FRET based monitoring of caspase activity in differentiated SH-SY5Y cells exposed to Aβ
. Differentiated neuroblastoma cells expressing FRET sensor molecules were treated with oligomer-enriched preparations of Aβ1-42 at concentrations known to induce apoptosis. (A) FRET intensity time-lapse imaging of the caspase-3 and -6 sensors in cells treated with 20 μM oligomer-enriched Aβ1-42. Time is indicated on top. The frame before the initial drop in FRET intensity was chosen as zero time point for the caspase-3 (DEVD) and -6 (VEID) sensors, whereas initiation of neurite retraction was chosen as zero time point for the control (LEVA) sensor. Corresponding graphs show the quantitative change in FRET intensity in the soma (blue squares) and neurites (red dots). (B) Western blot analysis using anti-GFP antibodies, of FRET sensor molecules in extracts of cells expressing the caspase-6 (VEID) sensor incubated for 18 hours in the absence (lane 1) or presence (lane 2) of 10 μM of oligomer-enriched Aβ1-42 preparation. Staurosporine treatment (2 μM, 18 h) is shown for comparison. (C) Image of cell expressing the caspase-6 (VEID) sensor treated with oligomer-enriched Aβ1-42 showing loss of FRET signal, but maintained distribution of EYFP fluorescence along microtubules. ECFP fluorescence was diffuse. (D) FRET intensity time-lapse imaging of the caspase-9 (LEHD) sensor and corresponding quantitative graphic representation. Note the maintained high FRET intensity before and after neurite retraction, which was chosen as zero time point.