Fig. 5.

Fas ligand overexpression in enriched cultured cortical neurons. A, Expression of Fas ligand and Fas receptor in cortical neurons transfected with a gene encoding Fas ligand (see Materials and Methods). E16 cortical neurons were transfected with a recombinant adenoviral vector with or without Fas ligand. At 48 hr after transfection, cell lysates were subjected to Western blot using an anti-Fas ligand antibody (N-20) or an anti-Fas antibody (M-20). Fas receptor was constitutively expressed and did not change, whereas Fas ligand was upregulated in neurons transfected with vector containing the gene encoding Fas ligand (rAd–FasL) but not by control vector (rAd–LacZ). B, Coimmunoprecipitation of Fas–procaspase-8 in cultured cortical neurons transfected with Fas ligand. E16 neurons were transfected with adenoviral vectors as inA. At 48 hr after transfection, cell lysates were incubated with anti-caspase-8 antibody (H-134), and the immunoprecipitants were separated by SDS-PAGE and probed with anti-Fas antibody (clone 13). Fas–procaspase-8 coimmunoprecipitation was increased in overexpressing cortical neurons. IP, Immunoprecipitation antibody. C, Neuronal death induced by Fas ligand in vitro is mediated in part by caspases. Cell death was assessed at 72 hr in cortical neurons overexpressing Fas ligand (trypan blue staining). Eighty percent of neurons transfected with Fas ligand died (vertical stripes) versus control vector (diagonal stripes) (*p < 0.05). Pretreatment with ZVAD-fmk (500 μm;open bar) reduced Fas ligand-induced cell death by ∼50% at 72 hr after transfection compared with vehicle (0.5% DMSO; solid bar) (**p < 0.05 vs control).