Fig. 1

Increased presynaptic colocalization of BDNF-GFP caused by IL-1β as an indicator of reduced endosome transport. Presynaptic colocalization of BDNF-GFP was examined by immunostaining of BDNF-GFP and synaptophysin. Neurons were fixed and analyzed after 1 h post-treatment to assess BDNF-GFP endosome dispersion through neurites (illustrated by traces) in culture. a–d Representative images that show neurons treated with BDNF-GFP (a and c) or BDNF-GFP + IL-1β (b and d) and stained with antibodies against GFP [34], synaptophysin [52], and tau-1 (blue). Scalebars: 10 μm in a–b and 2 μm in c–d. e Quantification of the colocalization of BDNF-GFP and synaptophysin in neurons treated with BDNF-GFP or BDNF-GFP + IL-1β. IL-1β treatment was associated with a 2.6-fold increase in BDNF-GFP/synaptophysin colocalization. Graph depicts mean ± SE (as fold relative to BDNF); BDNF = 2.6 ± 0.4, n = 3 vs BDNF + IL-1β = 1.0 ± 0.2, n = 3 independent cultures. f P2 fractions were isolated from three independent cultures and pooled as an alternate means of colocalization. By flow cytometry analysis, forward-Side (FSC-SSC) density plot shows the size-complexity profile of particles in the P2 fraction isolated from hippocampal cultures. The gate (inside rectangle) selects according to size (0.75 < gated particles < 3.0 μm), relative to calibrated beads (f, lower panel) [34, 52]. g The population of size-gated synaptosomes was probed for the presence of the presynaptic marker synapsin-1 and BDNF-GFP. The presence of GFP signal in synapsin-1-positive events was used as an indicator BDNF-GFP retained in presynaptic compartments. BDNF + IL-1β groups exhibited a fourfold increase in GFP events colocalized with synapsin-1 (n = 3 independent cultures pooled). Thresholds for endogenous/non-specific fluorescence for each marker were set using untreated (no BDNF-GFP) synaptosomal fractions stained with an IgG isotype-matched conjugated to Alexa 647