Fig. 1

LRRK2 kinase activity modulates synaptic vesicle fusion. a We recorded synaptophluorin fluorescence from DIV14 wild-type cortical neurons treated with DMSO (control) or treated with LRRK2 inhibitor GSK2578215A (GSK in, 0.2 μM, 2 h) and from cortical neurons obtained from BAC hG2019S mice (hG2019S). Representative snapshots were taken at DIV16 from 1 Hz recordings at rest (0), after 40 action potential stimulation (40AP), after 300 action potential stimulation (300AP) and upon neutralization with 50 mM NH₄Cl to reveal total fluorescence (Fmax). Panels size is 113 × 113 μm. b The graph shows representative pattern of fluorescence. Y-axis reports ΔF/F0 at the given time point (second). c The graphs report the increase in fluorescence after 40AP (ΔF40/F0) and 300AP (ΔF300/F0) and the kinetic of signal after 300AP expressed as time constant describing the increase (tau upstroke) and decay (tau decay) of fluorescence. Data are expressed as mean ± SEM, n = 4, at least 50 boutons from minimum five neurons were analyzed for experiment (* p < 0.05; ** p < 0.01 versus control, ANOVA). d The exo/endocytotic assay was performed at DIV14 on wild-type and BAC hG2019S cortical neurons infected at DIV4 with virus expressing GFP. Cycling SV appears as synaptotagmin (s-tagmin) positive clusters along neuron processes. Total SV pool was revealed by staining with anti-synaptophysin antibodies upon fixation and permeabilization. Images show signals acquired for synaptotagmin, synaptophysin and their superimposition plus GFP (merge). Panel size is 28 × 4 μm. (e) The percentage of s-tagmin and s-physin positive clusters within the totality of s-physin positive clusters reflects the pool of cycling vesicles. Data are expressed as mean ± SEM, n = 4, at least 7 neurons were analyzed for experiment (** p < 0.01 Student’s t-test)