A. Emission spectrum analysis of the FRET substrate incubated in the absence or presence of calpain. As expected, the peak of emission was at ~480 nm. In the presence of activated calpain, a clear decrease in FRET was observed.
B. FRET substrate (arrows) was internalized into HEK-TrkB cells (lower panel). Upper panel shows phase-contrast image of the same field.
C. Kinetic of calpain activation by BDNF and EGF in HEK-TrkB cells. BDNF (50 ng/mL) or EGF (20 ng/mL) was added in cells preloaded with the FRET substrate, and fluorescence at 480 nm recorded at various times. Results are expressed as percent of T₀ and are means ± S.E.M. of 3 experiments.
D. BDNF-elicited calpain activation in HEK-TrkB cells is suppressed by MAPK inhibition or TrkB receptor inhibition. Fluorescence was measured 20 min after BDNF (50 ng/mL) addition in HEK-TrkB cells preloaded with the FRET substrate and pretreated with U0126 (10 μM) or K252a (0.5 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 6 experiments with a total of 8–18 readings/condition. ** p < 0.01 as compared to control; ‡ p < 0.01 as compared to BDNF control samples.
E. EGF-elicited calpain activation in HEK-TrkB cells is inhibited by MAP kinase inhibition or ErbB1receptor inhibition. Fluorescence was measured 20 min after EGF (100 ng/mL) addition in HEK-TrkB cells preloaded with the FRET substrate and pretreated with PD98059 (25 μM) or PD153035 (0.5 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 6 experiments with a total of 8–18 readings/condition. * p < 0.05 as compared to control; ‡ p < 0.01 as compared to EGF control samples.
F. BDNF- and EGF-induced calpain activation is blocked by calpain inhibitor III. Fluorescence was measured 20 min after BDNF (50 ng/mL) or EGF (100 ng/mL) addition in HEK-TrkB cells preloaded with the FRET substrate and pretreated with calpain inhibitor III (10 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 4 experiments with 3–4 readings/experiment. ** p < 0.01 as compared to control; ‡ p < 0.01 as compared to BDNF AND EGF control samples.

A. Hippocampal neurons were preloaded with the FRET substrate and treated with BDNF (50 ng/mL) for 10 min or EGF (20 ng/mL) for 2 min. They were then fixed, stained with phalloidin-Alexa Fluor594 and processed for confocal microscopy. Decrease in FRET signal (increased fluorescence) was observed in dendrites as well as in dendritic spine-like structures labeled with phalloidin-Alexa Fluor594 (arrows).
B. Hippocampal neuronal cultures were pretreated with calpain inhibitor III (10 μM) for 20 min before adding vehicle or BDNF (50 ng/mL) for 20 min. At the end of incubation, actin polymerization was analyzed as described under Materials and Methods with the rhodamine-phalloidin fluorescence enhancement assay. Results of the rhodamine-phalloidin fluorescence were normalized (by subtracting control fluorescence signals) and represent means ± S.E.M. of 5 experiments. * p < 0.001 (ANOVA followed by Bonferroni test).

A. BDNF-elicited calpain activation in cortical neurons is blocked by inhibitors of MAP kinase and TrkB receptor. Fluorescence was measured 20 min after BDNF (50 ng/mL) addition to cortical neurons preloaded with the FRET substrate and pretreated with U0126 (10 μM) or K252a (0.5 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 5 experiments with a total of 6–14 readings/condition. * p < 0.05 as compared to control; ‡ p < 0.01 as compared to BDNF control samples.
B. EGF-elicited calpain activation in cortical neurons is inhibited by MAP kinase inhibition or ErbB1receptor inhibition. Fluorescence was measured 20 min after EGF (20 ng/mL) addition to cortical neurons preloaded with the FRET substrate and pretreated with PD98059 (25 μM) or PD153035 (0.5 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 6 experiments. * p < 0.05 as compared to control; ‡ p < 0.01 as compared to EGF control samples.
C. BDNF- and EGF-induced calpain activation in cortical neurons is blocked by calpain inhibitor III. Fluorescence was measured 20 min after BDNF (50 ng/mL) or EGF (20 ng/mL) addition to cortical neurons preloaded with the FRET substrate and pretreated with calpain inhibitor III (10 μM). Results are expressed as normalized fluorescence (percent of untreated control values) and are means ± S.E.M. of 6–8 experiments. ** p < 0.01 as compared to control; ‡ p < 0.01 as compared to BDNF or EGF control samples.

