Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

Abstract

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin(-/-) neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin(-/-) phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca(2+) sensor, we demonstrate that snapin-dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca(2+) sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.

Keywords: BLOC‐1; dynein motor; dysbindin; endosomal sorting and transport; snapin.

Figure 1

Snapin-deficient neurons display enlarged presynaptic terminals retaining various degradative organelles

1. A, B Representative electron micrographs (A) and quantitative analysis (B) showing aberrant accumulation of degradative organelles within enlarged presynaptic terminals of snapin^−/− cortical neurons at DIV14. Colored arrowheads indicate various types of degradative organelles: red, AVs; blue, endosomal vacuoles; and green, MVBs/LEs. All these organelles were not readily observed at WT presynaptic terminals.

2. C, D Representative images (C) and quantitative analysis (D) showing enhanced intensity of synaptophysin in snapin^−/− neurons. Cortical neurons at DIV14 were co-immunostained with antibodies against SV protein synaptophysin (green) and dendritic marker MAP2 (red).

3. E Sequential immunoblots of synaptosomal fractions (Syn), cytosolic fractions (Cytosol), and total brain lysates (Total) showing elevated endolysosomal marker LAMP-1 and autophagy marker LC3-II (red boxes) in synapse-enriched preparations from snapin cKO mice at P40. Snapin is relatively enriched in synaptosomal fractions in WT mouse brains and is almost absent in synaptosomes from snapin cKO animals (green box). Note that synaptic protein levels (i.e., synaptotagmin) are not necessarily increased in synaptosomal preparations from adult snapin cKO mice, while EM and immunocytochemistry revealed increased SVs levels in snapin^−/− presynaptic terminals in in vitro culture conditions. A possible explanation for this discrepancy is that oversized and dysfunctional terminals might be eliminated by microglia in adult snapin cKO animals to optimize network activity and survival.

Data information: Data were analyzed from the total number of electron micrographs (B) or neurons (D) indicated in parentheses (n) above bar graphs (B) or within bars (D) taken from three pairs of mice for each genotype and expressed as means ± s.e.m. with Student’s t-test (B) and Mann–Whitney U-test (D). P-values: *0.01–0.05; ***0.0001 to 0.001; **** < 0.0001. Scale bars: 200 nm (A) and 20 μm (C).

Figure 2

Snapin mutants disturb LE retrograde transport in axons

1. A Representative GST-DIC pull-down illustrating distinct binding capacities of WT and mutant snapin with dynein intermediate chain (DIC). Note that the L99K mutation abolished snapin binding to DIC and the S50D mutation robustly decreased snapin–DIC coupling. The same membranes were sequentially blotted with anti-His and anti-GST antibodies. Lower panels demonstrate similar amount of GST or GST-tagged DIC used for the pull-down.

2. B, C Representative kymographs (B) and quantitative analysis (C) showing the relative motility of axonal LEs during 5-min time-lapse imaging from cortical neurons at DIV14. Neurons were co-transfected with GFP-Rab7 and a pcDNA vector alone as a control or expressing WT or mutant snapin as indicated. Vertical lines correspond to stationary organelles; oblique traces reflect retrograde (leftward) or anterograde (rightward) transport. Motile or stationary organelles were normalized to the total number of organelles per axon segment selected for recording and averaged from the total number of axons indicated in parentheses. Retrograde transport of Rab7-labeled LEs was robustly inhibited by expressing snapin-L99K and reduced by expressing snapin-S50D. Data are means ± s.e.m., Kruskal–Wallis test with Dunn’s post hoc test. P-values: *0.01–0.05; **0.001 to 0.01; **** < 0.0001. (See also Supplementary Fig S1.)

