Fig. 3

LRRK2 kinase activity regulates propagation of α-synuclein. a-d The accumulation of internalized α-synuclein in Triton X-100 soluble fraction (Tx sol) (a, b) and Triton X-100 insoluble fraction (Tx insol) (c, d). The α-synuclein monoclonal antibody Syn-1 from BD Biosciences was used for detection of α-synuclein. NT represents non-treated control. Arrowheads in a and c indicate α-synuclein monomer. Bar on the right side of blot in c represents the α-synuclein aggregates. Asterisk in a represents non-specific binding of antibody. Quantified regions were indicated on the right as an arrowhead in a and a line in (c). Black open circle: Mock, blue closed square: LRRK2 WT, black closed circle: LRRK2 G2019S (GS), red closed triangle: LRRK2 D1994A (DA). b, d Relative levels of internalized α-synuclein in Tx sol (b) and Tx insol (d). b N = 3, Transgene F(3,24)=60.79, Time F(2,24)=264.8, Interaction F(6,24)=28.42, ** p < 0.01, **** p < 0.001. d N = 3, Transgene F(3,24)=13.23, Time F(2,24)=352.2, Interaction F(6,24)=7.237, * p < 0.05, *** p < 0.005, Graphs in b, d were analyzed by two-way ANOVA with Dunnet’s post hoc test. e, f Alterations in LRRK2 kinase activity regulate propagation of α-synuclein. e The effects of LRRK2 kinase activity on the propagation of α-synuclein. Arrowhead: Venus BiFC puncta. Blue: Nuclei. Scale: 20 μm. f The number of Venus BiFC (+) cell. The color bars in graph (f) represent the number of Venus BiFC puncta in Venus BiFC fluorescence (+) cells. Five independent experiments were performed. Two hundred cells were analyzed per each experiment. Y axis represents the number of Venus BiFC(+) cells out of 200 cells analyzed. Transgene F(3,48)=3.09, Puncta number F(2,48)=172.1, Interaction F(6,48)=10.4, ns: not significant, *** p < 0.005, **** p < 0.001, #### p < 0.001 by two-way ANOVA with Tukey’s post hoc test. g Effects of LRRK2 kinase activity on the secondary secretion of Venus (+) α-synuclein aggregates. N = 6, F(3,20)=44.77, ns: not significant, *** p < 0.005, **** p < 0.001, #### p < 0.001 by one-way ANOVA with Tukey’s post hoc test. h–j Pharmacological inhibition of LRRK2 kinase activity diminishes LRRK2-induced α-synuclein propagation. h The autophosphorylation of LRRK2. i The propagation of α-synuclein. Arrowhead: Venus BiFC puncta. Blue: Nuclei. Scale: 20 μm. j Quantification of Venus BiFC fluorescence (+) cell (%). Four independent experiments were performed. Two hundred cells were analyzed per each experiment. Treatment F(1,24)=24.12, Transgene F(3,24)=41.28, Interaction F(3,24)=13.35, ns: not significant, *** p < 0.005, ++++ p < 0.0001, # p < 0.05, ### p < 0.005, #### p < 0.0001 by two-way ANOVA with Tukey’s post hoc test. Data are represented as mean ± SEM