Figure 6.

Motoneuron cell death triggered by Fas activation in the presence of trophic factors involves caspases-3 and -8. (A) Activation of caspase-3 in cultured mouse motoneurons treated with anti-Fas was visualized using the CM1 antibody, which specifically recognizes the activated form of caspase-3. The motoneuron illustrated was undergoing apoptosis, as visualized by the fragmented chromatin of the nucleus stained with DAPI (inset). Note the intense perinuclear staining for activated caspase-3. (B) Increase in the fraction of CM1-positive mouse motoneurons 30 h after treatment with anti-Fas antibodies in the presence of neurotrophic factors. After immunostaining, CM1-positive motoneurons were counted along two diameters of 14-mm coverslips (∼100 motoneurons counted for each). All CM1-positive cells showed pyknotic nuclei by DAPI staining. Values are means ± SEM of five coverslips, and are typical of two independent experiments (asterisks indicate P = 0.001 by t test). Total survival was not significantly different in the two conditions (P > 0.5; data not shown). (C) Death of E14 rat motoneurons triggered by sFasL (10 ng/ml) in the presence of BDNF (1 ng/ml) is inhibited in a dose-dependent fashion by the caspase-3 peptide inhibitor DEVD-fmk. (D) Death of mouse motoneurons triggered by sFasL (10 ng/ml) and enhancer (1 μg/ml), or anti-Fas antibodies (10 ng/ml), is blocked by DEVD-fmk (10 μM). Survival is expressed as the percentage of the number of motoneurons surviving in the presence of BDNF alone. (E) Death of rat motoneurons triggered by sFasL (10 ng/ml, in the presence of enhancer) is inhibited by the caspase-8 inhibitor IETD-fmk (1 μM). C, D, and E show combined mean values (± S.D.) from duplicate wells in two independent experiments.