Fig 2: Presynaptic localization of SNAP47 in the CA3 area of mouse and rat hippocampus. (A) Confocal images of the co-localization of SNAP47 (red pseudocolor, left) and the presynaptic marker ZnT3 (in green, middle left) in the CA3 area of the mouse hippocampus. Note that SNAP47 shows weak labeling and weak co-localization with ZnT3 in the stratum lucidum corresponding to the mossy fiber (MF) terminals (see the superimposed signals and the colocalization on the rigth). (B) Confocal images of the co-localization of SNAP47 and the presynaptic marker ZnT3 in the CA3 area of rat hippocampus. The labeling for SNAP47 is strong in the stratum lucidum and shows substantial overlap with ZnT3. (C) Sections double stained against SNAP47 (red pseudocolor) and the presynaptic marker vesicular glutamate transporter 1 (VGLUT1; in green) in the CA3 area of the mouse hippocampus. (D) Sections double stained against SNAP47 and the presynaptic marker VGLUT1 in the CA3 area of the rat hippocampus. Scale bar represent, (A–D), 50 μm.

Fig 3: TRIM67 alters exocytosis by modulating SNAP47 protein levels(A) Immunoblot of SNAP47 and GAPDH from lysate of Trim67+/+ and Trim67−/− cortical neurons at 2 DIV. Quantification of percent increase of SNAP47/GAPDH from 4 blots (right; log-paired t test, analysis in STAR methods).(B) Frequency of VAMP2-pHluorin exocytosis ± tagRFP or tagRFP-SNAP47 (n = 16 neurons per condition, n = 3 biological experiments, Welch’s t test).(C) Relative proportions of exocytic modes in Trim67+/+ neurons, Trim67−/− neurons, or Trim67+/+ neurons expressing tagRFP-SNAP47 (n = 16 neurons per condition; n = 3 biological replicates; multivariate linear regression, error bars report standard error of the percent).(D) tagRFP-SNAP47-normalized fluorescence intensity at fusion sites (n = 16 neurons, n = 3 biological replicates; Welch’s t test with Benjamini-Hochberg correction).(E) Quantification of time after peak ΔF/F before fluorescence decay onset of KNRd and FVFd events (n = 17 neurons; n = 4 biological replicates; Welch’s t test).(F) t1/2 of each exocytic class ± TagRFP-SNAP47 (n = 17 neurons per condition; Welch’s t test with Benjamini-Hochberg correction).(G) Measured neuronal surface areas at indicated times and conditions.(H) Example SNAP47 immunocytochemistry images in neurons expressing siRNA for SNAP47 knockdown (KD) (GFP+, arrow) or without (*). Cells were transfected with an eGFP expression plasmid and SNAP47 siRNA, followed by immunostaining of SNAP47; GFP-positive neurons did not stain for SNAP47, suggesting KD of SNAP47. (H’) Immunoblot and quantification of SNAP47 and βIII tubulin protein levels in SNAP47 KD compared with control (n = 3 experiments; Welch’s t test).(I) Frequency of VAMP2-pHluorin exocytosis ± SNAP47 siRNA (n = 12 neurons per condition; Welch’s t test).(J) Relative proportions of exocytic classes in Trim67+/+ neurons and Trim67−/− neurons, with SNAP47 KD (n = 12 neurons per condition, n = 3 biological replicates; multivariate linear regression, error bars report standard error of the percent).

Fig 4: Immunolabeling intensities for SNAP47 in YFP-positive INs and surrounding neuropil in the mouse and the rat hippocampus. Summary bar charts of the immunolabeling intensity for SNAP47 in the somata of YFP-positive INs and surrounding neuropil in the CA1 area (A), the CA3 area (B) and the DG (C) of the mouse and the rat hippocampus. *P = 0.04, ****P < 0.0001.

Fig 5: Ultrastructural localization of SNAP47 in axon terminals and dendrites of GABAergic INs of the mouse hippocampus. (A) Postembedding immunogold labeling for SNAP47 (10 nm gold, arrowheads) in the cytoplasm of a putative interneuron cell body (icb) in the CA3 stratum radiatum associated to the endoplasmic reticulum (ER). Scale bar: 500 nm. (B) A putative GABAergic basket cell terminal (GABAergic terminals, gt) forming symmetric contact onto a pyramidal cell body (pcb) in the CA3 stratum pyramidale. Note that postembedding immunogold labeling for SNAP47 (10 nm gold; arrowheads) was preferentially found postsynaptically in the pyramidal cell body and only at lower level presynaptically in the inhibitory axon terminals. Scale bar: 200 nm. (C) Putative gt making symmetric contacts with the axon initial segment (AIS) of a pyramidal cell in the CA3 region. Arrowheads indicate immunogold labeling for SNAP47 (10 nm gold) found at low level in the axon terminals. Scale bar: 500 nm. (D) Putative gt making symmetric contact with a dendritic shaft (pd) of a putative pyramidal cell in the CA3 region. Arrowheads indicate immunogold labeling (10 nm gold) for SNAP47 found in pyramidal dendritic shaft and at low level in the axon terminal. Scale bar: 200 nm. (E) A putative interneuron dendritic shaft (id) receiving convergent asymmetrical, presumably excitatory synapses with small axon terminals (at) in the CA1 stratum radiatum. Arrowheads indicate immunogold labeling (10 nm gold) for SNAP47 found in the dendritic shaft and in the axon terminal. Scale bar: 500 nm.