Amyloid-Beta Induced Changes in Vesicular Transport of BDNF in Hippocampal Neurons

Figure 5

Release of BDNF was not affected after expression of mutated hAPP. Dissociated hippocampal neurons from 5xFAD mice and wild-type littermates, respectively, were transfected at 10 DIV with a BDNF-GFP plasmid. Depolarization-induced release of BDNF-GFP as well as the incidence of fusion event of BDNF-containing vesicles was analyzed by live cell imaging. (a) Representative single vesicles of a hippocampal neuron transfected with BDNF-GFP showing fusion events as indicated by the change in fluorescence intensity (green, yellow, and blue) compared to a single vesicle showing no fusion pore opening (red). For most events, BDNF-GFP fluorescence increased due to the neutralisation of the intragranular pH after fusion. BDNF release was observed as decrease in fluorescence intensity after fusion event. At the end of the release measurement, neurons were superfused with BPB (0.3 mM) to differentiate between vesicles in an open or closed state at this time point (scale bar = 1 µm). (b) Graph shows time course of fluorescence intensity of color coded vesicles shown in (a). Change in fluorescence intensity indicates neutralisation of intravesicular pH after fusion pore opening of BDNF-GFP-containing vesicles (fluorescence increase) and depolarization-induced release of BDNF-GFP (fluorescence decrease), respectively. (c) Average time course of GFP-fluorescence intensity of single hippocampal neurons. Note the absence of any difference in release efficiency or time course between neurons from 5xFAD animals and wild-type littermates. (d) Quantification of the fluorescence decrease after fusion event. The maximal fluorescence intensity after fusion was set to 100% and the remaining fluorescence intensity 300 s after fusion pore opening was analyzed. BDNF release amplitude was unaltered in hippocampal neurons derived from 5xFAD mice. (e) Quantification of depolarization-induced fusion events. Bar diagram shows the percentage of fusion events of vesicles in hippocampal neurons from 5xFAD animals and wild-type littermates (n.s. = no significant difference; one-way ANOVA). Error bars represent SEM.

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