Fig 1: AP1-mediated sorting of VAMP4 to endolysosomes regulates its abundance in the SV pool.(A, C, and E) Representative images display hippocampal neurons transfected with VAMP4-EGFP and mCherry, a-synuclein A53T, or red fluorescent protein (RFP)–bassoon (Bsn) (A), VAMP4-EGFP–transfected neurons with and without 30 µM SMIFH2 (C) and VAMP4-EGFP cotransfected with either AP1 shRNA (AP1 KD) or a scrambled (Scr) control (E). Scale bars, 5 µm. (B, D, and F) Synaptic VAMP4 expression under all conditions described in (A), (C) and (E). (B) n = 40 (mCherry), n = 51 (a-synuclein A53T), and n = 53 (bassoon) cells from three independent preparations, ****P < 0.001 and **P = 0.0038, Kruskal-Wallis test with Dunn’s multiple comparisons test (P values adjusted). (D) n = 47 (control) and n = 61 (SMIFH) cells from three independent preparations (F), n = 18 coverslips (scrambled and AP1 KD) from four independent preparations, ***P = 0.0009 and **P = 0.0085, Mann-Whitney test. (G and H) Hippocampal neurons were transfected with either wild-type or VAMP4 L25A–FT. (G) Representative images display fluorescence in the blue (new protein), red (old protein), or merged channel. Scale bar, 5 µm. (H) Blue/red ratio [n = 30 (wild-type) and n = 39 (L25A) cells from four independent preparations, ****P < 0.001, Mann-Whitney test]. (I to K) Hippocampal neurons cotransfected with VAMP4-pHluorin and or shRNAs against either AP1 or CHC (or scrambled). Representative images (I) (scale bar, 5 µm) and quantification of VAMP4-pHluorin expression (J) in either scrambled or CHC KD synapses [n = 12 (scrambled), n = 14 (CHC KD) coverslips from three independent preparations, *P = 0.011, Mann-Whitney test]. (K) VAMP4-pHluorin fusion events [n = 12 (scrambled), n = 10 (AP1 KD), and n = 14 (CHC KD) coverslips from three independent preparations, ****P < 0.0001 and **P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons test].
Fig 2: Endolysosomal sorting of VAMP4 regulates its abundance in the SV pool.(A and B) Hippocampal neurons cotransfected with VAMP4-pHluorin and either wild-type (WT), constitutively active (Q67L), or dominant-negative (T22N) mCherry-rab7. Scale bar, 5 µm. Representative images (A) and bar graph (B) display synaptic VAMP4-pHluorin expression. n = 23 (mCherry), n = 12 (mCherry-rab7), n = 13 (Q67L), and n = 18 (T22N) coverslips from four independent preparations, ****P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons test. (C) Effect of T22N mCherry-rab7 overexpression on sypHy synaptic expression. n = 21 (mCherry) or n = 16 (T22N mCherry-rab7) coverslips from four independent preparations, ***P < 0.0002, Mann-Whitney test. (D) Fusion events reported by VAMP4-pHluorin upon stimulation with 400 APs at 40 Hz under the conditions shown in (A) and (B), n = 23 (mCherry), n = 12 (mCherry-rab7), n = 13 (Q67L), and n = 18 (T22N) coverslips from four independent preparations, *P = 0.0129, Kruskal-Wallis test with Dunn’s multiple comparisons test.
Fig 3: VAMP4 targeting to endolysosomes controls Pr.(A) Strong synaptic stimulation triggers ADBE, which recycles SV membranes and proteins, such as syb2 and VAMP4, following SV fusion. ADBE forms transient bulk endosomes from which vesicles bud to (i) refill the recycling SV pool (ii) or fuse with (mature to) endolysosomes for long-range trafficking and degradation. Proteins are sorted at bulk endosomes to either endolysosomes or SVs by different mechanisms. VAMP4 is predominantly sorted to endolysosomes via an AP1-dependent mechanism, whereas syb2 and, to a lesser extent, VAMP4 are sorted into SVs by different adaptor proteins. Both sorting pathways require clathrin for their function. (B) In wild-type nerve terminals, inhibition of either endolysosomal trafficking or AP1 function abolishes VAMP4 sorting to endolysosomes and results in its increased sorting in SVs. VAMP4 accumulation in the SV pool reduces SV fusion capacity and Pr, although increased numbers of VAMP4 molecules visit the surface during stimulation. (C) In VAMP4 KO terminals, loss of VAMP4 causes an increase in Pr under basal conditions due to loss of inhibitory control. In addition, blocking of endolysosomal trafficking and function does not affect Pr, highlighting the essential role of VAMP4 in converting endolysosomal dysfunction into changes of presynaptic release properties.
