Fig. 2.

The signaling ability of p75 is required for NGF-dependent apoptosis in oligodendrocytes. A, Schematic diagram of the mutant-p75 lacking the cytoplasmic domain. The cytoplasmic domain of this mutant receptor was replaced with the corresponding domain of the kinase-dead EGF receptor. Thearrows indicate two independent CMV promoters, one directing expression of the mutant p75 and the other directing the expression of GFP. All the adenoviruses used in this study coexpress GFP. B, The mutant p75 binds ¹²⁵I-NT3. Cos cells were infected with the full-length (FL) p75 or the mutant (Mut) p75 adenovirus and subjected to cross-linking with ¹²⁵I-NT3. PC12 cells were used as a positive control (lane 1), and uninfected Cos cells were used as a negative control (lane 2). The FL-p75 was immunoprecipitated with 9992 antibody (lane 3), and the mutant p75 was immunoprecipitated with HA antibody (lane 4). C, The mutant p75 protects oligodendrocytes from NGF-dependent apoptosis. Oligodendrocytes were infected with GFP control or the mutant p75 adenovirus in four-well slide dishes for 24 hr at 150 pfu/cell. After 4 hr of NGF treatment, cells were stained for TUNEL. The number of TUNEL⁺cells was determined among GFP⁺ cells. The quantitation data are from three to five independent experiments, each with 200–300 cells counted for a total of 600–1500 cells.D, The mutant-p75 inhibits JNK activation in oligodendrocytes. Twenty-four hours after infection with the viruses, oligodendrocytes were treated with NGF at 100 ng/ml for 4 hr. The changes in JNK activity were measured by solid-phase kinase assays. The presence of the mutant-p75 was detected with anti-HA antibody, and the presence of the JNK protein was detected with anti-JNK antibody.E, Representative picture of oligodendrocytes quantified after infection with adenoviruses and NGF treatment. The cells expressing the mutant p75 were identified by GFP fluorescence, because the virus also expresses GFP as well as the mutant p75 cDNA. Thearrows indicate the TUNEL⁺ cells among GFP⁺ cells. Scale bars, 20 μm.EGFR-kin⁻, A kinase-dead EGF receptor;Ext, extracellular domain.