Fig. 4

RAB35 mediates LRRK2-induced propagation of α-synuclein. a-c Localization of transferred α-synuclein in the co-culture of α-synuclein overexpressing SH-SY5Y cells (donor cells) and naive SH-SY5Y cells (recipient cells labeled with Q tracker). a The localization of transmitted α-synuclein was analyzed by co-immunostaining with RAB proteins (RAB1, RAB8, and RAB35). Red: RAB proteins, Blue: Qdot 585, recipient cell marker, arrowhead: transmitted α-synuclein, colocalized with RAB proteins, scale bar: 20 μm. The percentage of RAB (+) transmitted α-synuclein was quantified in b. Three independent experiments were performed. Three hundred cells were analyzed per each experiment. F(2,6)=16.09, * p < 0.05 by one-way ANOVA with Dunnet’s post hoc test. The pearson’s coefficient was calculated in (c). Three independent experiments were performed. Fifty cells were analyzed per each experiment. F(2,6)=68.1, *** p < 0.005 determined by one-way ANOVA with Dunnet’s post hoc test. d–g Interaction between LRRK2 and RAB35. WT: LRRK2 WT, GS: LRRK2 G2019S. d Pull-down assay of 6X histidine conjugated LRRK2 with nickel beads. The relative levels of eluted RAB35 were calculated in e. N = 3, F(3,8)=39.08, ns: not significant, **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test. f Immunoprecipitation of RAB35 with 3F10, monoclonal antibody against HA tag. Mouse IgG was used as a control. The relative levels of eluted LRRK2 were quantified in g. N = 3, F(3,8)=66.84, ns: not significant, **** p < 0.0001 by one-way ANOVA with Tukey’s post hoc test. h, i Dominant-negative mutant RAB35 S22N (RAB35 DN) ameliorated LRRK2-induced-propagation of α-synuclein. Propagation of α-synuclein aggregates were calculated by measuring the Venus BiFC (+) cell (%). Arrowhead: Venus BiFC (+) puncta, red: RAB35, blue: nuclei, scale: 20 μm. The percentage of Venus BiFC (+) cells was calculated in (i). Five independent experiments were performed. Three hundred cells were analyzed per each experiment. F(4,20)=15.79, ns: non-significant, ** p < 0.01 by one-way ANOVA with Dunnet’s post hoc test. Data are represented as mean ± SEM