Fig. 4

Inhibition of LRRK2 kinase activity in vivo and synaptosomal presynaptic distribution of LRRK2 and α-synuclein. A C57BL/6 J mice (3 months of age) received 7 days access to control chow (n = 4) or chow with 175 mg/kg (n = 5) or 350 mg/kg (n = 5) PF-360. Forebrain homogenates were immunoblotted using antibodies to pS935-LRRK2 or total LRRK2. Total LRRK2 was quantified by normalizing to total protein on the stain free Biorad gel imaged on Chemdoc. Data is expressed as an average percentage (± SEM) of LRRK2 from control mice. pS935-LRRK2 was quantified by normalizing the signal to total LRRK2 and data is expressed as an average percentage (± SEM) of LRRK2 from control mice. One-way ANOVA revealed significant inhibition of LRRK2 kinase activity. F (2, 13) = 5.6. ***P < 0.001, ****P < 0.0001. B A separate cohort of C57BL/6 J mice were given access to control chow (n = 2) or 175 mg/kg PF-360 chow (n = 3) for 7 days. Whole brain lysates were immunoblotted for LRRK2, pS1292-LRRK2, presynaptic vGLUT1 and DAT, α-synuclein, and loading control vinculin. Relative protein expression was quantified by normalizing to (vinculin) followed by normalization to control treatment and is indicated as mean values (± SEM). pS1292-LRRK2 levels were reduced upon PF-360 treatment. C Forebrains were fractionated into total homogenate, P2 (crude synaptosomes), LP1 (synaptosome membrane fraction) and LP2 synaptic vesicle enriched fraction. Fractions were immunoblotted for LRRK2, α-synuclein, VAMP2. The right panel shows quantitation of α-synuclein relative to presynaptic VAMP2