Figure 8.

Regulation of FasL and FLIP by neurotrophic factors. (A and B) Levels of molecules involved in Fas signaling were followed using semiquantitative RT-PCR on motoneurons cultured for 1 and 3 DIV in the presence or absence of neurotrophic factors. (A) FasL mRNA is upregulated in motoneurons cultured in the absence of trophic factors. (B) Regulation of fas and FLIP mRNAs. mRNA samples were incubated with (+) or without (−) reverse transcriptase. Fas was expressed at relatively constant levels when normalized to actin, whereas FLIP was strongly upregulated in motoneurons cultured for 3 DIV in the presence of NTFs. (C) Strong upregulation of FLIP protein in the presence of neurotrophic factors. Western blots are shown of extracts of purified motoneurons cultured for 1 and 3 DIV in the presence of NTFs; two major forms of FLIP at 55 and 52 kD are observed. The loading control was neurofilament-68. (D) Immunolabeling of Fas-resistant mouse motoneurons cultured for 3 d in the presence of NTFs, using the blot-purified polyclonal antibodies against Fas characterized in Fig. 2. Fas continues to be expressed at the cell membrane. (E) Motoneurons treated for 3 d with NTFs also continue to express caspase-8. (F) Summary diagram showing the potential involvement of the Fas system in PCD of motoneurons. The main experimental strategies adopted in our study of the role of Fas are depicted. In addition, we have indicated the three levels at which neurotrophic factors may potentially act to block Fas-related motoneuron death.