Fig 4: Synaptic accumulation of VAMP4 lowers SV fusion competence.Primary cultures of hippocampal neurons were cotransfected with syp-mOr2 and either VAMP4-pHluorin or syb2-pHluorin. Neurons were stimulated with 400 APs (40 Hz) and pulsed with NH4Cl imaging buffer after 200 s. (A and B) Representative images of the fluorescent response of syp-mOr2 (A and B), VAMP4-pHluorin (A), and syb2-pHluorin (B) are displayed at rest, during stimulation, or during exposure to NH4Cl [white arrows, nerve terminals that display a syp-mOr2 response (active synapses); yellow arrows, those that do not (silent synapses)]. Scale bar, 5 µm. (C and D) Hippocampal neurons transduced with PSD95-EGFP after a prior transfection with syp-mOr2 were subjected to an identical protocol as above. (C) Representative images of syp-mOr2 and PSD95-EGFP at rest, during stimulation, or during NH4Cl exposure (white arrows, active synapses; yellow arrows, silent synapses). Scale bar, 5 µm. (D) Number of active or silent syp-mOr2 puncta colocalizing with PSD95-EGFP (n = 13 coverslips from four independent preparations, P = 0.82, Mann-Whitney test). (E and F) Number of synapses with fusion events reported by pHluorin (E) or the expression level of pHluorin at synapses that show an activity-dependent syp-mOr2 response (F). n = 15 coverslips, each from four independent preparations, ****P < 0.001 and ***P = 0.001, Mann-Whitney test. (G to I) Expression level of syb2-pHluorin (G), sypHy (H), and VAMP4-pHluorin (I) at active and silent synapses. n = 10 (G) or n = 14 (H and I) coverslips from four independent preparations, ***P = 0.0005, **P = 0.002, and *P = 0.039, either Wilcoxon matched-pairs signed-rank test (G and I) or paired t test (H). (J to L) Correlation between the extent of the sypHy response with the expression of sypHy (J) or the syp-mOr2 response with the expression of syb2-pHluorin (K) and VAMP4-pHluorin (L). ns, not significant.
Fig 5: VAMP4 is retrieved constitutively from axons through the endolysosomal system.(A) Representative kymographs display the axonal mobility and directionality of traffic of VAMP4-EGFP and syb2-EGFP. (B and C) Frequency of anterograde, retrograde, or stationary trajectories (B) or the run length of retrograde trajectories (C) for both syb2-EGFP and VAMP4-EGFP. n = 25 (syb2) or n = 16 coverslips (VAMP4) from four independent preparations, ****P < 0.001 and **P = 0.0002, two-way ANOVA with Fisher’s least significant difference (B), and **P = 0.0012, unpaired t test (C). (D and E) Hippocampal neurons were transfected with syb2, synaptophysin, or VAMP4 tagged with an FT protein. (D) Representative images display FT protein fluorescence in either the blue (new protein), red (old protein), or merged channel. Scale bar, 5 µm. (E) Blue/red ratio of FT proteins. n = 24 (syb2 and synaptophysin) or n = 23 cells (VAMP4) from four independent preparations, ****P < 0.001 and ***P = 0.0004, Kruskal-Wallis test with Dunn’s multiple comparisons test. (F to H) Hippocampal neurons were transfected with mCherry-rab7 and either syb2-EGFP or VAMP4-EGFP. (F and G) Kymographs show the retrograde cotrafficking of syb2-EGFP and VAMP4-EGFP with mCherry-rab7. (H) Fraction of retrograde trajectories where syb2-EGFP or VAMP4-EGFP cotraffic with mCherry-rab7. n = 16 (syb2) or n = 18 (VAMP4) coverslips from four independent preparations, ****P < 0.001, Mann-Whitney test.
Supplier Page from Abcam for Anti-VAMP4 antibody