Figure 3

Snapin-mediated LE retrograde transport regulates total SV pool size

1. A, B Representative images (A) and quantitative analysis (B) showing the impact of snapin and its mutants on the intensity of SV and active zone markers. WT cortical neurons co-transfected with HA-synaptophysin or GFP-bassoon and snapin constructs at DIV8, followed by immunostaining with antibodies against MAP2 and HA-tag at DIV14. Relative fluorescence intensities of HA-synaptophysin and GFP-bassoon were normalized to control neurons. Note that over-expressing WT snapin decreases, whereas the L99K and S50D mutants increase synaptophysin puncta intensities. As an internal control, expressing snapin or its mutant has no detectable effect on the intensity of GFP-bassoon.

2. C, D Representative images (C) and quantitative analysis (D) showing the rescue of snapin-deficient boutons by snapin and its mutants. snapin^−/− cortical neurons were co-transfected with HA-synaptophysin and snapin constructs as indicated at DIV3, and immunostained with antibodies against MAP2 and HA-tag at DIV14. Relative fluorescence intensities of HA-synaptophysin were normalized to snapin^−/− neurons. WT snapin, but not the L99K or S50D mutants, rescues HA-synaptophysin-tagged SV fluorescence intensity to the wild-type level.

3. E, F Sample kymographs (E) and quantitative analysis (F) illustrating the dynamic trafficking of recycling SV cargoes along axons of cortical neurons co-transfected with VGluT-pHluorin-mCherry and snapin or the L99K mutant. Active axons were selected based on the pHluorin response to 100 APs, and mobile VGluT-labeled SV cargoes were tracked through mCherry during 2.5-min dual-channel recordings. Note that over-expressing snapin enhances SV cargoes dynamics while the L99K mutant decreases trafficking.

4. G Sample kymographs illustrating co-trafficking of Tomato-synaptophysin with GFP-LAMP-1-labeled LEs along axons from cortical neurons at DIV14. Axonal trafficking was monitored in neurons co-expressing the SV marker Tomato-synaptophysin and endolysosomal marker GFP-LAMP-1 during 5-min dual-channel time-lapse acquisitions. Arrowheads indicate retrogradely moving organelles containing both synaptophysin and LAMP-1.

5. H Sample kymographs illustrating co-trafficking of VGluT-pHluorin with mApple-LAMP-1-labeled LEs along axons from cortical neurons at DIV14. VGlut-pHluorin fluorescence was elicited by 600 APs at 10 Hz in the presence of 1 μM bafilomycin to prevent SV re-acidification; axonal transport was monitored during 5-min dual-channel acquisitions. Arrowheads indicate retrogradely moving organelles containing both SV recycling material (VGluT-pHluorin) and LAMP-1.

Data information: Scale bars: 10 μm. The total number of images (B, D) or time-lapse videos (F) analyzed is indicated within bars. Data are means ± s.e.m., one-sample t-test, where each value is compared to the normalized control value (B, D); or ordinary one-way ANOVA with Dunnett’s post hoc test (F). P-values: *0.05–0.01; **0.001 to 0.01; *** < 0.001. Live imaging data were recorded during 2.5 (E) or 5 min (G, H). (See also Supplementary Fig S2.)

Figure 4

Altered SV exocytosis occurs at snapin-deficient presynaptic terminals

1. A Sample pHluorin images from synapses expressing VGluT-pHluorin-mCherry before and during a 10-Hz, 10-s stimulation (100 APs) in cortical neurons at DIV14. Exposure of pHluorin to the alkaline extracellular media during exocytosis gives rise to green fluorescence emission.

2. B, C Average pHluorin traces (B) and peak amplitudes (C) elicited by 20 (20 Hz, 1 s), 100 (10 Hz, 10 s), or 1,500 (5 Hz, 300 s) APs. Deleting snapin decreases total releasable pool size without affecting SVs recycling rate during a prolonged (300 s) stimulation. The total number of boutons analyzed is indicated within the columns. Data are means ± s.e.m., Student’s t-test. P-value: **** < 0.0001.

