in the compound eye results in a progressive loss of pigmentation compared to expression of UAS-Nhtt(18Q)EGFP, UAS-Nhtt(18Q)NLS (A). Carrying an extra UAS transgene does not noticeably alter this phenotype (B). Female flies carrying a UAS-CG1109 RNAi transgene on the X chromosome show a clear suppression of the mutant Htt-induced toxicity phenotype, whereas no clear modification is seen in male flies lacking the UAS-CG1109 RNAi transgene (C). Expression of UAS-CG5537 RNAi on the X chromsome (D), and an autosomal UAS-CG4738 RNAi transgene (E) also suppress the mutant Htt-induced degenerative phenotype, with a rescue of pigmentation in these flies. Note: both males and females carry a copy of the for the UAS-CG5537 RNAi transgene as explained in Materials and Methods. Flies carrying a UAS-hiw RNAi transgene showed a slight rough eye phenotype that was also observed when coexpressed with normal Htt (F). Overexpression of Rheb resulted in a drastic enhancement of the Htt phenotype with an increase in black necrotic patches, further loss of pigmentation and a rough eye with bristle disorganization (G). Overexpression of Rheb in flies expressing normal Htt also resulted in a rough eye phenotype with a mild loss of pigmentation. The chaperone molecule, dhdJ1 clearly suppressed Htt-induced loss of pigmentation when overexpressed (H). All flies were aged between 21 and 22 days.

Mentions: In order to follow up interesting candidates in vivo, we established transgenic flies for expression of EGFP-tagged Nhtt with either a normal (18Q) or expanded (152Q) polyglutamine tract and either lacking or containing a nuclear localization signal (NLS). We established UAS-NhttEGFP lines for driving transgene expression in various cell types. We have found that heterozygous expression of non-nuclear mutant Htt using the GMR-Gal4 driver does not produce a strong external eye phenotype, compared to a moderate loss of pigmentation phenotype caused by NLS-containing mutant Htt (data not shown). For genetic modification of Htt-induced cellular toxicity, we established a Drosophila line that expresses mutant Htt in both the cytoplasm and nucleus of all neurons and other cell types of the developing and adult compound eye using the GMR-Gal4 driver. We confirmed the presence of Nhtt(48Q)EGFPNLS inclusions in the nucleus and Nhtt(152Q)EGFP inclusions in the cytosol by confocal microscopic imaging of 3rd instar larval eye discs (Figure S6). These flies produce a degenerative eye phenotype characterized by loss of pigment cells and an increased occurrence of dark necrotic spots on the external eye while flies expressing Nhtt(18Q)EGFP/Nhtt(18Q)EGFPNLS have a normal eye phenotype at the same age (Figure 4A). There was no noticeable alteration of this phenotype in flies carrying an extra UAS insertion (Figure 4B).


RNAi screening in Drosophila cells identifies new modifiers of mutant huntingtin aggregation.

Doumanis J, Wada K, Kino Y, Moore AW, Nukina N - PLoS ONE (2009)

Selected screen candidates modify mutant Htt-induced toxicity in the fly eye.GMR-Gal4-driven expression of UAS-Nhtt(152Q)EGFP, UAS-Nhtt(48Q)NLS in the compound eye results in a progressive loss of pigmentation compared to expression of UAS-Nhtt(18Q)EGFP, UAS-Nhtt(18Q)NLS (A). Carrying an extra UAS transgene does not noticeably alter this phenotype (B). Female flies carrying a UAS-CG1109 RNAi transgene on the X chromosome show a clear suppression of the mutant Htt-induced toxicity phenotype, whereas no clear modification is seen in male flies lacking the UAS-CG1109 RNAi transgene (C). Expression of UAS-CG5537 RNAi on the X chromsome (D), and an autosomal UAS-CG4738 RNAi transgene (E) also suppress the mutant Htt-induced degenerative phenotype, with a rescue of pigmentation in these flies. Note: both males and females carry a copy of the for the UAS-CG5537 RNAi transgene as explained in Materials and Methods. Flies carrying a UAS-hiw RNAi transgene showed a slight rough eye phenotype that was also observed when coexpressed with normal Htt (F). Overexpression of Rheb resulted in a drastic enhancement of the Htt phenotype with an increase in black necrotic patches, further loss of pigmentation and a rough eye with bristle disorganization (G). Overexpression of Rheb in flies expressing normal Htt also resulted in a rough eye phenotype with a mild loss of pigmentation. The chaperone molecule, dhdJ1 clearly suppressed Htt-induced loss of pigmentation when overexpressed (H). All flies were aged between 21 and 22 days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2748703&req=5

