Fig. 4

LRRK2 inhibition does not reduce pathological α-synuclein in wildtype hippocampal neurons. a Primary cortical neurons were treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, at 5 DIV and fed with media containing inhibitors each week for 16 days. Cell lysate was run by Western blot to detect LRRK2 and pS935 LRRK2, which is indicative of LRRK2 activity. Images shown are representative of n = 4–12 biological replicates. b Quantification of Western blot of LRRK2 and pS935 LRRK2. LRRK2 levels were not significantly altered by one-way ANOVA, but pS935 levels were reduced to near undetectable levels (*p < 0.05, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparison test). c Primary hippocampal neurons from CD1 pups were transduced with α-synuclein PFFs and allowed to age a further 14 days prior to fixation and staining for pS129 α-synuclein (magenta), MAP2 (gray) and NeuN (blue). The neurons were additionally treated with LRRK2 inhibitors PF-475, PF-360, or MLi-2, 2 days prior to transduction and fed with media containing inhibitors each week thereafter. No large differences can be observed in the type or abundance of α-synuclein pathology. d Quantification of α-synuclein pathology reveals no change in response to LRRK2 inhibition, while PBS-treated neurons have no pathology (****p < 0.0001 by Dunnett’s multiple comparison test). MAP2 area (e) and neuron number (f) are also not altered in response to LRRK2 inhibition. No significant response was seen when compared with vehicle-treated neurons by one-way ANOVA with Dunnett’s multiple comparison test (d) or by Kruskal-Wallis test followed by Dunn’s multiple comparison test (e) and (f). (N = 9 biological replicates). Means + s.e.m.; all values are normalized to neurons treated with α-synuclein PFFs and DMSO. Scale bars = 50 μm