Figure 5

Snapin mutations differentially affect presynaptic activity

1. A, B Average pHluorin traces (A) and corresponding peak amplitudes (B) elicited by 20 (left), 100 (middle), or 1,500 (right) APs from cortical neurons at DIV14. Note that over-expressing snapin or snapin-L99K decreases releasable pool size without affecting the recycling rate. Conversely, expressing snapin-S50D accelerates decay during a prolonged stimulation without affecting the releasable pool size. Data are means ± s.e.m., ANOVA with Dunnett’s post hoc test, where each value is compared to the control. The total number of boutons imaged is indicated within the columns. P-value: **** < 0.0001.

2. C, D Average pHluorin traces elicited by 1,500 APs in the presence of 1 μM of the V-ATPase inhibitor bafilomycin (C) and sample kymographs illustrating pHluorin responses from control cells in the absence (left) or presence (right) of 1 μM bafilomycin (D). Note that in the presence of bafilomycin, trafficking of the pHluorin signal out of boutons becomes readily detectable. “n” indicates the total number of boutons imaged and averaged. Error bars represent s.e.m. (See also Supplementary Figs S3 and S4.)

Figure 6

A snapin mutant defective in dysbindin binding impairs SV positional priming

1. A Schematic illustration of Ca²⁺ imaging using the high-sensitivity green fluorescent Ca²⁺ sensor GECO. The synaptophysin-GECO chimera selectively reports SV exposure to activity-triggered Ca²⁺ influx, while soluble GECO serves as a reporter for global Ca²⁺ levels within terminals.

2. B Representative images (left) and average traces of Ca²⁺ imaging and SV cycling (right) during a 100-AP stimulation. Dual-channel time-lapse imaging of synaptophysin-GECO and pH-sensitive VGluT-mOrange demonstrates that the newly developed probe is targeted to active synapses. Average traces illustrate the relative timing of Ca²⁺ influx with exo- and endocytosis. Data are means ± s.e.m. from 60 boutons. Scale bar: 5 μm.

3. C, D Average synaptophysin-GECO or GECO traces (C) and peak values (D) recorded from cortical neurons at DIV14 in response to 100 APs. Neurons were co-transfected with either synaptophysin-GECO or GECO and snapin or its mutants, as indicated. Note that SVs experience higher Ca²⁺ levels in terminals over-expressing snapin and lower Ca²⁺ levels in the ones over-expressing snapin-L99K. The global Ca²⁺ waveform monitored through soluble GECO was unchanged.

4. E, F Average pHluorin traces (E) and peak amplitudes (F) elicited by 20 (left), 100 (middle), or 1,500 (right) APs in the presence of 6 mM extracellular Ca²⁺. Elevating extracellular Ca²⁺ levels rescues release in synapses expressing snapin-L99K that impairs positional priming, but fails to rescue snapin-over-expressing boutons where the release defect is attributable to reduced SV pool size by enhancing SV transport into the endolysosomal pathway.

Data information: Data are means ± s.e.m., ANOVA with Dunnett’s post hoc test, where each value was compared to the control. P-values: **** < 0.0001. The total number of boutons analyzed is indicated within columns. (See also Supplementary Fig S5.)

Figure 7

Deleting snapin affects SV molecular identity with BLOC-1/AP-3 inhibition

1. A, B SVs were immuno-isolated from synaptosomal preparations of wild-type and snapin cKO (flox/flox; thy/Cre) adult mouse brains using magnetic beads coated with an antibody against synaptophysin. Sequential immunoblotting was conducted on the same membrane after stripping between each antibody application (A). Quantification of protein intensities from three independent experiments (B). Note the reduced amounts of Rab3a and the AP-3-dependent cargo Ti-VAMP in purified SVs from snapin cKO mouse brains (red boxes). Data are means ± s.e.m.

2. C Model illustrating the bipartite regulation of synaptic activity by the endolysosomal sorting and trafficking pathways. Late endosomal transport regulates SV pool size by shuttling SV components into the endolysosomal pathway (Route 1), while BLOC-1/dysbindin/AP-3-dependent endosomal sorting regulates SV composition, and consequently positional priming and release probability (Route 2). By balancing these two dynamic pathways, snapin coordinates the releasable pool size and Ca²⁺ sensitivity of neurotransmitter release.