SNX4 is knocked-out in primary hippocampal neurons using a novel conditional knock-out mouse line.

(a) Schematic overview of conditional knock-out design of SNX4. (b) Schematic overview of experimental design using cre intervention at different DIVs. (c) Typical example of western blot of SNX4 and actin at 14 days, 18 days and 21 days of cre and control (delta cre) expression. (d) Quantification of western blot SNX4 protein levels for control (dcre), 14 days of cre, 18 days of cre and 21 days of cre, normalized to control. (e) Quantification of immunocytochemistry SNX4 intensity normalized to control. (f) Typical example of primary hippocampal neurons stained for SNX4 (magenta) and map2 (green) for control and 21 days of cre. Scale bar = 20 μm. Zoom-in scale bar = 3 μm. (g) Dendrite length for control and KO hippocampal neurons. (h) Number of synapses per neuron (MAP2 mask) based on VAMP2 puncta (shown in Fig. 6) ****p=<0.0001, ***p=0.0003, **p=0.0005, *p=0.0019. Ordinary one-way ANOVA with Tukey’s multiple comparisons test for (d) with N=3 animals. Mann-Whitney test for (e) with n=32-42 neurons per group and N=6 animals and. Data points represent individual western blots/neurons; bar graph represents mean with SEM.

SNX4 depletion increases sustained synaptic vesicle release.

(a) Schematic overview of experimental design using cre intervention at DIV0, SNX4 rescue at DIV14 and recording at DIV21. (b) Representative traces of the first evoked response for control, KO and KO+SNX4 upon stimulation. (c) Amplitude of first evoked response. (d) Charge of first evoked response. (e) Paired pulse ratios of amplitude at inter pulse intervals of 25, 50, 100 and 200 msec. (f) Mean EPSC charge trace for 100 AP at 40 Hz train. (g) Average charge at pulse 90-100. (h) Cumulative EPSC charge for 100 AP at 40 Hz train with back extrapolation linear fit to AP 80-100. (i) Estimate of RRP based on back extrapolation (h). (j) Estimate of slope based on based on back extrapolation (h). Multilevel ANOVA with n = 25-38 neurons per group of N = 4 animals. ***p < 0.0001, **p < 0.001. Data points represent individual neurons; bar graph represents mean with SEM.

Statistics overview

EPSC charge for 5 Hz and 10 Hz train stimulation.

(a) Mean EPSC charge trace for 100 AP at 5 Hz train. (b) Mean EPSC charge trace for 100 AP at 10 Hz train.

SNX4 depletion does not affect synaptic vesicle recycling at room temperature and physiological temperatures.

(a) Stimulation paradigm with a single vesicle fluorescence intensity trace. (b, Left) Representative image of a Syphy-pHluorin–infected neuron during Tyrode’s NH4 application (Scale bar: 20 μm.). (Right) Neurite before (baseline) and during (100 AP at 40 Hz) stimulation and during NH4 application (NH4) (Scale bar: 10 μm.). (c) The average fluorescence normalized from baseline to maximum Syphy intensity during Tyrode’s NH4 application. (d) Maximum response amplitude during the first electrical stimulation normalized to maximum NH4 response plotted as ΔFmax/FNH4. (e) Maximum response amplitude during the second electrical stimulation normalized to maximum NH4 response plotted as ΔFmax/FNH4. (f) Fluorescence decay time constant tau in the 60 s after electrical stimulation representing SV endocytosis and acidification (g) Percentage active synapses. (h) Stimulation paradigm with a single vesicle fluorescence intensity trace. Recycling SVs are labelled by 60s-incubation with FM4-64 in 60mM K+ at 37C, followed by a 10min washout. Electrical field stimulation (100AP, 40Hz) triggers SV exocytosis recorded as decreased FM4-64 fluorescence intensity. (i, Left) Representative image of FM64-dye endocytosed by a neuron during baseline (Scale bar: 20 μm.). (Right) Neurite before (baseline) and during (100 AP at 40 Hz) stimulation (Scale bar: 10 μm.). (j) The average FM4-64 fluorescence traces, normalized to control baseline, over time showing initial SV loading, followed by release after electrical field stimulation. (k) FM4-64 release upon electrical field stimulation (l) FM4-64 uptake (upon 60 mm K+ incubation). Multilevel ANOVA with N=3 animals and n=21-24 neurons per group for Syphy-pHluorin experiment and N=4 animals with n=28-36 neurons per group for FM64-dye experiment. Bar graph represents mean with SEM. Data points represent individual fields of view.

SNX4 depletion does not decrease VAMP2 levels and localization.

(a) Typical example immunocytochemistry images of primary hippocampal neurons stained for SNX4 (magenta), VAMP2 (red) and MAP2 (green) from control and SNX4 cKO mice. Scale bar = 20 μm. Merge zoom-in scale bar = 2 μm. (b) VAMP2 puncta per μm dendrite for control and KO. (c) VAMP2 intensity measured in dendrites. (d) Sholl analysis of average VAMP2 intensity over distance from the soma for control and KO. I Western blot of SNX4, VAMP2, and actin for control and KO. Data points represent individual neurons, N=6 animals and n=35-41 neurons for VAMP2 data, bar graphs represent mean with SEM.

SNX4 depletion increases the number of docked vesicles at the active zone.

(a) Example micrographs of control and SNX4 cKO synapses (Scale bar: 200 nm) with zoom in on the active zone region indicating docked (blue) and non-docked vesicles (pink) (Scalebar : 100 nm) (b) Schematic overview of docked and undocked vesicles. (c & d) The average vesicle size, distinguished between synaptic vesicles (<50 nm) and larger clear core vesicles (>50 nm). (e) The total amount of synaptic vesicles in the synaptic cloud. (f) The number of docked synaptic vesicles in control and SNX4 cKO synapses. (g) The active zone length. (h) The number of docked vesicles normalized to active zone length. (i) The post synaptic density (PSD) length. (j) The area of the vesicle cloud in control and SNX4 cKO synapses. Bar graphs represent mean with SEM with n = 174-175 synapses per group of N = 3 animals. Data points are individual synapses. ***p < 0.0001, **p < 0.001 using Mann-Whitney test.

Overview of uncropped blots used for western blot analysis.

(a,b,c) SNX4 at 50Kda for control (21 days of dcre), 21 days of cre, 18 days of cre and 14 days of cre. Repeated for three different animals, wk1 is normalized to actin and wk2 and wk3 are normalized to unspecific band on the blot. Fold of change is determined for all weeks by normalizing to control per week. (d) Actin at 42 kD for control, 21 days of cre, 18 days of cre and 14 days of cre. Same animal and transfer as SNX4 wk1. (e) VAMP2 at 15 kD for control, 21 days of cre, 18 days of cre and 14 days of cre. Same animal and transfer as SNX4 wk1. (f) Actin at 42 kD for control and 21 days of cre for three different animals on one blot.