pone-0007275-g004: Selected screen candidates modify mutant Htt-induced toxicity in the fly eye.GMR-Gal4-driven expression of UAS-Nhtt(152Q)EGFP, UAS-Nhtt(48Q)NLS in the compound eye results in a progressive loss of pigmentation compared to expression of UAS-Nhtt(18Q)EGFP, UAS-Nhtt(18Q)NLS (A). Carrying an extra UAS transgene does not noticeably alter this phenotype (B). Female flies carrying a UAS-CG1109 RNAi transgene on the X chromosome show a clear suppression of the mutant Htt-induced toxicity phenotype, whereas no clear modification is seen in male flies lacking the UAS-CG1109 RNAi transgene (C). Expression of UAS-CG5537 RNAi on the X chromsome (D), and an autosomal UAS-CG4738 RNAi transgene (E) also suppress the mutant Htt-induced degenerative phenotype, with a rescue of pigmentation in these flies. Note: both males and females carry a copy of the for the UAS-CG5537 RNAi transgene as explained in Materials and Methods. Flies carrying a UAS-hiw RNAi transgene showed a slight rough eye phenotype that was also observed when coexpressed with normal Htt (F). Overexpression of Rheb resulted in a drastic enhancement of the Htt phenotype with an increase in black necrotic patches, further loss of pigmentation and a rough eye with bristle disorganization (G). Overexpression of Rheb in flies expressing normal Htt also resulted in a rough eye phenotype with a mild loss of pigmentation. The chaperone molecule, dhdJ1 clearly suppressed Htt-induced loss of pigmentation when overexpressed (H). All flies were aged between 21 and 22 days.
Mentions: In order to follow up interesting candidates in vivo, we established transgenic flies for expression of EGFP-tagged Nhtt with either a normal (18Q) or expanded (152Q) polyglutamine tract and either lacking or containing a nuclear localization signal (NLS). We established UAS-NhttEGFP lines for driving transgene expression in various cell types. We have found that heterozygous expression of non-nuclear mutant Htt using the GMR-Gal4 driver does not produce a strong external eye phenotype, compared to a moderate loss of pigmentation phenotype caused by NLS-containing mutant Htt (data not shown). For genetic modification of Htt-induced cellular toxicity, we established a Drosophila line that expresses mutant Htt in both the cytoplasm and nucleus of all neurons and other cell types of the developing and adult compound eye using the GMR-Gal4 driver. We confirmed the presence of Nhtt(48Q)EGFPNLS inclusions in the nucleus and Nhtt(152Q)EGFP inclusions in the cytosol by confocal microscopic imaging of 3rd instar larval eye discs (Figure S6). These flies produce a degenerative eye phenotype characterized by loss of pigment cells and an increased occurrence of dark necrotic spots on the external eye while flies expressing Nhtt(18Q)EGFP/Nhtt(18Q)EGFPNLS have a normal eye phenotype at the same age (Figure 4A). There was no noticeable alteration of this phenotype in flies carrying an extra UAS insertion (Figure 4B).

Bottom Line: 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.Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.

View Article: PubMed Central - PubMed

Affiliation: Lab for Structural Neuropathology, RIKEN Brain Science Institute, Wako, Saitama, Japan.

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.

Show MeSH
Related in: MedlinePlus