Figure 7: WSB1 modulates LRRK2 aggregation, neuronal toxicity, and climbing behaviour in LRRK2 Drosophila models.
From: Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1
(a) Anti-WSB1 (green) and anti-elav (red) immunostaining of whole-mount adult brains derived from transgenic flies expressing wild type human WSB1 driven by the elav-promoter. Inset, Enlarged images from boxed region showing the localization of WSB1 and elav signals to the cytoplasm and nucleus, respectively. Scale bar, 50 μm. (b) Climbing score of Ddc-driven WT, G2019S, G2019S+WSB1 (lines 1–4 and 1–7) and G2019S+GFP-expressing flies at different age post-eclosion, as indicated. These data show that WSB1 rescues the climbing phenotype of G2019S Drosophila. (c) Quantification of TH+ neurons in the PPL1 cluster of WT, G2019S, G2019S+WSB1 (lines 1–4 and 1–7) and G2019S+GFP expressing flies at day 60. These data demonstrate that WSB1 rescues the neuronal toxicity in G2019S flies. (d) Immunocytochemistry of WT, G2019S, WT+WSB1, G2019S+WSB1 and G2019S+RNAi-WSB1-expressing Drosophila at day 60. Confocal images show aggregate formation in G2019S+WSB1 Drosophila (white arrow). Scale bar, 10 μm. (e) Quantification of d, indicating that there is a significant increase in aggregation in the G2019S+WSB1-expressing Drosophila and a significant decrease in aggregation in the G2019S+RNAi-WSB1-expressing Drosophila. These data together demonstrate that WSB1 modulates LRRK2 aggregation, toxicity and behavioral phenotype in an in vivo model. (f,g) WSB1 knockdown Drosophila mated to LRRK2 Drosophila. (f) Climbing score of WT, G2019S, RNAi-WSB1, WT+RNAi-WSB1, G2019S+RNAi-WSB1, expressing flies at day 60. (g) Quantification of TH+ neurons in the PPL1 cluster of WT-, G2019S-, RNAi-WSB1-, WT+RNAi-WSB1- and G2019S+RNAi-WSB1-, expressing flies at day 60. These data demonstrate that there is a significant increase in toxicity with G2019S+WSB1 RNAi compared to G2109S alone. In addition, there is a decrease in aggregation when WSB1 is knocked down (d,e). This further suggests that WSB1 regulates G2019S LRRK2 toxicity through protein aggregation. Data are the mean±s.d. *P<0.05, **P<0.001 using a one-way analysis of variance test.
