Tetanus insensitive VAMP2 differentially restores synaptic and dense core vesicle fusion in tetanus neurotoxin treated neurons

摘要

TeNT efficiently cleaves VAMP1, 2 and 3, and abolishes DCV exocytosis in hippocampal neurons. ( ) Schematic representation of VAMP1, 2 and 3 proteins with TeNT cleavage site. Indicated are the transmembrane (TM, blue) and SNARE (red) domains. ( ) Western blot of cortical neurons (DIV 16), infected with a control construct or TeNT at DIV 14, incubated with antibodies against VAMP1 or VAMP2. Actin was used as loading control (full length gels are shown in Figure S2). ( ) Western blot of whole brain and cortical neurons (DIV 13), incubated with antibodies against VAMP1, VAMP2 or VAMP3 (original blots are shown in Figure S3). ( ) Representative images of a neurite stretch at DIV 14 of control construct and TeNT infected (DIV 10) hippocampal neuron, stained for dendritic marker MAP2 (magenta) and VAMP2 (green). ( ) Schematic representation of the method to detect DCV exocytosis in neurons infected with NPY-pHluorin. Electrical stimulation (16 trains of 50 AP at 50 Hz (blue bars) interspaced by 0.5 s) elicits DCV fusion with the plasma membrane, de-quenching NPY-pHluorin through an increase from pH 5.5 (in the DCV lumen) to pH 7.4. Before stimulation, NPY-pHluorin is quenched (a). During stimulation DCVs fuse with the plasma membrane visualized by a rapid increase in fluorescence (b) followed by a rapid decrease through cargo release or fusion pore closure and re-acidification (c). Scale bar 1 µm. Trace indicates F/F0 of this event. ( ) Histogram of DCV fusion events in control (black) and TeNT infected (at DIV 9–10, red) hippocampal neurons imaged at DIV 14 (blue bars indicate 16 trains of 50 APs at 50 Hz interspaced by 0.5 s). ( ) Cumulative plot of DCV fusion events in control construct and TeNT infected neurons. Shaded area represents SEM. ( ) Average DCV fusion events per cell in control (n = 21, N = 3) and TeNT (n = 21, N = 3) infected neurons. Mann–Whitney test: ***  = 4.5* 10 . Bars represent mean + SEM. Detailed statistics are shown in Supplementary Table .