Fig. 6. Modulating AMPK-PAK-myo6 Axis Affects Presynaptic Calcium Clearance.

a, Syn-GECO F/F₀ curves and quantitative analysis showing that presynaptic mitochondria accelerate Ca²⁺ clearance under intensive synaptic activity. Cortical neurons were co-transfected with DsRed-Mito and presynaptic calcium indicator Syn-GECO at DIV7, followed by time-lapse imaging of presynaptic [Ca²⁺]_i transients evoked by a train of stimulation (100 Hz, 10 sec) at DIV12–14. The peak value of F/F₀ over baseline was averaged. b-d, Representative presynaptic kymographs (b), Syn-GECO F/F₀ curves (c), and quantitative analyses (d) showing that activating or inhibiting the AMPK-PAK-myo6 axis alters presynaptic calcium clearance capacity during intensive synaptic activity. Cortical neurons at DIV7 were co-transfected with Syn-GECO along with myo6, myo6-T405A, myo6-T405E, PAK3-KD or PAK3-CA, or were treated with AICAR (1 mM) or CC (10 μM) for 2 hr, followed by time-lapse imaging of [Ca²⁺]_i transients evoked by a train of stimulation (100 Hz, 10 sec) at DIV12–14. Vertical white lines in kymographs (b) represent dynamic [Ca²⁺]_i transients at individual presynaptic terminals during a train of stimulation, where the peak value of F/F₀ over baseline were normalized (c) and averaged (d). Data were quantified from the total number of boutons (a) or neurons indicated in parentheses (a, c) or bars (d), expressed as mean ± SEM with dots as individual values (a, d), and analyzed by two-sided unpaired Student’s t-test (a), or one-way ANOVA with post hoc testing by the Holm-Sidak’s multiple comparisons test (d), where ** P <0.01 and *** P < 0.001 by comparing each condition with control (blue bar). Scale bars: 10 μm.