Lejeune et al. BMC Genomics 2012, 13:91 http://www.biomedcentral.com/1471-2164/13/91 signals, considered as noise, in the context of gene con- nectivity. The logic behind this procedure is that highly inter-connected genes may be implicated in the same pathway and may fluctuate in the same direction [32]. Starting from the raw RNAi screen data, we iteratively removed the high-frequency components 2.5% at a time, resulting in 40 increasingly smoothed versions of the data. Given a smoothed gene activity data, we consid- ered modules as groups of connected genes with a signal > mean S-score + SD or < mean S-score-SD. Addition- ally, and to generate an even more relevant signal and robust information, we selected the modules that were present after the 4th attenuation and that showed a stable gene content (≥ 40% of the genes with a value over the threshold) for at least 2 successive attenuations. The resulting modules were filtered for redundant gene content and functional content of the remaining mod- ules analyzed using Ontologizer v 2.0 [22]. Additionally, we used hypergeometric tests to annotate for signaling pathways (gene lists extracted from Wormbook, KEGG and Panther), druggable genes [33], htt partners [7,34] and genes involved in autophagy, mitochondrial function and synaptic activity (gene lists kindly provided by the CHDI Foundation). Information on conservation in humans was derived from Inparanoid clusters [35]. In addition to tables included herein, the results of net- work-boosted data analysis are displayed in a freely- accessible database (see http://www.broca.inserm.fr/ EHDN2/RNAiscreen) for the vizualisation of modules. This database allows the users to browse the results using GO/KEGG/Panther annotations and other annota- tions of interest, and it uses VisANT 3.5 for graph visualization [36]. We used confidence graphs for dis- play of modules by normalizing the statistical weights for functional interactions as available in Wormnet v 2.0 [26]. Results The touch receptor neurons of F1 128Q;rrf-3 animals are sensitive to RNAi To construct polyQ strains with enhanced RNAi sensi- tivity in neurons, we crossed the enhanced RNAi strain YY13 which carries the rrf-3(mg373)II mutation [19] with integrated strains that co-express N-terminal (amino acids 1-57) htt under the control of mec3-p and YFP under the control of mec7-p [14]. This resulted in strain ID448 (19Q;rrf-3) and strain ID447(128Q;rrf-3). The efficiency of gene inactivation in touch receptor neurons was tested by incubating strain ID447 L1 larvae in 96-well plates with bacteria expressing either RNAi against GFP (plasmid L4417) or empty vector (plasmid L4440) until F1 young adults are produced. Treatment with RNAi against GFP induced an overall reduction of Page 4 of 14 YFP expression levels in mec7-p target cells as illustrated in Figure 1A/B. The level of YFP mRNA expression was strongly reduced (about 80%) by RNAi against GFP compared to empty vector (Figure 1C). Additionally, the level of YFP expression in the mechanosensory neurons that control the response to touch at the tail (PLM neu- rons) was reduced by RNAi against GFP compared to empty vector (Figure 1D). Collectively, these results indicated that the touch receptor neurons of F1 young adults are sensitive to RNAi. Consistent with previous reports [37], no or little reduction of YFP levels was detected in F0 young adults (data not shown). A large-scale functional RNAi screen identifies 662 genes that modulate neuron dysfunction in expanded- polyglutamine nematodes Using 128Q nematodes in the rrf-3 sensitized back- ground, we then conducted a primary RNAi screen for the modulation of neuronal dysfunction. This screen was based on the manual quantification of response to light touch at the tail of F1 young adult animals. We screened 6260 RNAi clones equaling 6034 genes in the C. elegans genome (Figure 2). To decrease the risk for false positive, we applied a stringent threshold where the modification of touch response was considered sig- nificant if the score for touch response was outside the mean baseline ± SD*2.6 (Additional file 1: Figure S1). This screen identified 2243 RNAi clones (2211 genes) that caused toxicity phenotypes such as lethality, larval arrest, egg-laying defect, developmental delay and other abnormalities such as morphological abnormalities, 3101 RNAi clones (3054 genes) that showed no effect and 916 RNAi clones (769 genes) that modulated neuronal dysfunction (Figure 2, Additional file 2: Table S1; Addi- tional file 3: Table S2). The latter class of RNAi clones was subjected to a second screen in 128Q nematodes, which led to the identification of 664 RNAi clones con- firmed to modulate neuronal dysfunction. These clones corresponded to 662 genes, consisting of 399 genes that reduced neuronal dysfunction when knocked-down by RNAi and 263 genes that enhanced neuronal dysfunc- tion when knocked-down by RNAi (Additional file 4: Table S3). As previously reported, 128Q nematodes show at least 80% decreased touch response at the tail with a strong level of axonal dystrophy, whereas 19Q nematodes show a 45-50% reduction of touch response at the tail without significant axonal dystrophy [14]. Since our screen was based on the manual quantifica- tion of touch response in 70-90 animals/RNAi clone, which is considerably more labour-intensive and time- consuming compared to faster RNAi screens such as those looking for modifiers of mutant htt aggregation [38,39], we evaluated whether the modifiers of 128Q-