Top. Representative images of BDNF (50 ng/mL)- or EGF (20 ng/mL)-induced changes in FRET in hippocampal neurons pre-incubated with BAPTA-AM (BAPTA) 10 min after addition of BDNF or EGF (see Materials and Methods for details). Calpain activation is still observed in dendrites (arrows).
Bottom. Primary cortical neurons pre-loaded with the FRET substrate were incubated with BAPTA-AM (50 μM) for 20 min before application of BDNF (50 ng/mL) or EGF (20 ng/mL) for 20 min. Normalized FRET signal was measured using spectrofluorometry. Results are expressed as normalized fluorescence and are means ± S.E.M. of 4 experiments with a total of 7–14 readings/condition. * p < 0.05 as compared to control.

A. HEK-TrkB cells were pretreated with vehicle or the MAP kinase inhibitor, PD98059 (25 μM) for 15 min before being treated with BDNF (50 ng/mL) for 60 min. Cell lysates were processed for immunoprecipitation with m-calpain or μ-calpain antibodies and immunoprecipitated proteins were immunoblotted with anti-m-calpain, anti-μ-calpain or anti-phosphoserine antibodies. Levels of serine-phosphorylated m-calpain and serine phosphorylated μ-calpain were quantified and results expressed as percent of control. Results are means ± S.E.M. of 4 experiments. * p < 0.01 as compared to control; † p < 0.05 as compared to BDNF-treated samples.
B. Cortical neurons were pretreated with vehicle or the MAP kinase inhibitor, PD98059 (25 μM) for 15 min before being treated with BDNF (50 ng/mL) for 60 min. Cell lysates were processed for immunoprecipitation with m-calpain antibodies and immunoprecipitated proteins were immunoblotted with anti-m-calpain or anti-phosphoserine antibodies. Levels of serine-phosphorylated m-calpain were quantified and results expressed as percent of control. Results are means ± S.E.M. of 4 experiments. * p < 0.01 as compared to control; † p < 0.05 as compared to BDNF-treated samples.

Cultured hippocampal neurons were preloaded with the FRET substrate as described in Materials and Methods. A&B. FRET analysis of calpain activation by BDNF (50 ng/mL, A) or EGF (20 ng/mL, B) at the indicated time points. Arrows indicate spine-like structures.

Cortical neurons were preloaded with the FRET substrate and treated with BDNF (A, 50 ng/mL) or EGF (B, 20 ng/mL) for the indicated periods of time. They were pretreated without or with the PKA inhibitor, KT5720 (10 μM) for 20 min before BDNF or EGF treatment. At the indicated times, cells were trypsinized (PBS buffer containing 5 mg/mL trypsin and 2 mg/mL EDTA for 10 min) and cell lysates were processed for spectrofluorometric analysis as described in Materials and Methods. Fluorescence intensity was expressed as percent of values measured at time 0 and results are means ± S.E.M. of 6 experiments from independent cell dishes. KT5720 alone had no effect on fluorescence intensity (not shown).

Cultured cortical neurons (DIV14) were transfected with 40 μM siRNA diluted in Neurobasal media and 40 μl of HiPerfect, as described under Materials and Methods. After 5 days cells were preincubated with the FRET reagent for 3 h. They were then treated with vehicle (control), EGF (20 μg/ml) or BDNF (50 μg/ml) for 30 min. Cells were lysed and fluorescence in the lysates was determined (A). Aliquots of cell lysates were also processed for western blots with m- (B) or μ-(C) calpain antibodies (actin antibodies were used as loading control). Fluorescence intensity results are expressed as percent of values found in control-treated non-transfected neurons and are means ± S.E.M. of 3 experiments. ** p < 0.01 as compared to the respective control values. Levels of m- and μ-calpain were normalized and expressed as percent of values found in control-treated non-transfected neurons and are means ± S.E.M. of 9 experiments. ** p < 0.01 as compared to control values.

Cortical neurons were pre-treated with vehicle or the MAP kinase inhibitor, PD98059 (25 μM) for 15 min before being treated with BDNF (50 ng/mL) or EGF (20 ng/mL) for 60 min. Cell lysates were processed for western blotting with spectrin antibodies and actin antibodies (as a loading control). Levels of the 145–150 kD calpain specific breakdown products were quantified, and expressed as percent of values found in control cultures. Results are means ± S.E.M. of 6 independent experiments. * p < 0.01, as compared to control; ‡p < 0.05 as compared to BDNF-or EGF-treated samples.