Fig. 1: Pharmacological inhibition of γ-secretase slows synaptic vesicle endocytosis.
From: A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aβ synaptotoxicity
A Synaptophysin-pHluorin (SypHy) imaging of autapses in microisland cultures of cortical neurons reveals synaptic vesicle exo- and endocytosis elicited by electrical stimulation (400 stimuli at 20 Hz; indicated by blue bar). Time courses of SypHy fluorescence in control (black trace) and L-685,485 (γ-secretase inhibitor, 5 µM for 2 days; magenta trace) treated neurons. Individual SypHy puncta on a transfected neuron were averaged per cell and normalized to the NH4Cl signal. B The maximal SypHy fluorescence signal did not significantly differ between control and L-685,485 treated neurons. Left and middle panel: SypHy puncta of each cell (control: n = 10; L-685,485 treated: n = 16) were averaged. Individual values for each cell (left) and mean ± SEM (middle) are shown. Right panel: Cumulative distributions of individual SypHy puncta of all cells recorded (control: n = 108 puncta; L-685,485: n = 185 puncta). C Percent loss of SypHy signal (% endocytosis) 90 s after the end of stimulation was significantly reduced in L-685,485 treated neurons. Left and middle panel: SypHy puncta of each cell were averaged. Right panel: Cumulative distributions of individual SypHy puncta of all cells recorded. D The decay time constant of SypHy fluorescence decay was significantly increased in L-685,485 treated neurons. Monoexponential fit of the average time course of SypHy signals from all puncta of a given cell (A) was used. Individual values for each cell (left) and mean ± SEM (right) are shown. Student’s t test; **P < 0.01; n.s. non significant. E–G Western blot analysis of the major sheddase-dependent C-terminal fragments (CTF1) of the indicated synaptic adhesion proteins upon pharmacological inhibition of γ-secretase in cultured hippocampal neurons. Note the strong increases in CTF1 fragments of N-cadherin (E, Ncad-CTF1), neurexins (F, NRX-CTF1; anti-Neurexin 1/2/3 antibody), and neuroligin1 (G, NLG1-CTF1) following treatment with the γ-secretase inhibitor L-685,485 (GSI). DMSO: vehicle control. Native: no treatment. β-actin was used as loading control.
